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Abstraqcts: HGF and HCC

(Rogler and Chisari 1992; Shiota, Rhoads et al. 1992; Boix, Rosa et al. 1994; Selden, Farnaud et al. 1994; Shiota, Kawasaki et al. 1994; Shiota, Nakamura et al. 1994; Tameda and Horiguchi 1994; Yada 1994; Bouzahzah, Nishikawa et al. 1995; Shiota, Kawasaki et al. 1995; Shiota, Okano et al. 1995; Shiota, Umeki et al. 1995; Burr, Hillan et al. 1996; Hu, Evarts et al. 1996; Nakayama, Kashiwazaki et al. 1996; Shiota, Kawasaki et al. 1996; Yamaguchi, Nalesnik et al. 1996; Yamaguchi, Nalesnik et al. 1996; Chen, Seol et al. 1997; Ljubimova, Petrovic et al. 1997; Neaud, Faouzi et al. 1997; Newell, Alonso et al. 1997; Singh, Tsan et al. 1997; Tomiya, Hayashi et al. 1997; Ueki, Fujimoto et al. 1997; Ueki, Fujimoto et al. 1997; Hakawa, Kouda et al. 1998; Kamiyama, Une et al. 1998; Murakami, Matsuura et al. 1998; Shiota and Kawasaki 1998; Terada, Nakanuma et al. 1998; Bell, Chen et al. 1999; Hu, Lee et al. 1999; Hu, Lee et al. 1999; Huang, Lee et al. 1999; Junbo, Li et al. 1999; Luo, Wu et al. 1999; Murakami, Sakukawa et al. 1999; Nakanishi, Fujimoto et al. 1999; Okano, Shiota et al. 1999; Suzuki, Mori et al. 1999; Utsunomiya, Iemura et al. 1999; Guirouilh, Castroviejo et al. 2000; Jo, Stolz et al. 2000; Neaud, Hisaka et al. 2000; Suzuki, Hayashida et al. 2000; Tavian, De Petro et al. 2000; Gil-Benso, Martinez-Lorente et al. 2001; Jiang, Xu et al. 2001; Nagata, Hirono et al. 2001; Rao, Gollin et al. 2001; Xie, Liu et al. 2001; Yamagami, Moriyama et al. 2001; Horiguchi, Takayama et al. 2002; Monvoisin, Bisson et al. 2002; Orian-Rousseau, Chen et al. 2002; Price, Kovach et al. 2002; Qin and Tang 2002; Radaeva, Jaruga et al. 2002; Varnholt, Asayama et al. 2002; Yamagamim, Moriyama et al. 2002; Daveau, Scotte et al. 2003; Hah and Lee 2003; Iimuro and Fujimoto 2003; Kataoka, Miyata et al. 2003; Mabed, Aref et al. 2003; Ozaki, Mizuta et al. 2003; Tanimizu, Nishikawa et al. 2003; Cheng, Imanishi et al. 2004; Efimova, Glanemann et al. 2004; Gujdar, Sipeki et al. 2004; Lai, Chien et al. 2004; Nowak, Stellbrink et al. 2004; Vejchapipat, Tangkijvanich et al. 2004; Yan, Wang et al. 2004; Heideman, Overmeer et al. 2005; Imai, Kubota et al. 2005; Kuroki, Tajima et al. 2005; Oosthuizen, Ndaba et al. 2005; Osada, Kanematsu et al. 2005; Park, Lee et al. 2005; Ranganathan, Tan et al. 2005; Sakaida, Kimura et al. 2005; Sripa, Leungwattanawanit et al. 2005; Ueno, Sakurai et al. 2005; Breuhahn, Longerich et al. 2006; Farazi, Zeisberg et al. 2006; Han, Michalopoulos et al. 2006; Llovet, Chen et al. 2006; Majka, Drukala et al. 2006; Nakanishi, Moriuchi et al. 2006; Wu, Zheng et al. 2006; Hsia, Huo et al. 2007; Hu, Ho et al. 2007; Kitisin, Pishvaian et al. 2007; Mas, Maluf et al. 2007; Orian-Rousseau, Morrison et al. 2007; Skopkova, Penesova et al. 2007; Tward, Jones et al. 2007; Yang, Magilnick et al. 2007; Yoshida, Hirano et al. 2007; Zhao, Yue et al. 2007; Abdel Aziz, El-Miligy et al. 2008; Absood, Hu et al. 2008; Chau, Lui et al. 2008; Kim, Lee et al. 2008; Martinez-Lopez, Varela-Rey et al. 2008; Nussbaum, Samarin et al. 2008; Osada, Kanematsu et al. 2008; Sagmeister, Eisenbauer et al. 2008; Shen, Wong et al. 2008; Shen, Yang et al. 2008; Sun, Wan et al. 2008; Zender and Kubicka 2008; Apte, Gkretsi et al. 2009; Huynh, Ngo et al. 2009; Kaposi-Novak 2009; Kaufmann, Oettel et al. 2009; Ke, Shi et al. 2009; Kogure, Ueno et al. 2009; Li, Fu et al. 2009; Mizuguchi, Nagayama et al. 2009; Patil, Lee et al. 2009; Salvi, Sabelli et al. 2009; Tung, Wong et al. 2009; Xie, Tang et al. 2009; Bae, Bissonette et al. 2010; Chang, Liu et al. 2010; Chen, Shi et al. 2010; Costantini, Capone et al. 2010; Fornari, Milazzo et al. 2010; Henry, Park et al. 2010; Hora, Romanque et al. 2010; Lee, Ladu et al. 2010; Liu, He et al. 2010; Malaguarnera, Giordano et al. 2010; Oliva, French et al. 2010; Osada and Yoshida 2010; Paranjpe, Bowen et al. 2010; Porta and Paglino 2010; Quiles-Perez, Munoz-Gamez et al. 2010; Trusolino, Bertotti et al. 2010; Whittaker, Marais et al. 2010; Xie, Xing et al. 2010; Zhuang, Zhang et al. 2010; Cornella, Alsinet et al. 2011; Hora, Romanque et al. 2011; Nagai, Arao et al. 2011; Wang, Xu et al. 2011)

Abdel Aziz, M. T., D. El-Miligy, et al.; (2008). "Molecular evaluation of apoptotic versus antiapoptotic angiogenic markers in hepatocellular carcinoma." Clin Biochem; 41(12); 1008-1014.

                OBJECTIVE: To assess the role of HO-1 in HCC progression and to study the expression of apoptotic factors represented by TNF-alpha, and Fas-L versus antiapoptotic and angiogenic factors represented by HO-1, TGF-beta, HGF, and VEGF in HCC compared to non cancerous cirrhotic liver. DESIGN AND METHODS: Liver biopsies were taken from twelve patients with grade II HCC confined to the liver and twelve patients with non cancerous liver cirrhosis (served as control). RT-PCR of previous genes was evaluated. RESULTS: HO-1, VEGF, HGF, and TNF-alpha genes were significantly increased (P0.05). HGF showed significant positive correlation with HO-1 (r=0.8217, P=0.001). CONCLUSION: HCC is associated with increased expression of VEGF, HGF, and TGF-beta, and with suppression of Fas-L. In addition, HO-1 is highly significantly expressed in HCC. The significant positive correlation between HO-1 and HGF was first reported in Egyptian human liver biopsies, and this suggests that it may play a role in the progression of hepatocellular carcinoma.

Absood, A., B. Hu, et al.; (2008). "VIP inhibits human HepG2 cell proliferation in vitro." Regul Pept; 146(1-3); 285-292.

                Hepatocellular carcinoma (HCC) is an aggressive and often fatal neoplasm. HepG2 cells are a cell line derived from HCC. This investigation shows that vasoactive intestinal peptide (VIP) inhibits HepG2 cell proliferation in vitro. In addition, VIP decreases the expression of signal transducers and activators of transcription-3 (STAT-3) and phosphorylated STAT-3 (pSTAT-3). Transfection of HepG2 cells with STAT-3 siRNA also dose-dependently inhibits proliferation. These findings suggest that VIP-mediated inhibition of HepG2 proliferation may be mediated by STAT-3. Further studies demonstrate that VIP increases HepG2 cAMP levels and 8-cl-cAMP inhibits HepG2 proliferation as well as pSTAT-3 and STAT-3 levels, suggesting that cAMP is also involved in the inhibition of HepG2 proliferation. VIP also attenuates the proliferative effects of hepatocyte growth factor (HGF) and interleukin-6 (IL-6) on HepG2 cells. These preliminary studies suggest that the antiproliferative actions of VIP may offer a new and promising means of suppressing HCC.

Apte, U., V. Gkretsi, et al.; (2009). "Enhanced liver regeneration following changes induced by hepatocyte-specific genetic ablation of integrin-linked kinase." Hepatology; 50(3); 844-851.

                Following liver regeneration after partial hepatectomy, liver grows back precisely to its original mass and does not exceed it. The mechanism regulating this "hepatostat" is not clear and no exceptions have been found to date. Although pathways initiating liver regeneration have been well studied, mechanisms involved in the termination of liver regeneration are unclear. Here, we report that integrin-linked kinase (ILK) (involved in transmission of the extracellular matrix [ECM] signaling by way of integrin receptors) and/or hepatic adaptations that ensue following ILK hepatocyte-targeted removal are critical for proper termination of liver regeneration. Following partial hepatectomy (PHx), mice with a liver-specific ILK ablation (ILK-KO-Liver) demonstrate a termination defect resulting in 58% larger liver than their original pre-PHx mass. This increase in post-PHx liver mass is due to sustained cell proliferation driven in part by increased signaling through hepatocyte growth factor (HGF), and the beta-catenin pathway and Hippo kinase pathways. Conclusion: The data indicate that ECM-mediated signaling by way of ILK is essential in proper termination of liver regeneration. This is the first evidence of a defect leading to impaired termination of regeneration and excessive accumulation of liver weight following partial hepatectomy.

Bae, M. H., G. B. Bissonette, et al.; (2010). "Hepatocyte growth factor (HGF) modulates GABAergic inhibition and seizure susceptibility." Exp Neurol; 221(1); 129-135.

                Disrupted ontogeny of forebrain inhibitory interneurons leads to neurological disorders, including epilepsy. Adult mice lacking the urokinase plasminogen activator receptor (Plaur) have decreased numbers of neocortical GABAergic interneurons and spontaneous seizures, attributed to a reduction of hepatocyte growth factor/scatter factor (HGF/SF). We report that by increasing endogenous HGF/SF concentration in the postnatal Plaur null mouse brain maintains the interneuron populations in the adult, reverses the seizure behavior and stabilizes the spontaneous electroencephalogram activity. The perinatal intervention provides a pathway to reverse potential birth defects and ameliorate seizures in the adult.

Bell, A., Q. Chen, et al.; (1999). "The five amino acid-deleted isoform of hepatocyte growth factor promotes carcinogenesis in transgenic mice." Oncogene; 18(4); 887-895.

                Hepatocyte growth factor (HGF) is a polypeptide with mitogenic, motogenic, and morphogenic effects on different cell types including hepatocytes. HGF is expressed as two biologically active isotypes resulting from alternative RNA splicing. The roles of each HGF isoform in development, liver regeneration and tumorigenesis have not yet been well characterized. We report the generation and analysis of transgenic mice overexpressing the five amino acid-deleted variant of HGF (dHGF) in the liver by virtue of an albumin expression vector. These ALB-dHGF transgenic mice develop normally, have an enhanced rate of liver regeneration after partial hepatectomy, and exhibit a threefold higher incidence of hepatocellular carcinoma (HCC) beyond 17 months of age. Moreover, overexpression of dHGF dramatically accelerates diethyl-nitrosamine induced HCC tumorigenesis. These tumors arise faster, are significantly larger, more numerous and more invasive than those appearing in non-transgenic littermates. Approximately 90% of female dHGF-transgenic mice had multiple macroscopic HCCs 40 weeks after injection of DEN; whereas the non-transgenic counterparts had only microscopic nodules. Liver tumors and cultured tumor cell lines from dHGF transgenics showed high levels of HGF and c-Met mRNA and protein. Together, these results reveal that in vivo dHGF plays an active role in liver regeneration and HCC tumorigenesis.

Boix, L., J. L. Rosa, et al.; (1994). "c-met mRNA overexpression in human hepatocellular carcinoma." Hepatology; 19(1); 88-91.

                This study was aimed at assessing the presence of c-met overexpression in human hepatocellular carcinoma and at determining whether this feature is associated with a definite clinical or pathological characteristic. Expression of c-met was determined by Northern-blot hybridization of a specific probe (human met proto-oncogene) in 18 tumoral and nontumoral liver samples obtained in 18 cirrhotic patients with hepatocellular carcinoma submitted to surgical treatment. Eight of the 18 hepatocellular carcinomas exhibited c-met overexpression, with an increase ranging between 2-fold and 10-fold when compared by densitometry with the surrounding liver. By contrast, in the remaining 10 cases c-met expression was almost identical to that of the surrounding nontumoral liver tissue. Overexpression of c-met was not related to either the age, sex, etiology or functional status of the underlying liver disease, or to the size of the tumor, to its differentiation degree or to the presence of pseudocapsule invasion and existence of additional neoplastic nodules. These data indicate that almost half of the human hepatocellular carcinomas exhibit c-met overexpression. Nevertheless, the biological relevance of this characteristic is not known.

Bouzahzah, B., Y. Nishikawa, et al.; (1995). "Growth control and gene expression in a new hepatocellular carcinoma cell line, Hep40: inhibitory actions of vitamin K." J Cell Physiol; 165(3); 459-467.

                The growth characteristics of a newly established cell line, Hep40, derived from a human hepatoma are described. An absolute requirement was found for serum to mediate cell growth. Neither EGF, TGF-alpha, nor HGF altered cell growth in the presence or absence of serum. A partial suppression of cell growth was achieved by several TGF-beta family proteins. Affinity crosslinking gels using 125I-labeled TGF-beta showed a significant decrease in the TGF-beta cell-surface type II receptor in Hep40 cells, compared to the TGF-beta-sensitive Hep3B cell line. However, growth could be completely suppressed by addition of vitamins K to the culture medium in both Hep40 and several other hepatoma cell lines. Growth suppression by vitamins K was accompanied by an increased level of transcripts for c-myc, c-jun, and prothrombin genes, in contrast to the actions of TGF-beta 1 protein, which caused a decrease in the level of c-myc transcripts. These data show that this new human hepatoma cell line has partial resistance to growth inhibition by TGF-beta with a unique TGF-beta receptor defect. However, growth was completely suppressed by vitamins K. The differing gene expression patterns in response to TGF-beta as compared to vitamin K suggest that these two growth inhibitors act through differing pathways.

Breuhahn, K., T. Longerich, et al.; (2006). "Dysregulation of growth factor signaling in human hepatocellular carcinoma." Oncogene; 25(27); 3787-3800.

                Dysregulation of pleiotropic growth factors, receptors and their downstream signaling pathway components represent a central protumorigenic principle in human hepatocarcinogenesis. Especially the Insulin-like Growth Factor/IGF-1 receptor (IGF/IGF-1R), Hepatocyte Growth Factor (HGF/MET), Wingless (Wnt/beta-catenin/FZD), Transforming Growth Factor alpha/Epidermal Growth Factor receptor (TGFalpha/EGFR) and Transforming Growth Factor beta (TGFbeta/TbetaR) pathways contribute to proliferation, antiapoptosis and invasive behavior of tumor cells. This review focuses on the relevant alterations in these pathways identified in human human hepatocellular carcinomas (HCCs). Resultant functional effects are modulated by multiple cross-talks between the different signaling pathways and additional tumor-relevant factors, such as cyclooxygenase-2 and p53. Several specific strategies are currently under development such as receptor kinase inhibitors, neutralizing antibodies and antagonistic proteins, which may improve the systemic treatment of human HCCs.

Burr, A. W., K. J. Hillan, et al.; (1996). "Hepatocyte growth factor levels in liver and serum increase during chemical hepatocarcinogenesis." Hepatology; 24(5); 1282-1287.

                Hepatocyte growth factor (HGF) is mitogenic for hepatocytes and some tumor cell lines. Elevations in plasma HGF levels have been detected in patients with hepatocellular carcinoma (HCC), and it is possible that HGF is involved in the promotion and/or progression of tumor growth. We measured serum and liver tissue HGF levels during chemically induced hepatocarcinogenesis. Wistar rats were given diethylnitrosamine (DEN) in drinking water for 10 weeks with controls receiving drinking water only. Animals were killed at 10, 16, and 19 weeks. Liver HGF levels were determined from immunoblotted protein by scanning densitometry, and serum HGF levels were measured by sandwich enzyme-linked immunosorbent assay (ELISA). HGF was also immunolocalized in fixed liver tissue sections. In DEN-treated animals, at 10 weeks, there was necroinflammation but no dysplasia. Serum HGF was elevated compared with controls (P

Chang, J., Y. Liu, et al.; (2010). "Hepatopoietin Cn suppresses apoptosis of human hepatocellular carcinoma cells by up-regulating myeloid cell leukemia-1." World J Gastroenterol; 16(2); 193-200.

                AIM: To investigate the role of hepatopoietin Cn (HPPCn) in apoptosis of hepatocellular carcinoma (HCC) cells and its mechanism. METHODS: Two human HCC cell lines, SMMC7721 and HepG2, were used in this study. Immunostaining, Western blotting and enzyme linked immunosorbent assay were conducted to identify the expression of HPPCn and the existence of an autocrine loop of HPPCn/HPPCn receptor in SMMC7721 and HepG2. Apoptotic cells were detected using fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide. RESULTS: The HPPCn was highly expressed in human HCC cells and secreted into culture medium (CM). FITC-labeled recombinant human protein (rhHPPCn) could specifically bind to its receptor on HepaG2 cells. Treatment with 400 ng/mL rhHPPCn dramatically increased the viability of HCC-derived cells from 48.1% and 36.9% to 85.6% and 88.4%, respectively (P

Chau, G. Y., W. Y. Lui, et al.; (2008). "Significance of serum hepatocyte growth factor levels in patients with hepatocellular carcinoma undergoing hepatic resection." Eur J Surg Oncol; 34(3); 333-338.

                BACKGROUND: Hepatocyte growth factor (HGF) is a potent hepatocyte mitogen and may stimulate the proliferation and invasiveness of human hepatocellular carcinoma (HCC) cells through the c-met receptor. This study evaluates the significance of serum HGF levels in patients undergoing HCC resection. STUDY DESIGN: The peripheral and portal sera and HCC and non-tumorous tissues of 40 HCC patients, with tumor TNM stage I (n=12), II (n=17), and III (n=11) diseases, who underwent hepatic resection were prospectively collected. Serum HGF levels were determined by enzyme-linked immunosorbent assay. The c-met protein expressions were examined by immunohistochemistry. Median follow-up time was 69 months. RESULTS: The prehepatectomy portal HGF levels (median, 622pg/mL) were significantly higher than peripheral HGF levels (564pg/mL) (P=0.026). The posthepatectomy portal HGF levels (699pg/mL) were significantly higher than prehepatectomy portal HGF levels (P699pg/mL (P699pg/mL and with a positive c-met expression in HCC tissue have the worst survival. CONCLUSIONS: In HCC patients, high peripheral and portal HGF serum levels related with poor prognosis after hepatic resection. Hepatocyte growth factor and c-met receptor can be targets of future HCC postoperative treatment.

Chen, J. A., M. Shi, et al.; (2010). "Angiogenesis: multiple masks in hepatocellular carcinoma and liver regeneration." Hepatol Int; 4(3); 537-547.

                Hepatocellular carcinoma (HCC) is naturally resistant to radiotherapy and cytotoxic chemotherapy, leaving surgery as the mainstream therapeutic approach. However, the 5-year recurrence rate after curative resection is as high as 61.5%. The background hepatitis B- or C-induced cirrhosis and the presence of micrometastases at the time of surgery have been regarded as two main causes of recurrence. Recently, accumulating evidence suggests that growth factors and cytokines released during the physiological process of post-surgical liver regeneration could induce the activation of dormant micrometastatic lesions. The establishment of neovasculature to support either liver regeneration or HCC growth involves multiple cell types including liver sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, and circulating endothelial progenitors. The crosstalks among these cells are driven by multiple molecules and signaling pathways, including vascular endothelial growth factors and their receptors, platelet-derived growth factor, the angiopoietin/Tie family, hepatocyte growth factor/c-Met signaling, and others. Anti-angiogenic agent targeting liver cancer vasculature has been reported to be able to generate limited survival benefit of the patients. In this review, discussions are focused on various angiogenic mechanisms of HCC and liver regeneration, as well as the prevailing anti-angiogenic strategies.

Chen, Q., D. W. Seol, et al.; (1997). "Co-expression and regulation of Met and Ron proto-oncogenes in human hepatocellular carcinoma tissues and cell lines." Hepatology; 26(1); 59-66.

                Met and ron proto-oncogenes encode the cell surface receptors for hepatocyte growth factor (HGF) and hepatocyte growth factor-like (HLP) protein, respectively, and induce mitogenesis, motogenesis, morphogenesis, and metastatic activity in various cell types. Overexpression of met in human carcinoma has been reported by several groups including ours; however, the mechanisms that control met gene expression are thus far unclear. The present study focuses on the expression and regulation of the Met and Ron receptors in human hepatocellular carcinoma (HCC). We report here that abnormal expression of met and ron proteins occurs in some cases of human HCC. Using several HCC cell lines as a model system, we show that HGF, as well as other cytokines, such as epidermal growth factor (EGF), interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha), induce met and ron expression. Using several chimeric constructs consisting of various lengths of the met promoter region fused to the reporter gene of chloramphenicol acetyl transferase (CAT), and by performing transient transfection of these constructs into HepG2 cells, we show that induction of met gene expression by HGF and other cytokines is, at least in part, through up-regulation of met gene promoter activity. The DNA region conferring responsiveness to cytokine induction was located within 0.2 kb of the met core promoter. Interestingly, EGF did not stimulate met promoter activity in any of the met-CAT chimeric constructs. These results provide evidence that met and ron are modulated in the liver by a similar cytokine network. In the case of met expression, the 0.2-kb region in the met gene promoter may play an important role in mediating its gene induction in response to HGF and other cytokines. Our results also suggest that unregulated expression of met and ron may be associated with pathological conditions, such as HCC, in the liver.

Cheng, J., H. Imanishi, et al.; (2004). "Involvement of cell cycle regulatory proteins and MAP kinase signaling pathway in growth inhibition and cell cycle arrest by a selective cyclooxygenase 2 inhibitor, etodolac, in human hepatocellular carcinoma cell lines." Cancer Sci; 95(8); 666-673.

                Recent studies have shown that selective cyclooxygenase-2 (COX-2) inhibitors induce growth inhibition and cell cycle arrest in hepatocellular carcinoma (HCC) cell lines. However, the mechanism by which COX-2 inhibitors regulate the cell cycle and whether or not growth signal pathways are involved in the growth inhibition remain unclear. In this study, we investigated the mechanisms of growth inhibition and cell cycle arrest by etodolac, a selective COX-2 inhibitor, in HCC cell lines, HepG2 and PLC/PRF/5, by studying cell cycle regulatory proteins, and the MAP kinase and PDK1-PKB/AKT signaling pathways. Etodolac inhibited growth and PCNA expression and induced cell cycle arrest in both HCC cell lines. Etodolac induced p21WAF1/Cip1 and p27Kip1 expression and inhibited CDK2, CDK4, CDC2, cyclin A and cyclin B1 expression, but did not affect cyclin D1 or cyclin E. HGF and 10% FBS induced ERK phosphorylation, but phosphorylation of p38, JNK and AKT was down-regulated by etodolac. PD98059, a selective inhibitor of ERK phosphorylation, induced growth inhibition, the expression of p27Kip1 and cell cycle arrest. In conclusion, p21WAF1/Cip1, p27Kip1, CDK2, CDK4, CDC2, cyclin A, cyclin B1 and the MAP kinase signaling pathway are involved in growth inhibition and cell cycle arrest by a selective COX-2 inhibitor in HCC cell lines.

Cornella, H., C. Alsinet, et al.; (2011). "Molecular Pathogenesis of Hepatocellular Carcinoma." Alcohol Clin Exp Res.

                Hepatocellular carcinoma (HCC) is one of the major causes of death among cirrhotic patients, being viral hepatitis and alcohol abuse, the main risk factors for its development. The introduction of highly sophisticated genomic technologies has spurred extensive research on the molecular pathogenesis of this devastating disease. Several signaling cascades have been consistently found dysregulated in HCC (e.g., WNT-beta-catenin, PI3K/AKT/MTOR, RAS/MAPK, IGF, HGF/MET, VEGF, EGFR, and PDGF). In addition, there have been numerous molecular classifications proposed for this disease, what provides an additional hint about its genomic complexity. The importance of knowing the molecular drivers of HCC is underscored by the positive results of a molecular targeted agent, sorafenib, able to improve survival in patients with advanced disease. This review will briefly outline key concepts in alcohol-related hepatocarcinogenesis, and provide some insight regarding current trends in translating HCC genomics into clinical management of the disease.

Costantini, S., F. Capone, et al.; (2010). "Serum cytokine levels as putative prognostic markers in the progression of chronic HCV hepatitis to cirrhosis." Eur Cytokine Netw; 21(4); 251-256.

                Hepatitis C virus (HCV) infection can present as an acute manifestation, and can lead to severe complications such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). It represents a global health problem because there is no vaccine currently available. Cytokines play an important role in viral clearance, infection control, inflammation, regeneration and fibrosis, and also are implicated in the pathological processes occurring in the liver during viral infection. Immunological markers of chronic HCV hepatitis progression as compared to cirrhosis and HCC would be extremely useful, particularly for distinguishing between the molecules produced during HCV-induced chronic inflammation and those secreted during cirrhosis and HCC. In this work, we evaluated the serum levels of several cytokines, chemokines and growth factors in 30 patients affected by chronic HCV (HC), 30 patients affected by HCV-related cirrhosis (LC) and 20 healthy, control subjects. We used a multiplex biometric ELISA-based immunoassay in order to identify molecules that might be useful for monitoring the progression of HCV to liver cirrhosis and, possibly, to cancer. Our results show that some pro-inflammatory molecules are significantly up-regulated, and play a role as immunological markers in the intermediate steps towards liver cancer, and that hepatocyte growth factor (HGF) is a specific marker of liver cirrhosis. Finally, these data will be used to define a cytokinome profile, which might prove useful for studies involving the transition of chronic inflammation to neoplastic processes.

Daveau, M., M. Scotte, et al.; (2003). "Hepatocyte growth factor, transforming growth factor alpha, and their receptors as combined markers of prognosis in hepatocellular carcinoma." Mol Carcinog; 36(3); 130-141.

                A change in the balance between proliferation and apoptosis in the course of hepatocellular carcinoma (HCC) development and progression has been suspected. We wanted to identify related genes whose mRNA levels could provide markers of severity and prognosis after resection. The extent of cell apoptosis, proliferation, and differentiation was measured with a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick-end labeling assay, and the Ki-67 index was determined in paired tumor and cirrhotic tissue samples from patients who had undergone HCC resection after diagnosis of hepatitis C-related or alcoholism-related cirrhosis. These patients included two groups with highly versus poorly differentiated tumor cells, and the latter was split into two subgroups of those with versus without early recurrence. The mRNA levels for various apoptosis-related or proliferation-related genes and those for the growth factor/receptor systems were measured by quantitative reverse transcriptase-polymerase chain reaction in paired tumor and cirrhotic liver samples from every patient, and some of the corresponding proteins were detected by immunohistochemistry. In all instances, protein expression was highly heterogeneous within groups and similar between groups. In contrast, some differences in mRNA level between tumor and cirrhotic tissues were quite informative. Low levels of hepatocyte growth factor and transforming growth factor alpha mRNAs were found concomitantly in highly differentiated tumors, whereas overexpression of mRNAs for the cognate receptors c-met and epidermal growth factor receptor were found in poorly differentiated tumors and primarily in patients with early tumor recurrence. These results argue for growth factor-dependent HCC development and provide novel and combined prognosis markers after HCC surgery.

Efimova, E. A., M. Glanemann, et al.; (2004). "Effects of human hepatocyte growth factor on the proliferation of human hepatocytes and hepatocellular carcinoma cell lines." Eur Surg Res; 36(5); 300-307.

                BACKGROUND: Hepatocyte growth factor (HGF) has been suggested to initiate both hepatocyte and tumor cell proliferation after partial hepatectomy, thereby supporting local tumor recurrence. The aim of this study was to clarify the role of HGF in the regeneration of human hepatocyte and the growth of residual hepatocellular carcinoma cells after liver resection. PATIENTS/METHODS: 36 patients who underwent partial hepatectomy for hepatocellular carcinoma (HCC) or living liver donation have been analyzed for HGF serum levels at day -1 through day 5 following surgery using an enzyme-linked immunosorbent assay. Isolated human hepatocytes and HCC cell lines (Hep 3B, Hep G2) were treated either with recombinant human (rh)-HGF, or sera from the 36 patients in the presence or absence of anti-HGF in order to measure their proliferative capacity using (3)H-thymidine incorporation. RESULTS: Basal HGF levels were significantly higher in HCC than in healthy patients (1,573 +/- 131 vs. 778 +/- 64 pg/ml; p

Farazi, P. A., M. Zeisberg, et al.; (2006). "Chronic bile duct injury associated with fibrotic matrix microenvironment provokes cholangiocarcinoma in p53-deficient mice." Cancer Res; 66(13); 6622-6627.

                Intrahepatic cholangiocarcinoma (CCA) is a lethal malignancy of the biliary epithelium associated with p53 mutations, bile duct injury, inflammation, and fibrosis. Here, to validate these processes in CCA, we developed a liver cirrhosis model driven by chronic intermittent toxin exposure, which provokes bile duct injury/necrosis and proliferation, fibroblast recruitment, and progressive extracellular matrix (ECM) changes. Fibrotic changes in the matrix microenvironment, typified by increased type I and III collagens and fibroblast recruitment, were shown to stimulate biliary epithelium hyperplasia with subsequent progression to malignant intrahepatic CCA only in mice harboring a p53 mutant allele. These murine CCAs bear histologic and genetic features of human intrahepatic CCA, including dense peritumoral fibrosis, increased inducible nitric oxide synthase, nitrotyrosine, and cyclooxygenase-2 expression, c-Met activation, cErbB2 overexpression, down-regulation of membrane-associated E-cadherin, and p53 codon 248 mutation. Thus, p53 deficiency, chronic bile duct injury/proliferation, and the fibrotic matrix microenvironment cooperate to induce intrahepatic CCA, highlighting the key role of the ECM microenvironment in this common liver cancer.

Fornari, F., M. Milazzo, et al.; (2010). "MiR-199a-3p regulates mTOR and c-Met to influence the doxorubicin sensitivity of human hepatocarcinoma cells." Cancer Res; 70(12); 5184-5193.

                MicroRNAs (miRNA) have rapidly emerged as modulators of gene expression in cancer in which they may have great diagnostic and therapeutic import. MicroRNA-199a-3p (miR-199a-3p) is downregulated in several human malignancies including hepatocellular carcinoma (HCC). Here, we show that miR-199a-3p targets mammalian target of rapamycin (mTOR) and c-Met in HCC cells. Restoring attenuated levels of miR-199a-3p in HCC cells led to G(1)-phase cell cycle arrest, reduced invasive capability, enhanced susceptibility to hypoxia, and increased sensitivity to doxorubicin-induced apoptosis. These in vitro findings were confirmed by an analysis of human HCC tissues, which revealed an inverse correlation linking miR-199a-3p and mTOR as well as a shorter time to recurrence after HCC resection in patients with lower miR-199a-3p expression. These results suggest that tactics to regulate mTOR and c-Met by elevating levels of miR-199a-3p may have therapeutic benefits in highly lethal cancers such as HCC.

Gil-Benso, R., A. Martinez-Lorente, et al.; (2001). "Characterization of a new rat cell line established from 2'AAF-induced combined hepatocellular cholangiocellular carcinoma." In Vitro Cell Dev Biol Anim; 37(1); 17-25.

                A rat cell line-nominated CC-62 derived from a combined hepatocellular and cholangiocellular carcinoma obtained by administration of 2-acetylaminofluorene to male Wistar rats, has been established. Using light and electron microscopy it was determined that morphologically the tumor consisted of a mixed population of hepatocytes and cholangiolar neoplastic cells, intermingled with small, undifferentiated oval-like cells. The CC-62 line has been maintained through 90 passages in culture adopting a paving stone arrangement. Doubling time at the 12th passage was 23 h. Immunostaining with a panel of antisera was performed to identify the cytological profiles of the cell line. There was no k-ras or p53 expression by immunohistochemistry, and molecular biology failed to detect mutations. Molecular analysis by reverse transcriptase-polymerase chain reaction revealed transcripts for c-met but no expression of HGF messenger ribonucleic acid. Three cell lines cloned from CC-62 showed the same immunohistochemical and molecular pattern as the parental line. Cytogenetic analysis revealed a chromosome number ranging from 74 to 82 with a modal number of 79 but no clonal structural abnormalities were found. Deoxyribonucleic acid ploidy analysis showed an aneuploid peak. CC-62 caused tumors 1 mo after subcutaneous transplantation into nude mice, with morphological patterns of mucosecretory solid and spindle-shaped carcinoma. This cell line is the first established from a primary rat combined hepatocellular and cholangiocellular neoplasm. The resulting cells expressed biological and morphological markers of hepatocytes and cholangiolar cells. Therefore this cell line may contribute to a better understanding of the histogenesis of liver cancer.

Guirouilh, J., M. Castroviejo, et al.; (2000). "Hepatocarcinoma cells stimulate hepatocyte growth factor secretion in human liver myofibroblasts." Int J Oncol; 17(4); 777-781.

                Hepatocellular carcinoma (HCC), the main type of primary liver cancer, is characterized by a high rate of intra-hepatic invasion. The stroma of HCC is infiltrated by myofibroblasts. We have previously shown that hepatocyte growth factor (HGF) secreted by human liver myofibroblasts greatly increased the in vitro invasiveness of 3 human HCC cell lines. In this study we show that the conditioned medium (CM) from the same HCC cell lines dose-dependently stimulates HGF secretion by myofibroblasts. This effect was post-transcriptional as no increase in HGF mRNA was observed. We show that the effect of CM is not due to IL-1, IL-6, IGF-1, bFGF or PDGF, previously shown to stimulate HGF synthesis in other models. Our data demonstrate that HCC cells increase HGF secretion by liver myofibroblasts in a paracrine way that could act to enhance invasion.

Gujdar, A., S. Sipeki, et al.; (2004). "Protein kinase C modulates negatively the hepatocyte growth factor-induced migration, integrin expression and phosphatidylinositol 3-kinase activation." Cell Signal; 16(4); 505-513.

                Previously, we reported that, in hepatocyte growth factor (HGF)-induced HepG2 cells, protein kinase C (PKC) decreased the duration of intensive Erk1/Erk2 MAP kinase activation. This study shows that the inhibition of PKC enhanced significantly the HGF-induced integrin expression. Beside the prolonged activation of Erk1/Erk2, the activity of phosphatidylinositol 3-kinase (PI 3K) was required for growth factor-induced integrin expression. PI 3-kinase was activated to a higher extent in response to HGF than to epidermal growth factor (EGF), though the activation was transient in both cases. In EGF-induced cells, PI 3K activation was terminated by the loss of phosphotyrosine docking sites for PI 3K. To the contrary, the decrease of PI 3K activation, which followed the HGF-induced increase was not accompanied by the loss of phosphotyrosine docking sites and was prevented by the inhibition of PKC. The negative modulator effects of PKC on integrin expression and PI 3-kinase activation correlated with its ability to limit the HGF-induced motogen response.

Hah, N. and S. T. Lee; (2003). "An absolute role of the PKC-dependent NF-kappaB activation for induction of MMP-9 in hepatocellular carcinoma cells." Biochem Biophys Res Commun; 305(2); 428-433.

                Matrix metalloproteinases (MMPs) play an important role in inflammation, tumor cell invasion, and metastasis. We found that phorbol-12-myristate-13-acetate (PMA)-stimulated invasion of the hepatocellular carcinoma (HCC) SNU-387 and SNU-398 cells and that PMA induced the secretion of MMP-9 in the cells, but did not induce the secretion of MMP-2. The PMA-induced MMP-9 secretion was abolished by treatment of a pan-protein kinase C (PKC) inhibitor, GF109203X, and an inhibitor of NF-kappaB activation, sulfasalazine, and partly inhibited by treatment of inhibitors of ERK pathway, PD98059 and U0126. In addition, the PMA-stimulated activation of the MMP-9 promoter was completely inhibited by a mutation of the NF-kappaB site within the MMP-9 promoter, but not completely by mutations of two AP-1 sites. Moreover, the MMP-9 induction by HGF and TNF-alpha was also completely inhibited by GF109203X and sulfasalazine, but not by PD98059 and U0126. These data demonstrate that the PKC-dependent NF-kappaB activation is absolute for MMP-9 induction and that the PKC-dependent ERK activation devotes to increase the expression level of MMP-9, in HCC cells.

Hakawa, S. K., K. Kouda, et al.; (1998). "[Stimulation of asialoglycoprotein receptor activity after transcatheter arterial embolotherapy in clinical study and continuous infusion of human hepatocyte growth factor in rat]." Gan To Kagaku Ryoho; 25(9); 1352-1354.

                The concentration of hepatocyte growth factor (HGF) was increased immediately after transcatheter arterial embolization (TAE) for hepatocellular carcinoma (HCC). We measured the changes in serum HGF levels and those of the asialoglyco-protein receptors (ASGP) in hepatocytes using 99mTc-galactosyl human serum albumin (GSA) on 22 patients with HCC after TAE. HGF and Rmax were increased 33% +/- 32, 40% +/- 28, respectively. Effects of HGF administration were examined in normal rats. Liver weight, hepatocyte nuclear size, and number of hepatocytes (cells/mm2) were not altered by HGF, but GSA blood clearance per hepatocyte was significantly increased over the preinfusion rate. We conclude that increased HGF stimulates a hepatocytic function of the receptor-mediated uptake of ASGP.

Han, C., G. K. Michalopoulos, et al.; (2006). "Prostaglandin E2 receptor EP1 transactivates EGFR/MET receptor tyrosine kinases and enhances invasiveness in human hepatocellular carcinoma cells." J Cell Physiol; 207(1); 261-270.

                Recent evidence indicates that cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) are involved in hepatocarcinogenesis. This study was designed to evaluate the possible interaction between the COX-2 and EGFR signaling pathways in human hepatocellular carcinoma (HCC) cells. Immunohistochemical analysis using serial sections of human HCC tissues revealed positive correlation between COX-2 and EGFR in HCC cells (P

Heideman, D. A., R. M. Overmeer, et al.; (2005). "Inhibition of angiogenesis and HGF-cMET-elicited malignant processes in human hepatocellular carcinoma cells using adenoviral vector-mediated NK4 gene therapy." Cancer Gene Ther; 12(12); 954-962.

                NK4 is an hepatocyte growth factor (HGF)-antagonist and a broad angiogenesis inhibitor. NK4 gene therapy has confirmed antitumor efficacy on cancers with intact HGF-cMET signaling pathway. However, the feasibility to treat tumors in which the effect of the HGF-cMET signaling pathway is less unambiguous or may even be inhibitory on carcinogenesis, such as hepatocellular carcinoma (HCC) with NK4 needs further assessment. Therefore, we evaluated the effects of adenoviral vector-mediated expression of NK4 on the biological behavior of a series of HCC cell lines in vitro and on HepG2 xenografts in vivo. In vitro, transduction of HCC cell lines with the replication-deficient recombinant adenoviral vector AdCMV.NK4 resulted in significant inhibition of proliferation over and above the antimitogenic effects of HGF. In addition, HGF-induced scattering and invasion through matrigel were inhibited effectively. Moreover, transduced HCC cells produced sufficient amounts of NK4 protein to achieve bystander effects involving reduced migration of nontransduced tumor cells and reduced proliferation of endothelial cells. Finally, treatment of established HepG2 xenografts with AdCMV.NK4 resulted in significant tumor growth delay and significant reduction of intratumoral microvessel density. In conclusion, NK4 gene therapy is a promising strategy to treat HCC based on the pleiotropic functions of NK4 interfering with tumor growth, invasion, metastasis and angiogenesis.

Henry, J. C., J. K. Park, et al.; (2010). "miR-199a-3p targets CD44 and reduces proliferation of CD44 positive hepatocellular carcinoma cell lines." Biochem Biophys Res Commun; 403(1); 120-125.

                Previous work by us and others reported decreased expression of miR-199a-3p in hepatocellular carcinoma (HCC) tissues compared to adjacent benign tissue. We report here a significant reduction of miR-199a-3p expression in 7 HCC cell lines. To determine if miR-199a-3p has a tumor suppressive role, pre-miR-199a-3p oligonucleotides were transfected into the HCC cell lines. Pre-miR-199a-3p oligonucleotide reduced cell proliferation by approximately 60% compared to control oligonucleotide in only two cell lines (SNU449 and SNU423); the proliferation of the other 5 treated cell lines was similar to control oligonucleotide. A pre-miR-199a-3p oligonucleotide formulated with chemical modifications to enhance stability while preserving processing, reduced cell proliferation in SNU449 and SNU423 to the same extent as the commercially available pre-miR-199a-3p oligonucleotide. Furthermore, only the duplex miR-199a-3p oligonucleotide, and not the guide strand alone, was effective at reducing cell viability. Since a CD44 variant was essential for c-Met signaling [V. Orian-Rousseau, L. Chen, J.P. Sleeman, P. Herrlich, H. Ponta, CD44 is required for two consecutive steps in HGF/c-Met signaling, Genes Dev. 16 (2002) 3074-3086] and c-Met is a known miR-199a-3p target, we hypothesized that miR-199a-3p may also target CD44. Immunoblotting confirmed that only the two HCC lines that were sensitive to the effects of pre-miR-199a-3p were CD44+. Direct targeting of CD44 by miR-199a-3p was confirmed using luciferase reporter assays and immunoblotting. Transfection of miR-199a-3p into SNU449 cells reduced in vitro invasion and sensitized the cells to doxorubicin; both effects were enhanced when hyaluronic acid (HA) was added to the cell cultures. An inverse correlation between the expression of miR-199a-3p and CD44 protein was noted in primary HCC specimens. The ability of miR-199a-3p to selectively kill CD44+ HCC may be a useful targeted therapy for CD44+ HCC.

Hora, C., P. Romanque, et al.; (2010). "Effect of sorafenib on murine liver regeneration." Hepatology.

                Hepatocellular carcinoma (HCC) is a common cause of cancer-related death. Sorafenib prolongs survival of patients with advanced disease and is approved for the systemic treatment of unresectable HCC. It possesses antiangiogenic and antiproliferative properties by way of inhibition of the receptor tyrosine kinases vascular endothelial growth factor receptor 2 (VEGFR-2) and platelet-derived growth factor receptor-beta 1/2 (PDGFR-beta) and the kinase RAF. Sorafenib represents a candidate compound for adjuvant therapy in HCC patients. The aim of our study was to investigate whether sorafenib affects liver regeneration. C57BL6 mice received sorafenib orally at 30 mg/kg/day or its vehicle either for 14 days until the day before hepatectomy or starting the day after surgery or both. Animals were sacrificed 24, 72, and 120 hours after hepatectomy. Liver regeneration was calculated as a percent of initial liver weight. Bromodeoxyuridine (BrdU) incorporation and phospho-extracellular signal-regulated kinase (pERK1/2) were determined by immunohistochemistry on liver sections. VEGF-A, PDGF-BB, and hepatocyte growth factor (HGF) levels were measured in liver tissue homogenates. Histological analysis of scar tissue was performed. Treatment stopped 1 day before surgery had no impact on liver regeneration. Continuous sorafenib treatment and treatment started 1 day after surgery had statistically significant effects on liver regeneration at 120 hours compared to vehicle-treated control animals (72% +/- 12 versus control 88% +/- 15 and 70% +/- 13 versus control 86% +/- 5 at 120 hours, both P

Hora, C., P. Romanque, et al.; (2011). "Effect of sorafenib on murine liver regeneration." Hepatology; 53(2); 577-586.

                Hepatocellular carcinoma (HCC) is a common cause of cancer-related death. Sorafenib prolongs survival of patients with advanced disease and is approved for the systemic treatment of unresectable HCC. It possesses antiangiogenic and antiproliferative properties by way of inhibition of the receptor tyrosine kinases vascular endothelial growth factor receptor 2 (VEGFR-2) and platelet-derived growth factor receptor-beta 1/2 (PDGFR-beta) and the kinase RAF. Sorafenib represents a candidate compound for adjuvant therapy in HCC patients. The aim of our study was to investigate whether sorafenib affects liver regeneration. C57BL6 mice received sorafenib orally at 30 mg/kg/day or its vehicle either for 14 days until the day before hepatectomy or starting the day after surgery or both. Animals were sacrificed 24, 72, and 120 hours after hepatectomy. Liver regeneration was calculated as a percent of initial liver weight. Bromodeoxyuridine (BrdU) incorporation and phospho-extracellular signal-regulated kinase (pERK1/2) were determined by immunohistochemistry on liver sections. VEGF-A, PDGF-BB, and hepatocyte growth factor (HGF) levels were measured in liver tissue homogenates. Histological analysis of scar tissue was performed. Treatment stopped 1 day before surgery had no impact on liver regeneration. Continuous sorafenib treatment and treatment started 1 day after surgery had statistically significant effects on liver regeneration at 120 hours compared to vehicle-treated control animals (72% +/- 12 versus control 88% +/- 15 and 70% +/- 13 versus control 86% +/- 5 at 120 hours, both P

Horiguchi, N., H. Takayama, et al.; (2002). "Hepatocyte growth factor promotes hepatocarcinogenesis through c-Met autocrine activation and enhanced angiogenesis in transgenic mice treated with diethylnitrosamine." Oncogene; 21(12); 1791-1799.

                Hepatocyte growth factor (HGF) is a mitogen for hepatocytes, but it is not clear whether HGF stimulates or inhibits hepatocarcinogenesis. We previously reported that HGF transgenic mice under the metallothionein gene promoter developed benign and malignant liver tumors spontaneously after 17 months of age. To elucidate the role of HGF in hepatocarcinogenesis, diethylnitrosamine (DEN) was administered to HGF transgenic mice. HGF overexpression accelerated DEN-induced hepatocarcinogenesis, often accompanied by abnormal blood vessel formation. In this study, 59% of transgenic males (versus 20% of wild-type males) and 39% of transgenic females (versus 2% of wild-type females) developed either benign or malignant liver tumors by 48 weeks (P

Hsia, C. Y., T. I. Huo, et al.; (2007). "Evaluation of interleukin-6, interleukin-10 and human hepatocyte growth factor as tumor markers for hepatocellular carcinoma." Eur J Surg Oncol; 33(2); 208-212.

                AIM: Serum alpha-fetoprotein (AFP) is the most important tumor marker for hepatocellular carcinoma (HCC). Previous reports indicated that HCC was also associated with increased levels of interleukin (IL)-6, IL-10 and hepatocyte growth factor (HGF). This study investigated the role of these cytokines as tumor markers for HCC. METHOD: A total of 128 adults were prospectively enrolled and categorized into four groups: normal subjects (n=29), chronic hepatitis B or C (n=50), non-HCC tumors (n=23) and HCC (n=26). Serum AFP, IL-6, IL-10 and HGF levels were determined in all subjects. RESULTS: The expression of IL-6 or IL-10 (> or =3 pg/ml), or high level of HGF (>1000 pg/ml) or AFP (>20 ng/ml) was observed in only 0-3% of normal subjects. Patients with HCC more frequently had higher IL-6 and IL-10 levels (p5 cm) HCC more often had increased IL-6, IL-10 or AFP levels (p values all

Hu, R. H., M. C. Ho, et al.; (2007). "Peritoneal fluid contains high concentration of hepatocyte growth factor and vascular endothelial growth factor after resection for hepatocellular carcinoma." Hepatogastroenterology; 54(75); 862-865.

                BACKGROUND/AIMS: Surges in blood growth factor levels is a typical response to surgical stress. However, growth factor profiles in peritoneal fluid after hepatectomy for hepatocellular carcinoma (HCC) have not been clarified. We evaluated hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) in blood and peritoneal fluid preoperatively and postoperatively. METHODOLOGY: Serial blood and peritoneal fluid samples were collected from 47 patients who underwent curative hepatectomy for primary HCC at a university hospital. Blood samples were collected postoperative days 0, 1, 3, 5, and 7; peritoneal fluid was collected at the same time except day 0. VEGF and HGF concentrations were determined, and correlations between concentrations and clinical parameters of HCC were analyzed. RESULTS: HGF surges appeared postoperatively in blood and peritoneal fluid; concentrations paralleled each other. There was no postoperative surge of VEGF in blood, but peritoneal fluid VEGF concentration was remarkably high after hepatectomy. Both HGF and VEGF levels in blood and peritoneal fluid correlated with some clinical parameters. The difference in HGF and VEGF patterns in blood and peritoneal fluid imply that the origins of these two growth factors differ. CONCLUSIONS: Post-hepatectomy, VEGF and HGF levels surged in peritoneal fluid, whereas only HGF surged in blood, suggesting independent origins for the two growth factors. Both VEGF and HGF concentrations correlated with some clinical parameters.

Hu, R. H., P. H. Lee, et al.; (1999). "Secretion of acute-phase proteins before and after hepatocellular carcinoma resection." J Formos Med Assoc; 98(2); 85-91.

                Hepatocyte growth factor (HGF), interleukin-6 (IL-6), and C-reactive protein (CRP) are acute-phase reactants that are usually present at high concentrations in the serum of patients with liver disease. However, the origin of these high serum concentrations is not completely understood, and whether hepatocellular carcinoma (HCC) tissue is a contributing factor is a controversial issue. The purpose of this study was to investigate the profiles of these three proteins in patients with HCC before and after tumor resection, and to study factors that might affect the serum concentrations of these proteins. A retrospective cohort study was performed in 34 consecutive patients who underwent HCC resection at the National Taiwan University Hospital. Blood samples were collected before surgery and on days 1, 3, 5, 7, and 14 postoperatively for serum concentration determinations of these three proteins. Twenty-three patients admitted for health examinations were enrolled as normal controls. Multiple regression analysis was conducted to evaluate the correlations between the pre- and postoperative cytokine concentrations and various clinical parameters. Compared with normal controls, the HCC patients had a significantly higher preoperative concentration of HGF (1,472 +/- 73 vs 948 +/- 54 ng/mL, p

Hu, R. H., P. H. Lee, et al.; (1999). "Serum hepatocyte growth factor before and after resection for hepatocellular carcinoma." Hepatogastroenterology; 46(27); 1842-1847.

                BACKGROUND/AIMS: Hepatocyte growth factor (HGF) is the most potent hepatocyte proliferation stimulator. Serum HGF levels are high in various liver disease states such as cirrhosis, hepatocellular carcinoma (HCC) and hepatitis. But the role HGF plays in HCC is not clear at present. The purposes of this study are: 1) to reveal the HGF profile pre- and post-HCC resection, which has not been well-described before; and, 2) to analyze the relationships between the pre- and post-operative HGF levels and various clinical parameters. METHODOLOGY: We performed a retrospective cohort study to check the HGF profiles before and after curative resections for HCC and to analyze the relationships between them by using clinical parameters from 35 consecutive patients at the Department of Surgery, National Taiwan University Hospital. Blood samples collected from another 23 healthy individuals admitted for health check-ups were used as normal controls. Serum HGF was determined with an ELISA kit. RESULTS: The baseline HGF concentration in HCC patients was significantly higher than that in normal controls (1743+/-73 vs. 948+/-54 pg/ml, p

Hu, Z., R. P. Evarts, et al.; (1996). "Expression of transforming growth factor alpha/epidermal growth factor receptor, hepatocyte growth factor/c-met and acidic fibroblast growth factor/fibroblast growth factor receptors during hepatocarcinogenesis." Carcinogenesis; 17(5); 931-938.

                It is widely believed that abnormal production of polypeptide growth factors, together with other molecular alterations, play an important role in neoplastic development. Transforming growth factor alpha (TGFalpha), hepatocyte growth factor (HGF) and acidic fibroblast growth factor (aFGF) are the three major growth factors that contribute to liver regeneration occurring via both hepatocyte replication and oval cell proliferation. It is not clear, however, whether and to what extent these growth factors are also involved in hepatocarcinogenesis. In the present study, the gene expression of TGFalpha, HGF and aFGF and their corresponding receptors was examined by Northern blotting and in situ hybridization during hepatocarcinogenesis induced by the Solt-Farber protocol. All three growth factor/receptor systems, TGFalpha/epidermal growth factor receptor (EGFR), HGF/c-met and aFGF/FGF receptors (flg and bek) were significantly elevated at early time points when oval cells were proliferating. Their respective expression decreased after 1 month and remained at a low level until the development of liver tumors. In all hepatocellular carcinomas (HCC) examined, the transcripts of TGFalpha and aFGF were highly expressed, while those of HGF were low. With regard to the receptor expression in the tumors, EGFR was present at varying levels, c-met was expressed at higher levels and flg increased significantly, whereas bek remained at low levels. These data suggest that TGFalpha and aFGF are the major growth factors involved in the progression of HCC, and that the signal of aFGF is mainly transduced by the receptor flg in HCC. Furthermore, HCC cells were phenotypically very similar to oval cells with regard to the gene expression of growth factor/receptor systems. These results, along with the finding that all the HCC cells are positive for the oval cell antigen OV6, and that cytokeratin 19 is heavily expressed in both tumor and oval cells, strongly suggest that at least some of the HCC induced by the Solt-Farber protocol may be derived from oval cells.

Huang, G. T., H. S. Lee, et al.; (1999). "Tissue hepatocyte growth factor and proliferating cell nuclear antigen in hepatocellular carcinoma." J Formos Med Assoc; 98(2); 92-96.

                To better understand the roles of hepatocyte growth factor (HGF) and proliferating cell nuclear antigen (PCNA) in hepatocellular carcinoma (HCC), 37 surgically resected HCCs and corresponding nontumorous liver tissue specimens were collected and the expression of these two factors was quantified by Western blot analysis. Both HGF and PCNA expression levels were significantly higher in tumor tissue than in nontumorous liver tissue. However, their expression levels in HCC tissue and nontumorous tissue did not show any significant correlation with the recurrence of HCC. In addition, HGF and PCNA did not correlate with Edmondson's grade, invasiveness of tumor, presence of tumor capsule, or tumor size. No correlation was found between the expression levels of HGF and PCNA in HCC tissue. We conclude that, although both HGF and PCNA are present at higher levels in HCC tissue than in nontumorous liver tissue, they play little role in the clinicopathologic manifestations of this tumor. HGF appears to contribute little, if at all, to the proliferative activity of HCC cells.

Huynh, H., V. C. Ngo, et al.; (2009). "Sorafenib and rapamycin induce growth suppression in mouse models of hepatocellular carcinoma." J Cell Mol Med; 13(8B); 2673-2683.

                Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. Vascular endothelial growth factor, platelet derived growth factor and the Raf/mitogen-activated protein kinase/extracellular signal regulated kinase (Raf/MEK/ERK) signalling pathway regulates the growth, neovascularization, invasiveness and metastatic potential of HCC. In this study, we investigated the in vivo antitumour activity and mechanisms of action of sorafenib tosylate on four patient-derived HCC xenografts. Sorafenib dosed at 50 mg/kg and 100 mg/kg inhibited tumour growth by 85% and 96%, respectively. Sorafenib-induced growth suppression and apoptosis were associated with inhibition of angiogenesis, down-regulation of phospho-platelet-derived growth factor receptor beta Tyr1021, phospho-eIF4E Ser209, phospho-c-Raf Ser259, c-Raf, Mcl-1, Bcl-2, Bcl-x and positive cell cycle regulators, up-regulation of apoptosis signalling kinase-1, p27 and p21. Expression of IGF-1Rbeta and phosphorylation of c-Raf Ser338, MEK1/2 Ser217/221 and ERK1/2 Thr202/Tyr204 were increased by sorafenib treatment. Phosphorylation of mammalian target-of-rapamycin (mTOR) targets (p70S6K, S6R and 4EBP1) was reduced by sorafenib in sorafenib-sensitive lines but activated in sorafenib-less-sensitive 10-0505 xenograft. Sorafenib-induced phosphorylation of c-met, p70S6K and 4EBP1 was significantly reduced when 10-0505 cells were co-treated with anti-human anti-HGF antibody, suggesting that treatment with sorafenib leads to increased HGF secretion and activation of c-met and mTOR targets. Treatment of 10-0505 tumours with sorafenib plus rapamycin resulted in growth inhibition, inhibition of vascular endothelial growth factor receptor-2 phosphorylation, increased apoptosis and completely blocked sorafenib-induced phosphorylation of mTOR targets and cyclin B1 expression. These data also provide a strong rationale for clinical investigation of sorafenib in combination with mTOR inhibitors in patients with HCC.

Iimuro, Y. and J. Fujimoto; (2003). "Strategy of gene therapy for liver cirrohosis and hepatocellular carcinoma." J Hepatobiliary Pancreat Surg; 10(1); 45-47.

                BACKGROUND/PURPOSE: Liver cirrhosis and hepatocellular carcinoma (HCC) are major causes of morbidity and mortality worldwide, without effective therapies. Our aim was to establish a novel approach for the diseases utilizing in-vivo gene therapy. METHODS: To achieve effective gene expression in vivo, we employed a well-established transfection method, hemagglutinating virus of Japan (HVJ)-liposome. Liver cirrhosis, characterized by parenchymal collapse, was induced by dimethylnitrosamine (DMN) in rats, leading to accumulation of fibrous tissue. Muscle-directed gene transfer of hepatocyte growth factor (HGF) was performed in this model. As an approach for HCC therapy, suicide gene therapy using ganciclovir (GCV) with transfer of the herpes thymidine kinase (HSVtk) gene was tested. Alpha-fetoprotein (AFP) is highly expressed in many cases of HCC. To achieve specific gene expression of HSVtk in AFP-producing tumors, we employed the HSVtk gene driven by the AFP promoter (AFP-TK1), encapsulated in the HVJ-liposome. APF-producing HUH7 tumors in vivo were established in the livers of severe combined immunodeficiency (SCID) mice, by the injection of HUH7 cells into the portal vein. RESULTS: All cirrhotic rats died of liver dysfunction by 7 weeks after the initial injection of DMN. After repeated transfection with the HGF gene, increased concentrations of HGF in plasma and tyrosine phosphorylation of the c-Met/HGF receptor in the liver were detected, and the established massive hepatic fibrosis had almost totally disappeared and all cirrhotic rats survived. Repeated in-vivo transfection with AFP-TK1, using the HVJ-liposome, followed by GCV treatment, achieved abrogation of tumors in the liver, and improved survival. CONCLUSIONS: Our data indicate that the gene therapy may hold promise for the treatment of patients with liver cirrhosis and HCC.

Imai, Y., Y. Kubota, et al.; (2005). "Neutrophils enhance invasion activity of human cholangiocellular carcinoma and hepatocellular carcinoma cells: an in vitro study." J Gastroenterol Hepatol; 20(2); 287-293.

                BACKGROUND AND AIM: Tumor-mesenchymal interactions are involved in the mechanism of tumor invasion in several types of carcinoma. Mutual interactions between carcinoma cells and neutrophils, however, have been poorly understood. In the present study we examined the effect of neutrophils on invasion activities of carcinoma cells in vitro. Role of hepatocyte growth factor (HGF) as a mediator was also evaluated. METHODS: Using a Matrigel invasion chamber, invasion activities of HuCC-T1 human cholangiocellular carcinoma cells and HepG2 hepatocellular carcinoma cells in response to recombinant HGF or neutrophils were evaluated. RESULTS: Recombinant HGF dose-dependently increased invasion activities of HuCC-T1 and HepG2 cells. Neutrophils significantly enhanced invasion activities of these cells, which were suppressed to the respective basal levels with anti-HGF antibody. The carcinoma cells did not secrete HGF. Neutrophils cultivated in tumor condition medium (TCM) of HuCC-T1 or HepG2 cells secreted a significant level of HGF protein without increasing HGF mRNA expression. Treatment with heat or ultrafiltration of TCM of HuCC-T1 or HepG2 cells suggested carcinoma cell-derived HGF inducer(s) to be certain protein(s) with a molecular weight of more than 30 000. CONCLUSIONS: The present study suggests the presence of mutual interactions between HuCC-T1/HepG2 carcinoma cells and neutrophils in tumor invasion via paracrine regulation mediated by neutrophil-derived HGF.

Jiang, Y., W. Xu, et al.; (2001). "Invasiveness of hepatocellular carcinoma cell lines: contribution of hepatocyte growth factor, c-met, and transcription factor Ets-1." Biochem Biophys Res Commun; 286(5); 1123-1130.

                To understand the mechanism of invasion and metastasis of hepatocellular carcinoma (HCC), the expression of c-met and Ets-1, and the effect of HGF on these cell's motility and invasion ability were examined in four hepatoma cell lines. The analysis revealed that the overexpression of c-met and Ets-1 is closely connected with the motility and invasion ability of the HCC cell lines. Invasion activity of HepG2 and HLE cells were enhanced by the addition of HGF to medium. HGF regulated c-met transcription in HepG2 and Bel-7402 cells, HGF also induced Ets-1 transcription in Bel-7402 cell. Bel-7402 cells stably transduced with the human Ets-1 gene showed significantly increased invasion potentials compared to parental and mock-transfected cells. The expression level of c-met, MMP1, MMP9, and u-PA in Bel-7402 cells transfected with Ets-1 were markedly increased, and as a consequence of c-met expression increase. Bel-7402 cells transfected with Ets-1 were more responsive to exogenous HGF stimulation in invasiveness and motility ability. In addition, conditioned by antisense Ets-1 oligonucleotide-treat-Bel-7402 cells transfected with Ets-1 gene and HLE hepatoma cells showed markedly reduced invasion activity, and down-regulated the transcription of Ets-1, c-met, u-PA, MMP-1, and MMP-9. These results strongly suggest that Ets-1 has a crucial role in the invasive property in hepatoma cell lines, and there may exist a loop to enhance the invasive ability of hepatoma cell lines.

Jo, M., D. B. Stolz, et al.; (2000). "Cross-talk between epidermal growth factor receptor and c-Met signal pathways in transformed cells." J Biol Chem; 275(12); 8806-8811.

                In rat liver epithelial cells constitutively expressing transforming growth factor alpha (TGFalpha), c-Met is constitutively phosphorylated in the absence of its ligand, hepatocyte growth factor. We proposed that TGFalpha and the autocrine activation of its receptor, epidermal growth factor receptor (EGFR), leads to phosphorylation and activation of c-Met. We found that there is constitutive c-Met phosphorylation in human hepatoma cell lines and the human epidermoid carcinoma cell line, A431 which express TGFalpha, but not in normal human hepatocytes. Constitutive c-Met phosphorylation in A431, HepG2, AKN-1, and HuH6 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR. Exposure to exogenous TGFalpha or EGF increased the phosphorylation of c-Met in the human epidermoid carcinoma cell line, A431. The increase of c-Met phosphorylation by TGFalpha in A431 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR and by the EGFR-specific inhibitor tyrphostin AG1478. These results indicate that constitutive c-Met phosphorylation, and the increase of c-Met phosphorylation by TGFalpha or EGF, in tumor cell lines is the result of the activation via EGFR. We found that c-Met in tumor cells co-immunoprecipitates with EGFR regardless of the existence of their ligands in tumor cells, but not in normal human hepatocytes. We conclude that c-Met associates with EGFR in tumor cells, and this association facilitates the phosphorylation of c-Met in the absence of hepatocyte growth factor. This cross-talk between c-Met and EGFR may have significant implications for altered growth control in tumorigenesis.

Junbo, H., Q. Li, et al.; (1999). "Increased level of serum hepatocyte growth factor/scatter factor in liver cancer is associated with tumor metastasis." In Vivo; 13(2); 177-180.

                Hepatocyte growth factor/scatter factor (HGF/SF) has been shown to play an important role in tumor migration and metastasis. We therefore investigated the relationship between HGF/SF expression and metastasis of human liver tumors. The serum HGF/SF levels in 41 patients with primary hepatocellular carcinoma (HCC; 22 patients with metastasis, 19 patients without metastasis); 4 patients with benign hepatic tumor, 4 patients with secondary hepatic carcinoma, and 12 healthy blood donors, were measured by sandwich enzyme-linked immunosorbent assay (ELISA) in this study. Our results show that liver tumor patients have significantly higher serum levels of HGF/SF compared to healthy blood donors. However, there is no difference between the serum HGF/SF levels in patients with primary HCC and patients with secondary HCC or benign hepatic tumors. In addition, we measured significantly higher levels of HGF/SF in serum from HCC patients with metastasis compared to HCC patients without metastasis, indicating that the elevations in serum HGF/SF level correlated positively with the tumor metastasis in human HCC. These findings appear to suggest that HGF/SF may be a useful serological biomarker for clinical diagnosis and follow-up of HCC metastasis, and suggest that larger scale studies in patients with hepatomas are warranted.

Kamiyama, T., Y. Une, et al.; (1998). "Hepatocyte growth factor enhances the invasion activity of human hepatocellular carcinoma cell lines." Int J Oncol; 12(3); 655-659.

                We investigated whether hepatocyte growth factor (HGF) enhances the invasion activity of three human HCC cell lines, HLF, HLE, and HC-4, in vitro. The analysis of the invasiveness consisted of the production of u-PA and the chemotaxis for fibronectin. Invasion activity of all cell lines was enhanced by the addition of recombinant human hepatocyte growth factor (rhHGF) to the medium. HGF stimulated the production of u-PA in HLF cells. HGF accelerated the chemotaxis of HC-4 and HLE. These data suggest that HGF increase the invasion activity of human HCC cell lines by affecting the production of u-PA or the chemotaxis for fibronectin.

Kaposi-Novak, P.; (2009). "[Comparative genomic classification of human hepatocellular carcinoma]." Magy Onkol; 53(1); 61-67.

                Global transcriptome analysis has been successfully applied to characterize various human tumors, including hepatocellular carcinomas. This novel technology can facilitate early diagnosis, as well as prognostic and therapeutic diversification of cancer patients. To enhance access to the genomic information buried in archived pathology samples, we assessed RT-PCR amplification rates in paraffin-embedded tissues preserved in three different fixatives. Reliable amplification could be achieved from all paraffin-embedded specimens, when the amplicon size did not exceed 225 bp. A longer amplicon size resulted in rapid decrease of yield and reproducibility. In addition, formalin provided superior morphology and better reactivity with claudin-4 and -7 immunohistochemistry. Amplification of the initial sample is often required before transcriptome analysis of clinical specimens could be performed. We introduced a random nonamer primed T3 polymerase reaction into the conventional linear RNA amplification protocol. The modified T3T7 method generated a sense strand product ideal for synthesizing indirectly labeled cDNA templates. Microarray analysis of amplified frozen and laser-microdissected Myc and Myc/TGFalpha mouse liver tumors confirmed good reproducibility (r=0.9) of the reaction and conservation of original transcriptional patterns (r=0.78). Finally, we tested the utility of expression profiling for the classification of human HCC samples. By comparing expression data from HGF-treated c-Met conditional knock-out and control primary mouse hepatocytes, we identified 690 HGF/c-Met target genes. Functional analysis of the significant gene set implicated c-Met as key regulator of hepatocyte motility and oxidative homeostasis. Cross comparison of the c-Met-induced transcription signature with human HCC expression profiles revealed a group of tumors (27%) with potentially activated c-Met signaling (MET+). These tumors were characterized by higher vascular invasion rate, increased microvessel density, and shortened survival. A prediction model based on 111 cross-species conserved c-Met signature genes was able to diversify HCC patients into good and bad prognostic groups with 83-95% accuracy. Our results therefore demonstrate that careful experimental design and state-of-the-art laboratory methods could open the way for global expression profiling of archived and limited availability pathologic samples. Comparative functional genomics based analysis of the cancer transcriptome could lead to novel molecular classification systems which are essential for the introduction of individualized cancer therapeutics.

Kataoka, H., S. Miyata, et al.; (2003). "Roles of hepatocyte growth factor (HGF) activator and HGF activator inhibitor in the pericellular activation of HGF/scatter factor." Cancer Metastasis Rev; 22(2-3); 223-236.

                The activation of hepatocyte growth factor (HGF)/scatter factor (SF) in an extracellular milieu is a critical limiting step in HGF/SF-induced signaling that is believed to have important roles in invasive growth of tumor cells and regeneration of injured tissue. This activation is caused by a proteolytic cleavage at the bond between Arg494-Val495 in the single-chain HGF/SF precursor, generating an active two-chain heterodimeric form. The HGF activator (HGFA) is a coagulation factor XII-like serine proteinase critically involved in this process in injured tissues including tumor tissues. In the past several years, the identification of endogenous HGFA inhibitors (HAIs) has provided detailed knowledge of the regulation of HGFA activity. Currently, two types of HAIs, namely HAI-1 and HAI-2, have been reported. Both are Kunitz-type serine proteinase inhibitors and inhibit not only HGFA but also other serine proteinases, such as membrane-type serine protease 1 (matriptase), plasmin, trypsin and kallikreins. HAIs are of particular interest because they are synthesized as type-I transmembrane proteins. Therefore, HAIs must have important regulatory roles in a cell surface proteolytic reaction, which has emerged as an important mechanism for the generation of biologically active proteins mediating a diverse range of cellular functions. This review is a summary and interpretation of recent data regarding the regulation of pericellular HGF/SF activation mediated by HGFA and HAIs and includes a discussion of the possible role of the type I transmembrane Kunitz-type inhibitor in pericellular proteolysis.

Kaufmann, R., C. Oettel, et al.; (2009). "Met receptor tyrosine kinase transactivation is involved in proteinase-activated receptor-2-mediated hepatocellular carcinoma cell invasion." Carcinogenesis; 30(9); 1487-1496.

                The expression of proteinase-activated receptor (PAR)(2) in human hepatocellular carcinoma (HCC) was established by reverse transcription-polymerase chain reaction, confocal immunofluorescence and electron microscopy in permanent cell lines, primary HCC cell cultures and HCC tumor tissue. Stimulation of HCC cells with trypsin and the PAR(2)-selective activating peptide, 2-furoyl-LIGRLO-NH(2), increased cell invasion across Matrigel. Both effects were blocked by a PAR(2)-selective pepducin antagonist peptide (pal-PAR(2)) and by PAR(2) silencing with specific small interfering RNA (siRNA). PAR(2)-initiated HCC cell invasion was also blocked by inhibiting the hepatocyte growth factor receptor (Met receptor tyrosine kinase) with the receptor-targeted kinase inhibitors, SU 11274 and PHA 665752, or by downregulation of Met with specific siRNA. The involvement of Met in PAR(2)-mediated HCC invasive signaling was further supported by the finding that treatment of HCC cells with trypsin or the PAR(2)-selective agonist peptide, 2-furoyl-LIGRLO-NH(2), stimulated Met activation-phosphorylation. In addition, Met-dependent stimulation of p42/p44 mitogen-activated protein Kinases was found to be critical for the PAR(2)-Met receptor tyrosine kinase-invasive signaling axis in HCC cells. Our study establishes an important link between the PAR(2) and Met receptor tyrosine kinase signaling in promoting HCC cell invasion.

Ke, A. W., G. M. Shi, et al.; (2009). "Role of overexpression of CD151 and/or c-Met in predicting prognosis of hepatocellular carcinoma." Hepatology; 49(2); 491-503.

                It has been reported that tetraspanin CD151 acts as a promoter of metastasis in several tumors and plays an important role in c-Met/hepatocyte growth factor signaling. However, the role of CD151 alone and coexpression of CD151/c-Met in hepatocellular carcinoma (HCC) remains unclear. We found that expression of CD151 was positively related to metastatic potential of HCC cell lines, and modified cells with CD151(high) showed higher secretion of matrix metalloproteinase 9 and aggressiveness in vitro and higher metastatic ability in vivo. Furthermore, HCC patients with vascular invasion, large tumors, multiple tumors, high tumor-node-metastasis stage, and undifferentiated tumor were prone to have higher CD151 expression. The postoperative 3-, 5-, and 7-year overall survival (OS) of patients in HCCs with CD151(high) were significantly lower than those in the CD151(low) group, and correspondingly cumulative recurrence rates in HCCs with CD151(high) were significantly higher than those in the CD151(low) group. Both CD151 and c-Met were remarkably overexpressed in HCCs, compared with adjacent nontumorous and normal liver tissues. Pearson correlation analysis showed a slight correlation between CD151 and c-Met in HCCs. Importantly, the 5- and 7-year OS rates in CD151(high)/c-Met(high) patients were 50.5% and 37.8%, respectively, significantly lower than those of CD151(low)/c-Met(low) patients (63.9% and 54.6%, respectively). Five- and 7-year cumulative recurrence rates in CD151(high)/c-Met(high) patients were 53.3% and 71.9%, respectively, markedly higher than those of CD151(low)/c-Met(low) patients (39.0% and 52.5%, respectively). Multivariate analysis revealed that CD151 and combination of CD151/c-Met were independent prognostic indicators for OS and cumulative recurrence. Conclusion: CD151 is positively associated with invasiveness of HCC, and CD151 or combination of CD151/c-Met is a novel marker in predicting the prognosis of HCC and a potential therapeutic target.

Kim, S., U. J. Lee, et al.; (2008). "MicroRNA miR-199a* regulates the MET proto-oncogene and the downstream extracellular signal-regulated kinase 2 (ERK2)." J Biol Chem; 283(26); 18158-18166.

                MicroRNAs (miRNAs) constitute a class of small noncoding RNAs that play important roles in a variety of biological processes including development, apoptosis, proliferation, and differentiation. Here we show that the expression of miR-199a and miR-199a* (miR-199a/a*), which are processed from the same precursor, is confined to fibroblast cells among cultured cell lines. The fibroblast-specific expression pattern correlated well with methylation patterns: gene loci on chromosome 1 and 19 were fully methylated in all examined cell lines but unmethylated in fibroblasts. Transfection of miR-199a and/or -199a* mimetics into several cancer cell lines caused prominent apoptosis with miR-199a* being more pro-apoptotic. The mechanism underlying apoptosis induced by miR-199a was caspase-dependent, whereas a caspase-independent pathway was involved in apoptosis induced by miR-199a* in A549 cells. By employing microarray and immunoblotting analyses, we identified the MET proto-oncogene as a target of miR-199a*. Studies with a luciferase reporter fused to the 3'-untranslated region of the MET gene demonstrated miR-199a*-mediated down-regulation of luciferase activity through a binding site of miR-199a*. Interestingly, extracellular signal-regulated kinase 2 (ERK2) was also down-regulated by miR-199a*. Coordinated down-regulation of both MET and its downstream effector ERK2 by miR-199a* may be effective in inhibiting not only cell proliferation but also motility and invasive capabilities of tumor cells.

Kitisin, K., M. J. Pishvaian, et al.; (2007). "Liver stem cells and molecular signaling pathways in hepatocellular carcinoma." Gastrointest Cancer Res; 1(4 Suppl 2); S13-21.

                Hepatocellular carcinoma (HCC) is one of the most lethal cancers. Surgical intervention is the only curative option, with only a small fraction of patients being eligible. Conventional chemotherapy and radiotherapy have not been effective in treating this disease, thus leaving patients with an extremely poor prognosis. In viral, alcoholic, and other chronic hepatitis, it has been shown that there is an activation of the progenitor/stem cell population, which has been found to reside in the canals of Hering. In fact, the degree of inflammation and the disease stage have been correlated with the degree of activation. Dysregulation of key regulatory signaling pathways such as transforming growth factor-beta/transforming growth factor-beta receptor (TGF-beta/TBR), insulin-like growth factor/IGF-1 receptor (IGF/IGF-1R), hepatocyte growth factor (HGF/MET), Wnt/beta-catenin/FZD, and transforming growth factor-alpha/epidermal growth factor receptor (TGF-alpha/EGFR) in this progenitor/stem cell population could give rise to HCC. Further understanding of these key signaling pathways and the molecular and genetic alterations associated with HCC could provide major advances in new therapeutic and diagnostic modalities.

Kogure, T., Y. Ueno, et al.; (2009). "A novel third generation bisphosphonate, minodronate (YM529), prevented proliferation and migration of hepatocellular carcinoma cells through inhibition of mevalonate pathway." Hepatol Res; 39(5); 479-489.

                Aim: Skeletal metastases and bone metasitasis are a common occurrence in patients with advanced hepatocellular carcinoma (HCC). Bisphosphonates (BPs), which are used for the treatment of osteoporosis and tumor-associated hypercalcemia, have recently been reported to decrease skeletal morbidity in patients with metastatic bone disease. Several studies revealed that nitrogen-containing BPs (N-BPs) could inhibit tumor growth and migration, indicating the possibility that N-BPs have direct inhibitory effects. We aimed to determine the effects of novel a N-BP (YM529) on human HCC cells in vitro. Methods: HCC cells were treated with various concentrations of YM529 and the growth inhibition rate was determined. Apoptosis was evaluated by caspase-3/7 assay and caspase-9 cleavage detection. The effects of YM529 on the migration of HCC cells induced by hepatocyte growth factor (HGF) were determined by cell migration assay. To evaluate the involvement of the mevalonate pathway, farnesol (FOH) and geranylgeraniol (GGOH) were added. Results: YM529 inhibited the proliferation of HCC cells in a dose-dependent manner. The activation of caspase-3/7 and cleavage of caspase-9 demonstrated the involvement of apoptosis in cytotoxicity. GGOH reduced the growth inhibitory effect of YM529 and suppressed the induction of caspase-3/7 activities by YM529 on HCC cells. YM529 inhibited tumor cell migration induced by HGF and this effect was reduced by co-treatment with GGOH. Conclusion: YM529 inhibited the cell proliferation and migration of HCC cells, implicating the involvement of the mevalonate pathway. These results suggest that N-BPs are potential agents for the treatment of HCC skeletal metastases.

Kuroki, T., Y. Tajima, et al.; (2005). "Hepatolithiasis and intrahepatic cholangiocarcinoma: carcinogenesis based on molecular mechanisms." J Hepatobiliary Pancreat Surg; 12(6); 463-466.

                Hepatolithiasis is more frequently seen in East Asian countries than in Western countries, and it is well known to represent a high-risk state for intrahepatic cholangiocarcinoma. Intrahepatic cholangiocarcinoma is an aggressive tumor that shows a dismal outcome even after resection. Cancer results from multistep carcinogenesis; however, the precise molecular mechanisms involved in the genetic alterations in cancer remain unknown. The accumulation of alterations in cancer-related genes leads to disruptions in cell-cycle regulation and also to continuous cell proliferation. The present review provides an overview of cancer-related genes in intrahepatic cholangiocarcinogenesis arising in hepatolithiasis. Further study of molecular mechanisms in hepatolithiasis-related intrahepatic cholangiocarcinoma, and the delineation of the influence of the genes involved should lead to our understanding of cholangiocarcinogenesis.

Lai, J. P., J. R. Chien, et al.; (2004). "hSulf1 Sulfatase promotes apoptosis of hepatocellular cancer cells by decreasing heparin-binding growth factor signaling." Gastroenterology; 126(1); 231-248.

                BACKGROUND AND AIMS: The heparin-binding growth factors fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) are potent mitogens for hepatocellular carcinomas (HCCs). Heparin-binding growth factor signaling is regulated by sulfation of cell-surface heparan sulfate proteoglycans (HSPGs). We hypothesized that hSulf1, a recently described sulfatase, regulates growth signaling in HCCs. METHODS: Expression of hSulf1 in human HCC tumors was determined by real-time PCR. Down-regulation of hSulf1 expression was investigated by analyzing loss of heterozygosity (LOH) at the hSulf1 locus and the effect of the DNA methylation inhibitor 5-aza-deoxycytidine on hSulf1 expression. The subcellular location of hSulf1 and sulfation state of cell-surface HSPGs were assessed by immunocytochemistry. FGF and HGF signaling was examined by phospho-specific immunoblot analysis. Cell growth was measured by trypan blue exclusion, and the MTT assay and apoptosis were quantitated by fluorescence microscopy. RESULTS: hSulf1 expression was decreased in 29% of HCCs and 82% of HCC cell lines. There was LOH at the hSulf1 locus in 42% of HCCs. Treatment with 5-aza-deoxycytidine reactivated hSulf1 expression in hSulf1-negative cell lines. Low hSulf1-expressing cells showed increased sulfation of cell-surface HSPGs, enhanced FGF and HGF-mediated signaling, and increased HCC cell growth. Conversely, forced expression of hSulf1 decreased sulfation of cell-surface HSPGs and abrogated growth signaling. HCC cells with high-level hSulf1 expression were sensitive to staurosporine- or cisplatin-induced apoptosis, whereas low expressing cells were resistant. Transfection of hSulf1 into hSulf1-negative cells restored staurosporine and cisplatin sensitivity. CONCLUSIONS: Down-regulation of hSulf1 contributes to hepatocarcinogenesis by enhancing heparin-binding growth factor signaling and resistance to apoptosis.

Lee, S. A., S. Ladu, et al.; (2010). "Synergistic role of Sprouty2 inactivation and c-Met up-regulation in mouse and human hepatocarcinogenesis." Hepatology; 52(2); 506-517.

                Sprouty2 (Spry2), a negative feedback regulator of the Ras/mitogen-activated protein kinase (MAPK) pathway, is frequently down-regulated in human hepatocellular carcinoma (HCC). We tested the hypothesis that loss of Spry2 cooperates with unconstrained activation of the c-Met protooncogene to induce hepatocarcinogenesis via in vitro and in vivo approaches. We found coordinated down-regulation of Spry2 protein expression and activation of c-Met as well as its downstream effectors extracellular signal-regulated kinase (ERK) and v-akt murine thymoma viral oncogene homolog (AKT) in a subset of human HCC samples with poor outcome. Mechanistic studies revealed that Spry2 function is disrupted in human HCC via multiple mechanisms at both transcriptional and post-transcriptional level, including promoter hypermethylation, loss of heterozygosity, and proteosomal degradation by neural precursor cell expressed, developmentally down-regulated 4 (NEDD4). In HCC cell lines, Spry2 overexpression inhibits c-Met-induced cell proliferation as well as ERK and AKT activation, whereas loss of Spry2 potentiates c-Met signaling. Most importantly, we show that blocking Spry2 activity via a dominant negative form of Spry2 cooperates with c-Met to promote hepatocarcinogenesis in the mouse liver by sustaining proliferation and angiogenesis. The tumors exhibited high levels of activated ERK and AKT, recapitulating the subgroup of human HCC with a clinically aggressive phenotype. Conclusion: The occurrence of frequent genetic, epigenetic, and biochemical events leading to Spry2 inactivation provides solid evidence that Spry2 functions as a tumor suppressor gene in liver cancer. Coordinated deregulation of Spry2 and c-Met signaling may be a pivotal oncogenic mechanism responsible for unrestrained activation of ERK and AKT pathways in human hepatocarcinogenesis.

Li, N., H. Fu, et al.; (2009). "miR-34a inhibits migration and invasion by down-regulation of c-Met expression in human hepatocellular carcinoma cells." Cancer Lett; 275(1); 44-53.

                Several studies have shown that miR-34a represses the expression of many genes and induces G1 arrest, apoptosis, and senescence. In the present study, we identified the role of miR-34a in the regulation of tumor cell scattering, migration, and invasion. Down-regulation of miR-34a expression was highly significant in 19 of 25 (76%) human hepatocellular carcinoma (HCC) tissues compared with adjacent normal tissues and associated with the metastasis and invasion of tumors. Furthermore, resected normal/tumor tissues of 25 HCC patients demonstrated an inverse correlation between miR-34a and c-Met-protein. In HepG2 cells, ectopic expression of miR-34a potently inhibited tumor cell migration and invasion in a c-Met-dependent manner. miR-34a directly targeted c-Met and reduced both mRNA and protein levels of c-Met; thus, decreased c-Met-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). Taken together, these results provide evidence to show the suppression role of miR-34a in tumor migration and invasion through modulation of the c-Met signaling pathway.

Liu, Y., J. He, et al.; (2010). "Identification and confirmation of biomarkers using an integrated platform for quantitative analysis of glycoproteins and their glycosylations." J Proteome Res; 9(2); 798-805.

                Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver. However, accurate diagnosis can be difficult as most of the patients who develop this tumor have symptoms similar to those caused by longstanding liver disease. Herein we developed an integrated platform to discover the glycoprotein biomarkers in early HCC. At first, lectin arrays were applied to investigate the differences in glycan structures on serum glycoproteins from HCC and cirrhosis patients. The intensity for AAL and LCA was significantly higher in HCC, indicating an elevation of fucosylation level. Then serum from 10 HCC samples and 10 cirrhosis samples were used to screen the altered fucosylated proteins by a combination of Exactag labeling, lectin extraction and LC-MS/MS. Finally, 27 HCC and 27 cirrhosis serum samples were used for lectin-antibody arrays to confirm the change of these fucosylated proteins. C3, CE, HRG, CD14 and HGF were found to be biomarker candidates for distinguishing early HCC from cirrhosis, with a sensitivity of 72% and specificity of 79%. Our work gives insight to the detection of early HCC, and the application of this comprehensive strategy has the potential to facilitate biomarker discovery on a large scale.

Ljubimova, J. Y., L. M. Petrovic, et al.; (1997). "Expression of HGF, its receptor c-met, c-myc, and albumin in cirrhotic and neoplastic human liver tissue." J Histochem Cytochem; 45(1); 79-87.

                Hepatocellular carcinoma (HCC) is a common type of cancer, with approximately 260,000 new cases each year, and liver cirrhosis is generally considered a major predisposing factor for HCC. However, specific changes of gene expression in liver cirrhosis and HCC remain obscure. The expression of genes for hepatocyte growth factor (HGF), its receptor c-met proto-oncogene, c-myc proto-oncogene, and albumin was analyzed. Gene expression was studied by PCR in seven normal human livers, nine cases of hepatitis C cirrhosis, 12 cases of alcoholic cirrhosis, two cases of liver adenoma, and 12 cases of HCC. HGF and c-met protein were revealed by immunofluorescent staining. HGF mRNA was not expressed in normal livers but was detected in adenomas, in 80% of HCC, and in some cirrhoses. Paraffin-embedded and fresh-frozen tissue samples yielded similar results. Immunohistochemical data correlated with PCR results regarding the overexpression of the HGF/c-met system in HCC. Albumin gene expression was decreased in HCC vs normal livers, consistent with altered function of tumor hepatocytes. The elevated expression of the HGF/c-met system in HCC may play a role in tumor development and/or progression. Tissue localization studies of HGF and its receptor c-met protein support the existence of both autocrine and paracrine mechanisms of action of HGF in HCC vs only a paracrine mechanism in normal liver.

Llovet, J. M., Y. Chen, et al.; (2006). "A molecular signature to discriminate dysplastic nodules from early hepatocellular carcinoma in HCV cirrhosis." Gastroenterology; 131(6); 1758-1767.

                BACKGROUND & AIMS: Small liver nodules approximately 2 cm are difficult to characterize by radiologic or pathologic examination. Our aim was to identify a molecular signature to diagnose early hepatocellular carcinoma (HCC). METHODS: The transcriptional profiles of 55 candidate genes were assessed by quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) in 17 dysplastic nodules (diameter, 10 mm) and 20 early HCC (diameter, 18 mm) from HCV cirrhotic patients undergoing resection/transplantation and 10 nontumoral cirrhotic tissues and 10 normal liver tissues. Candidate genes were confirmed by quantitative RT-PCR in 20 advanced HCCs and by immunohistochemistry in 75 samples and validated in an independent set of 29 samples (dysplastic nodules [10] and small HCC [19; diameter, 20 mm]). RESULTS: Twelve genes were significantly, differentially expressed in early HCCs compared with dysplastic nodules (>2-fold change; area under the receiver operating characteristic curve > or =0.8): this included TERT, GPC3, gankyrin, survivin, TOP2A, LYVE1, E-cadherin, IGFBP3, PDGFRA, TGFA, cyclin D1, and HGF. Logistic regression analysis identified a 3-gene set including GPC3 (18-fold increase in HCC, P = .01), LYVE1 (12-fold decrease in HCC, P = .0001), and survivin (2.2-fold increase in HCC, P = .02), which had a discriminative accuracy of 94%. The validity of the gene signature was confirmed in a prospective testing set. GPC3 immunostaining was positive in all HCCs and negative in dysplastic nodules (22/22 vs 0/14, respectively, P

Luo, Y. Q., M. C. Wu, et al.; (1999). "Gene expression of hepatocyte growth factor and its receptor in HCC and nontumorous liver tissues." World J Gastroenterol; 5(2); 119-121.

                AIM:To study the changes of gene expression of hepatocyte growth factor (HGF) and hepatocyte growth factor receptor (HGFr) in hepatocellular carcinoma (HCC) tissue and nontumorous liver tissue and the relationship between these changes and the biological behavior of the tumor.METHODS:Gene expression of HGF and HGFr in 26 cases of HCC tissue and their adjacent nontumorous liver tissues was determined with digoxigenin labeled DNA probes.RESULTS:Positive expression of HGF in HCC tissue was similar to that in the adjacent nontumorous liver tissue, but positive rate of HGF expression was lower than HGFr gene expression. However, HGFr expression was higher in the metastatic cases than in those without metastasis. It was found that HGFr was overexpressed in HCC tissue as well as in the adjacent nontumorous liver tissue.CONCLUSION:There seems to be a close relationship between overexpression of HGFr gene and tumor metastasis, and the HGF and HGFr system plays an important role in regulating tumor growth and metastasis.

Mabed, M., S. Aref, et al.; (2003). "Hepatocellular carcinoma of a short malignant transformation time in a patient with acute myeloblastic leukemia." Ann Hematol; 82(5); 318-320.

                Few cases of hepatocellular carcinoma (HCC) have been described during the course of acute leukemia. The chemotherapy given may be responsible for the development of HCC in such cases. Associated hepatitis may also be responsible. Usually, cancer is a multistep process in which multiple genetic alterations must occur to have a cumulative effect on the control of cell differentiation, cell division, and growth control. This usually takes place over the span of years. Here, we present a case of a patient with acute myeloblastic leukemia who developed HCC of a short malignant transformation time, which does not seem to be related to associated hepatitis or to the chemotherapy given. This may draw attention to the possible contributory role of certain products secreted by the myeloid leukemic cells such as the hepatocyte growth factor (HGF) in increasing the risk of developing HCC.

Majka, M., J. Drukala, et al.; (2006). "SDF-1 alone and in co-operation with HGF regulates biology of human cervical carcinoma cells." Folia Histochem Cytobiol; 44(3); 155-164.

                Stromal Derived Factor-1 (SDF-1)-CXCR4 axis plays a pivotal role in biology and metastasis of several tumors. The aim of this study was to see if SDF-1 alone or in combination with Hepatocyte Growth Factor (HGF) affects biology of human cervical carcinoma (HCC) cells. We found that HCC cell lines investigated in our study highly express CXCR4 on their surface. CXCR4 was also expressed on tumor cells in tissue sections derived from cervical cancer patients. At the same time normal cervical epithelium was negative for CXCR4 expression what suggests a strong correlation between CXCR4 and malignant cell phenotype. Subsequently, we studied a potential role of the SDF-1-CXCR4 axis in HCC and noticed that SDF-1 (i) chemoattracted HCC cells, (ii) enhanced their scattering, (iii) stimulated nuclear localization of beta-catenins and upregulated their target gene cyclin D1 and (iv) at the molecular level induced calcium flux and activated RAS-MAPK, PI3-AKT and JAK-STAT pathways. SDF-1-mediated functions were additionally enhanced in the presence of HGF. Thus, our data show that the SDF-1-CXCR4 axis affects biology of HCC cells. Furthermore, we postulate that this axis might become a potential target to prevent progression of cervical cancer.

Malaguarnera, G., M. Giordano, et al.; (2010). "Serum markers of hepatocellular carcinoma." Dig Dis Sci; 55(10); 2744-2755.

                BACKGROUND: The hepatocellular carcinoma is one of the most common malignant tumors and carries a poor survival rate. The management of patients at risk for developing HCC remains intricate. METHODS: A literature search identified potential markers for hepatocellular carcinoma. These markers were analysed and justification was provided for these factors' inclusion to (or exclusion from) the markers of hepatocellular carcinoma (HCC). A search of the literature was made using cancer literature and the PubMed database for the following keywords: "markers and HCC," "Lens culinaris agglutinin reactive AFP (AFP-L3) and HCC," "Des-gamma-carboxy prothrombin (DCP) and HCC," "Glypican-3 and HCC," "Chromogranin A and HCC," "Transforming growth factor beta1(TGF) and HCC," "alpha-l-fucosidase (AFU) and HCC," "Golgi protein-73 (GP73) and HCC," "Hepatocyte growth factor (HGF) and HCC," "Nervous growth factor (NGF) and HCC." CONCLUSIONS: Despite the large number of studies devoted to the immunohistochemistry of HCC, at the present time, the absolute positive and negative markers for HCC are still lacking, and even those characterized by very high sensitivity and specificity do not have an universal diagnostic usefulness. Given the poor response to current therapies, a better understanding of the molecular pathways active in this disease could potentially provide new targets for therapy. However, AFP shows a low sensitivity, therefore other biomarkers have been developed to make an early diagnosis and improve patients' prognosis.

Martinez-Lopez, N., M. Varela-Rey, et al.; (2008). "S-adenosylmethionine and proliferation: new pathways, new targets." Biochem Soc Trans; 36(Pt 5); 848-852.

                SAMe (S-adenosylmethionine) is the main methyl donor group in the cell. MAT (methionine adenosyltransferase) is the unique enzyme responsible for the synthesis of SAMe from methionine and ATP, and SAMe is the common point between the three principal metabolic pathways: polyamines, transmethylation and transsulfuration that converge into the methionine cycle. SAMe is now also considered a key regulator of metabolism, proliferation, differentiation, apoptosis and cell death. Recent results show a new signalling pathway implicated in the proliferation of the hepatocyte, where AMPK (AMP-activated protein kinase) and HuR, modulated by SAMe, take place in HGF (hepatocyte growth factor)-mediated cell growth. Abnormalities in methionine metabolism occur in several animal models of alcoholic liver injury, and it is also altered in patients with liver disease. Both high and low levels of SAMe predispose to liver injury. In this regard, knockout mouse models have been developed for the enzymes responsible for SAMe synthesis and catabolism, MAT1A and GNMT (glycine N-methyltransferase) respectively. These knockout mice develop steatosis and HCC (hepatocellular carcinoma), and both models closely replicate the pathologies of human disease, which makes them extremely useful to elucidate the mechanism underlying liver disease. These new findings open a wide range of possibilities to discover novel targets for clinical applications.

Mas, V. R., D. G. Maluf, et al.; (2007). "Angiogenesis soluble factors as hepatocellular carcinoma noninvasive markers for monitoring hepatitis C virus cirrhotic patients awaiting liver transplantation." Transplantation; 84(10); 1262-1271.

                BACKGROUND: Physiological angiogenesis occurs during liver regeneration, leading to the formation of new functional sinusoids. Pathological angiogenesis occurs in hepatocellular carcinoma (HCC). We aimed to evaluate the expression of angiogenic factors in hepatitis C virus (HCV)-HCC tissues and the utility of angiogenesis soluble factors as noninvasive markers of HCC and tumor growth. METHODS: Thirty-eight HCV-HCC tumors with 10 corresponding nontumor cirrhotic tissues, as well as 42 independent HCV cirrhotic and 6 normal liver tissues were studied using high-density oligonucleotide arrays. Human angiogenesis microarray was used for the protein detection of EGF, TIMP-1, TIMP-2, HGF, angiopn-1, angiopn-2, VEGF-A, IP-10, PDGF, KGF, angiogenin, VEGF-D, ICAM-1, and FGF in plasma samples from 40 patients (30 HCCs and 10 HCV cirrhosis). RESULTS: From the gene expression analysis of the HCV-HCC tumors compared to normal livers, we found an important number of genes related to angiogenesis differentially expressed (alpha=0.01), including VEGF, PDGF, AGPTL2, ANG, EGFL6, EGFR, angiopn-1, angiopn-2, ICAM2, TIMP-2, among others. Moreover, angiogenic genes were also differentially expressed when HCV-HCC samples were compared to HCV cirrhotic tissues (alpha=0.01; VEGF, EGFL3, EGFR, VEGFB, among others). Ten out of 14 angiogenic proteins analyzed were statistically differentially expressed between HCV cirrhosis and HCV-HCC groups (TIMP-1, TIMP-2, HGF, angiopn-1, angiopn-2, VEGF-A, IP-10, PDGF, KGF, and FGF; P

Mizuguchi, T., M. Nagayama, et al.; (2009). "Prognostic impact of surgical complications and preoperative serum hepatocyte growth factor in hepatocellular carcinoma patients after initial hepatectomy." Journal of Gastrointestinal Surgery; 13(2); 325-333.

                INTRODUCTION: The relationship between postoperative complications and survival after hepatectomy is not completely understood. The purpose of this study was to determine if surgical complications would have a prognostic impact and to identify any difference of the prognostic factors between a complication group and complication-free group for hepatocellular carcinoma (HCC) patients after initial hepatectomy. PATIENTS AND METHODS: One hundred consecutive HCC patients were analyzed in this study. Operative variables and liver functional markers were compared between the complication group and complication-free group. The diagnostic accuracy for predicting complications was evaluated by the receiver operating characteristic (ROC) curve. The Kaplan-Meier method with log-rank test was employed for survival analysis. Univariate and multivariate analyses were performed to identify the prognostic factors in each group. RESULTS AND DISCUSSION: A total of 45 complications in 32 patients were observed according to the modified Clavien classification. The albumin, gamma-glutamyl transferase, choline esterase, indocyanine green retention rate at 15 min (ICGR(15)), hyaluronic acid, prealbumin, hepatocyte growth factor (HGF), HH15, and LHL15 levels before hepatectomy, operative time, and blood loss were significantly different between the two groups. Multivariate analysis revealed that gamma-glutamyl transferase, ICGR(15), and HGF were independent risk factors for postoperative complications. The values of the areas under the ROC curve for predicting complications proved the significance of the predictions. Although the recurrence-free survival rates were not significantly different, the overall survival rates were significantly different between the two groups. Univariate and multivariate analyses for the overall survival rate showed that the stage of the HCC and HGF for the complication group and tumor size for the complication-free group were independent prognostic factors for overall survival. CONCLUSION: Postoperative surgical complications could have a prognostic impact on overall survival in HCC patients after initial hepatectomy. Serum HGF could be a factor connected to complications and survival in this group.

Monvoisin, A., C. Bisson, et al.; (2002). "Involvement of matrix metalloproteinase type-3 in hepatocyte growth factor-induced invasion of human hepatocellular carcinoma cells." International Journal of Cancer; 97(2); 157-162.

                Intra-hepatic invasion is a key feature of hepatocellular carcinoma (HCC) progression. We have shown that human liver myofibroblasts induce invasion of HCC cells through Matrigel, via the secretion of hepatocyte growth factor (HGF). In our study, we investigated the role of matrix metalloproteinases (MMP) in HGF-induced HCC cells invasion. Marimastat, a synthetic MMP inhibitor, dose-dependently decreased HGF-induced invasion of HepG2 cells with a maximum of 82.7 +/- 13.3% at 20 microM. TIMP-2, a natural inhibitor, decreased invasion up to 51.2 +/- 11.2% at 200 ng/ml. To determine the target for these inhibitors, we examined MMP expression using RT-PCR. MMPs 1, 7-9 and 10 were not expressed in HepG2 cells either in the absence or in the presence of HGF. MMP-2 and MMP-13 transcripts were detected in unstimulated cells but their expression was unchanged after exposition to HGF. MMP-3 transcripts were undetectable in unstimulated HepG2 cells. They became clearly expressed in HGF-stimulated cells, however, and this was confirmed by Northern blot. By Western blot, HGF dose-dependently stimulated the secretion of pro-MMP-3 in the culture medium. The role of MMP-3 in HGF-induced invasion was directly confirmed by using an antibody to MMP-3, that blocked invasion. Finally, RT-PCR demonstrated MMP-3 expression in 10/16 human HCCs tested, but not in normal liver. In conclusion, our data demonstrate that MMPs, most likely MMP-3, mediate HGF-induced invasion of HCC cells. The in vivo expression of MMP-3 in HCC suggests a role for this protease in HCC progression.

Murakami, K., T. Matsuura, et al.; (1998). "4-[3,5-Bis(trimethylsilyl)benzamido] benzoic acid (TAC-101) inhibits the intrahepatic spread of hepatocellular carcinoma and prolongs the life-span of tumor-bearing animals." Clin Exp Metastasis; 16(7); 633-643.

                We examined the in vivo anti-tumor activity of the benzoic acid derivative, TAC-101 (4-[3,5-bis(trimethylsilyl)benzamido] benzoic acid), for intrahepatic spread of JHH-7 human hepatocellular carcinoma (HCC) cells and its mechanism of action. Oral administration of TAC-101 markedly inhibited liver tumor of JHH-7 cells and prolonged the life-span of tumor-bearing mice without affecting the body weight. The life-prolonging effect of TAC-101 was more effective than that of other anti-cancer agents including CDDP, 5-FU, and CPT-11 (T/C (%) of life-span; 181 to 219, 128, 133, and 142%, respectively). In vitro, TAC-101 at the concentration of more than 10 microM showed direct cytotoxicity against JHH-7 cells caused by induction of apoptosis. Hepatocyte growth factor (HGF) enhanced the invasive ability of JHH-7 cells without affecting the cell viability. Non-cytotoxic concentrations of TAC-101 inhibited the JHH-7 invasion induced by HGF and down-regulated the expression of c-MET protein in a concentration-dependent manner. In summary, these results suggest that TAC-101 would be useful for a new class of therapeutic agents and that it may improve the prognosis of patients with liver-tumors including metastasizing tumor and HCC.

Murakami, K., R. Sakukawa, et al.; (1999). "Invasiveness of hepatocellular carcinoma cell lines: contribution of membrane-type 1 matrix metalloproteinase." Neoplasia; 1(5); 424-430.

                Intrahepatic metastasis is one of the malignant features of hepatocellular carcinoma (HCC). Matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (u-PA)/plasmin, are known to be associated with the invasive properties of various types of tumor cells. In this study, we examined which proteinases play a role in the metastatic invasion of human HCC cell lines. JHH-5 and JHH-6 cells constitutively expressed mRNAs for both membrane-type 1 matrix metalloproteinase (MT1-MMP) and u-PA and invaded through reconstituted MATRIGEL in vitro, whereas JHH-7 cells expressed u-PA mRNA but not MT1-MMP and did not invade. However, hepatocyte growth factor (HGF) induced MT1-MMP expression on the surface of JHH-7 cells and markedly increased invasiveness of JHH-7 in a concentration-dependent manner. Moreover, cleavage activity for pro-MMP-2 was induced in HGF-treated JHH-7 cells. MMP inhibitor, rather than serine proteinase inhibitor, potently inhibited HCC cell invasion. Intrahepatic injection of HCC cell lines into athymic nude mice caused visible intrahepatic metastases in vivo. Moreover, JHH-7 tumors showed expression of MT1-MMP mRNA, while in vitro cultured JHH-7 cells did not. These findings suggest that MT1-MMP plays an important role in the invasive properties of HCC cells, and that HGF modifies the invasive properties of noninvasive HCC cells.

Nagai, T., T. Arao, et al.; (2011). "Sorafenib inhibits the hepatocyte growth factor-mediated epithelial mesenchymal transition in hepatocellular carcinoma." Mol Cancer Ther; 10(1); 169-177.

                The epithelial mesenchymal transition (EMT) has emerged as a pivotal event in the development of the invasive and metastatic potentials of cancer progression. Sorafenib, a VEGFR inhibitor with activity against RAF kinase, is active against hepatocellular carcinoma (HCC); however, the possible involvement of sorafenib in the EMT remains unclear. Here, we examined the effect of sorafenib on the EMT. Hepatocyte growth factor (HGF) induced EMT-like morphologic changes and the upregulation of SNAI1 and N-cadherin expression. The downregulation of E-cadherin expression in HepG2 and Huh7 HCC cell lines shows that HGF mediates the EMT in HCC. The knockdown of SNAI1 using siRNA canceled the HGF-mediated morphologic changes and cadherin switching, indicating that SNAI1 is required for the HGF-mediated EMT in HCC. Interestingly, sorafenib and the MEK inhibitor U0126 markedly inhibited the HGF-induced morphologic changes, SNAI1 upregulation, and cadherin switching, whereas the PI3 kinase inhibitor wortmannin did not. Collectively, these findings indicate that sorafenib downregulates SNAI1 expression by inhibiting mitogen-activated protein kinase (MAPK) signaling, thereby inhibiting the EMT in HCC cells. In fact, a wound healing and migration assay revealed that sorafenib completely canceled the HGF-mediated cellular migration in HCC cells. In conclusion, we found that sorafenib exerts a potent inhibitory activity against the EMT by inhibiting MAPK signaling and SNAI1 expression in HCC. Our findings may provide a novel insight into the anti-EMT effect of tyrosine kinase inhibitors in cancer cells.

Nagata, K., S. Hirono, et al.; (2001). "Expression of hepatocyte growth factor activator and hepatocyte growth factor activator inhibitor type 1 in human hepatocellular carcinoma." Biochem Biophys Res Commun; 289(1); 205-211.

                Hepatocyte growth factor activator inhibitor type 1 (HAI-1), a Kunitz-type serine protease inhibitor for hepatocyte growth factor activator (HGFA), is responsible for proteolytic activation of hepatocyte growth factor. We examined the expression of HGFA and HAI-1 in liver tissues of chronic liver diseases including hepatocellular carcinoma (HCC). HGFA expression was detected not only in the liver tissues of chronic hepatitis and cirrhosis and in the nontumorous liver tissues surrounding HCC, but also in HCC tissues. On the other hand, none of the liver tissues of hepatitis and cirrhosis and none of the nontumorous tissues surrounding HCC were stained with anti-HAI-1. However, 35% of HCC tissues were stained with anti-HAI-1, and HAI-1 positivity increased as the histological grade decreased and as serum alpha-fetoprotein increased. Transduction of antisense HAI-1 inhibited the growth of human hepatoma cells. These results suggest the possibility that HAI-1 plays an important role in the progression of HCC.

Nakanishi, C., A. Moriuchi, et al.; (2006). "Effect of hepatocyte growth factor on endogenous hepatocarcinogenesis in rats fed a choline-deficient L-amino acid-defined diet." Oncol Rep; 16(1); 25-31.

                Hepatocyte growth factor (HGF) is a promising agent for the treatment of intractable liver disease, due to its mitogenic, anti-apoptotic, and anti-fibrotic effects. We investigated the effect of recombinant human HGF (rh-HGF) on the development of both hepatocellular carcinoma (HCC) and preneoplastic nodules in rats fed a choline-deficient L-amino acid-defined (CDAA) diet, an animal model of hepatocarcinogenesis resembling human development of HCC with cirrhosis. From weeks 13 to 48 of the CDAA diet, rh-HGF (0.1 or 0.5 mg/kg/day) was administered intravenously to rats in four-week cycles, with treatment for five consecutive days of each week for two weeks, followed by a two-week washout period. Treatment with rh-HGF significantly inhibited the development of preneoplastic nodules in a dose-dependent manner at 24 weeks. Although the numbers and areas of the preneoplastic nodules in rats treated with rh-HGF were equivalent to those in mock-treated rats by 60 weeks, the incidence of HCC was reduced by HGF treatment. Although one rat treated with low-dose rh-HGF exhibited a massive HCC, which occupied almost the whole liver, and lung metastases, HGF treatment did not increase the overall frequency of HCC. Administration of high-dose rh-HGF, however, induced an increase in the urinary excretion of albumin, leading to decreased serum albumin at 60 weeks. These results indicate that long-term administration of rh-HGF does not accelerate hepatocarcinogenesis in rats fed a CDAA diet. However, these findings do not completely exclude the potential of HGF-induced hepatocarcinogenesis; this issue must be resolved before rh-HGF can be used for patients with intractable liver diseases, especially those with cirrhosis.

Nakanishi, K., J. Fujimoto, et al.; (1999). "Hepatocyte growth factor promotes migration of human hepatocellular carcinoma via phosphatidylinositol 3-kinase." Clin Exp Metastasis; 17(6); 507-514.

                Hepatocyte growth factor (HGF) is known to be a potent mitogen and motogen for epithelial cells. Hepatocellular carcinoma (HCC) often metastasizes, and the c-Met/HGF receptor is highly expressed by HCC cells. The aim of this study was to investigate the signaling pathways associated with the motogenic effect of HGF on HCC cells via c-Met. HCC cell lines (Hep3B, HepG2, PLC, and Huh-7) and HCC cells harvested from patients were used for the Boyden chamber assay of chemotactic activity as well as for immunoprecipitation and immunoblotting studies. HGF stimulated the motility of Hep3B, HepG2, and Huh-7 cells in a dose-dependent manner in association with tyrosine phosphorylation of c-Met and activation of phosphatidylinositol 3-kinase (PI3-K). A tyrosine kinase inhibitor (genistein) and a PI3-K inhibitor (wortmannin) prevented the migration of HCC cells. However, migration was not prevented by calphostin C, an inhibitor of protein kinase C (PKC), which is a downstream target of phospholipase Cgamma (PLCgamma). HGF also stimulated the migration of HCC cells obtained from three patients, while wortmannin prevented the migration of these cells. These results indicate that HGF stimulates the migration of HCC cells through the tyrosine phosphorylation of c-Met via activation of PI3-K.

Nakayama, N., H. Kashiwazaki, et al.; (1996). "Hepatocyte growth factor and c-met expression in Long-Evans Cinnamon rats with spontaneous hepatitis and hepatoma." Hepatology; 24(3); 596-602.

                The Long-Evans Cinnamon (LEC) rat is characterized by the spontaneous onset of acute and chronic hepatitis, followed by occurrence of liver cancer, and is thus able to provide a unique experimental model for human genetical liver disease, Wilson's disease. Hepatocyte growth factor (HGF) is a potent hepatotrophic factor in liver regeneration, and its expression is up-regulated in response to liver injuries. We found that the plasma HGF level in LEC rats rose markedly during the fulminant hepatitis phase, fell during the phase of chronic/cholangiofibrosis, and fluctuated during the hepatoma phase. Immunohistological staining of the liver revealed that the number of HGF-positive cells increased remarkably during the fulminant hepatitis phase, and that many of these cells were localized at the portal triads. Fewer HGF-positive cells were observed during the phase of chronic hepatitis. The surface of the hepatocellular carcinoma (HCC) cells and the cytoplasm of the nonepithelial cells in cancerous liver tissues were HGF-positive. The HGF-messenger RNA (mRNA) level in the liver rose in the fulminant hepatitis phase, fell in the chronic hepatitis phase, and was intermediate or high during the hepatoma phase. The expression of c-met mRNA was strong in the tissues of LEC rats with fulminant hepatitis and, especially, in the cholangiofibrosis tissues. c-met mRNA was also detected in HCCs. These results suggest that the HGF-c-met system may play an important role in the regeneration of hepatocytes as well as in the development of HCC in paracrine or autocrine mechanisms.

Neaud, V., S. Faouzi, et al.; (1997). "Human hepatic myofibroblasts increase invasiveness of hepatocellular carcinoma cells: evidence for a role of hepatocyte growth factor." Hepatology; 26(6); 1458-1466.

                The stroma of hepatocellular carcinomas (HCC) is infiltrated with myofibroblasts (MFs). Preliminary in vivo data have suggested that liver MF express hepatocyte growth factor (HGF), a cytokine that has been implicated in several tumor models. Our aim was to investigate the role of MF and HGF in HCC. Cultured liver MF expressed HGF messenger RNA (mRNA) and secreted HGF in their medium, as shown by Western blot, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Addition of MF-conditioned medium to the HepG2 HCC cell line induced cell scattering. This was associated with a decrease in cell proliferation. MF also increased about 100-fold the ability of HepG2 to invade Matrigel. Increased invasiveness was also shown for HuH7 cells, but no scattering was observed and cell proliferation was stimulated. All the effects of MF on both tumor cell types were blocked by addition of an antibody to HGF and they all could be reproduced by adding recombinant HGF to the tumor cells. RT-PCR and Western blot analysis confirmed that both tumor cell lines expressed c-met, the receptor for HGF. The effects of MF-conditioned medium were not reproduced by acidic fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor (EGF), transforming growth factor-beta1 (TGF-beta1), or platelet-derived growth factor (PDGF-BB). Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that HGF was expressed in human HCC. Our data show that human liver MF act on HCC cells to increase their invasiveness and suggest that MF-derived HGF could be involved in the pathogenesis of HCC.

Neaud, V., T. Hisaka, et al.; (2000). "Paradoxical pro-invasive effect of the serine proteinase inhibitor tissue factor pathway inhibitor-2 on human hepatocellular carcinoma cells." J Biol Chem; 275(45); 35565-35569.

                We have previously shown that human liver myofibroblasts promote in vitro invasion of human hepatocellular carcinoma (HCC) cells through a hepatocyte growth factor (HGF)/urokinase/plasmin-dependent mechanism. In this study, we demonstrate that myofibroblasts synthesize the serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2). Despite the fact that recombinant TFPI-2 readily inhibits plasmin, we show that it potentiates HGF-induced invasion of HCC cells and is capable of inducing invasion on its own. Furthermore, HCC cells stably transfected with a TFPI-2 expression vector became spontaneously invasive. HCC cells express tissue factor and specifically factor VII. Addition of an antibody to factor VII abolished the pro-invasive effect of TFPI-2. We suggest that TFPI-2 induces invasion following binding to a tissue factor-factor VIIa complex preformed on HCC cells. Our data thus demonstrate an original mechanism of cell invasion that may be specific for liver tumor cells.

Newell, K. A., E. M. Alonso, et al.; (1997). "Association between liver transplantation for Langerhans cell histiocytosis, rejection, and development of posttransplant lymphoproliferative disease in children." J PEDIATR; 131(1); 98-104.

                Objective: Langerhans cell histiocytosis (LCH) is an unusual indication for orthotopic liver transplantation in children. Data from limited case reports suggest that orthotopic liver transplantation for LCH is associated with excellent survival rates and a low incidence of disease recurrence. However, in our experience, children who hal transplantation for LCH appeared to experience a high incidence of refractory rejection and posttransplant lymphoproliferative disease (PTLD). Study design: Data from 398 liver transplants performed in 298 children younger than 15 years of age were reviewed to determine the presence of risk factors for PTLD in patients with LCH and other causes of liver failure. Results: The incidence of PTLD was significantly higher in children who received transplants for LCH compared with all indications (p

Nowak, B., C. Stellbrink, et al.; (2004). "Comparison of regional myocardial blood flow and perfusion in dilated cardiomyopathy and left bundle branch block: role of wall thickening." J Nucl Med; 45(3); 414-418.

                Heterogeneous perfusion in left bundle branch block (LBBB) has been demonstrated by (99m)Tc-methoxyisobutylisonitrile (MIBI) SPECT. Locally different contraction is also associated with LBBB. Quantitative analysis of myocardial SPECT is influenced by partial-volume effects depending on systolic wall thickening. Therefore, partial-volume effects may mimic perfusion heterogeneity in LBBB. METHODS: Fifteen patients with nonischemic dilated cardiomyopathy and LBBB underwent resting (15)O-water PET, (99m)Tc-MIBI SPECT, and gated (18)F-FDG PET for analysis of wall thickening. Myocardial blood flow corrected for rate-pressure product (corrMBF), (99m)Tc-MIBI uptake, and wall thickening were determined in 4 left ventricular wall areas. In 14 patients, M-mode echocardiographic recordings were available for comparison. RESULTS: Homogeneous distribution was found for corrMBF (1.09 +/- 0.41 to 1.19 +/- 0.31 mL x g(-1) x min(-1)). (99m)Tc-MIBI uptake and wall thickening were heterogeneous (P

Nussbaum, T., J. Samarin, et al.; (2008). "Autocrine insulin-like growth factor-II stimulation of tumor cell migration is a progression step in human hepatocarcinogenesis." Hepatology; 48(1); 146-156.

                The protumorigenic insulin-like growth factor (IGF)-II is highly expressed in a significant fraction of human hepatocellular carcinomas (HCC). However, a functional dissection that clarifies the contribution of IGF-II-binding receptors in tumor progression and a respective molecular characterization of IGF-II signaling has not been performed. Therefore, expression of IGF-II and its receptors IGF-receptor type I (IGF-IR) and insulin receptor (IR) was efficiently blocked using small interfering RNA (siRNA) in HCC cells. Despite functional IR-signaling, oncogenic IGF-II effects such as tumor cell viability, proliferation, and anti-apoptosis were solely transmitted by IGF-IR. Although IGF-II signaling was previously not described in the context of HCC cell migration, the IGF-II-dependent expression profile displayed a high percentage of genes involved in cell motility and adhesion. Indeed, IGF-II overexpression promoted HCC cell migration, especially in synergy with hepatocyte growth factor (HGF). The therapeutic relevance of IGF-II/IGF-IR signaling was tested in vitro and in a murine xenograft transplantation model using the IGF-IR inhibitor picropodophyllin (PPP). IGF-IR inhibition by small molecule treatment efficiently reduced IGF-II-dependent signaling and all protumorigenic properties of the IGF-II/IGF-IR pathway. CONCLUSION: In human HCC cells, IGF-IR but not IR is involved in oncogenic IGF-II signaling. Autocrine stimulation of IGF-II induces HCC motility by integration of paracrine signals for full malignant competence. Thus, activation of IGF-II/IGF-IR signaling is likely a progression switch selected by function that promotes tumor cell dissemination and aggressive tumor behavior.

Okano, J., G. Shiota, et al.; (1999). "Expression of hepatocyte growth factor (HGF) and HGF receptor (c-met) proteins in liver diseases: an immunohistochemical study." Liver; 19(2); 151-159.

                BACKGROUND: Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes in vivo as well as in vitro. Serum levels of HGF vary in liver diseases, reflecting liver damage and dysfunction. However there are no studies reporting expression of HGF and HGF receptor (c-met protein) simultaneously in various liver diseases. METHODS: To clarify the clinical significance of HGF/c-met protein expression in liver diseases, liver tissues from 62 patients consisting of 7 with acute hepatitis (AH), 20 with chronic hepatitis (CH), 9 with liver cirrhosis (LC) and 26 with hepatocellular carcinoma (HCC) were immunohistochemically examined. RESULTS: Intense staining of HGF was observed in patients from AH, CH and LC, although no immunoreactivity was seen in HCC. The expression of c-met protein was higher in patients with HCC and AH than in those with CH (p

Oliva, J., B. A. French, et al.; (2010). "The identification of stem cells in human liver diseases and hepatocellular carcinoma." Exp Mol Pathol; 88(3); 331-340.

                Liver stem cells are thought to preside in bile ducts and the canals of Hering. They extend into the liver parenchyma at a time when normal liver cell proliferation is suppressed and liver regeneration is stimulated. In the present study 69 liver biopsies and surgically excised liver tumors were studied for the presence of liver stem cells. It was found that human cirrhotic livers and hepatocellular carcinomas (HCC) frequently exhibited isolated single scattered hepatocyte stem cells within the liver parenchyma rather than in the portal tract, bile duct or the canal of Hering. These cells expressed liver stem cell markers. HCCs also contained isolated tumor cell which expressed the same stem cell markers. The markers used were GST-P, OV-6, CK-19, Oct-3/4 and FAT10. They were identified by immunofluorescent antibody staining. HGF, EGF, CK19, AIR, H19, Nanog, Oct-3/4 and FAT10 were identified by RNA-FISH. H19 is a non-coding RNA, which is expressed in most HCCs. Results: Immunohistochemistry and RNA-FISH performed on human livers identified isolated stem cells in liver parenchyma as follows: Stem cells identified by immunohistochemical markers (OV-6 and GST-P) and RNA-FISH markers (HGF, EGF, CK19 and H19) were found scattered in the liver parenchyma of cirrhotic livers and within hepatocellular carcinomas (HCCs). Precirrhotic ASH or NASH all stained negative for these stem cells. In HCCs, 13 out of 15 had stem cells located within the tumor (78%). In cirrhotic livers, 12 out of 28 (37%) had liver parenchymal stem cells present. In one case of stage 3 precirrhosis, stem cells were also found. Double staining for the markers showed colocalization of the markers in stem cells. Stem cells were found in 33% of HBV, 47% of HCV, 25% of alcoholic steatohepatitis (ASH) and 17% of non-alcoholic steatohepatitis (NASH). The frequency of stem cells found in the different disease categories correlates with the frequency of HCC occurring in these different diseases.

Oosthuizen, M. M., N. Ndaba, et al.; (2005). "Rat hepatoproliferin revealed the status of a complete hepatomitogen in human hepatoma cells." Transplant Proc; 37(1); 89-92.

                Hepatoproliferin (HPF), a liver regeneration factor isolated from rat hepatocytes, was assessed for its mitogenic status in the human hepatoma cell line PLC/PRF-5. HPF was able to enhance hepatoma cell growth on its own without the aid of the established complete mitogens EGF and TGF-alpha or the hepato-priming factor TNF-alpha. HPF therefore acted as a complete hepatomitogen and had no co-mitogenic properties since it did not augment proliferation when combined with EGF or TGF-alpha but showed only an additive effect in the presence of TGF-alpha. Rat HPF was phylogenetically unrestricted, because it was found active in human cells. When each of the established growth factors (GFs) was used alone, the hepatoma cells responded with the same kind of response profile, namely a bi-phasic bell-shaped dose-dependent response due to stimulation at low levels and inhibition at higher levels. However, hepatocyte growth factor (HGF) was an exception since it did not induce a growth response in hepatoma cells. On the contrary HPF, on its own, showed a progressive enhanced linear dose response at the levels used for the GFs (ie 1.0-15 ng/5 x 10(5) cells). The comparative potency (CP) (dpm x 10(3)/microg DNA/ng GF) of HPF (CP = 13) was in the same range as for the complete hepatomitogens EGF (CP = 12) and TGF-alpha (CP = 14), revealing that HPF has indeed the status of a complete mitogen.

Orian-Rousseau, V., L. Chen, et al.; (2002). "CD44 is required for two consecutive steps in HGF/c-Met signaling." Genes Dev; 16(23); 3074-3086.

                The tyrosine kinase receptor c-Met and its ligand HGF/SF, ezrin, and splice variants of CD44 have independently been identified as tumor metastasis-associated proteins. We now show that these proteins cooperate. A CD44 isoform containing variant exon v6 sequences is strictly required for c-Met activation by HGF/SF in rat and human carcinoma cells, in established cell lines as well as in primary keratinocytes. CD44v6-deficient tumor cells were unable to activate c-Met unless they were transfected with a CD44v6-bearing isoform. Antibodies to two v6-encoded epitopes inhibited autophosphorylation of c-Met by interfering with the formation of a complex formed by c-Met, CD44v6, and HGF/SF. In addition, signal transduction from activated c-Met to MEK and Erk required the presence of the cytoplasmic tail of CD44 including a binding motif for ERM proteins. This suggests a role for ERM proteins and possibly their link to the cortical actin cytoskeleton in signal transfer.

Orian-Rousseau, V., H. Morrison, et al.; (2007). "Hepatocyte growth factor-induced Ras activation requires ERM proteins linked to both CD44v6 and F-actin." Mol Biol Cell; 18(1); 76-83.

                In several types of cells, the activation of the receptor tyrosine kinase c-Met by its ligand hepatocyte growth factor (HGF) requires the coreceptor CD44v6. The CD44 extracellular domain is necessary for c-Met autophosphorylation, whereas the intracellular domain is required for signal transduction. We have already shown that the CD44 cytoplasmic tail recruits ezrin, radixin and moesin (ERM) proteins to the complex of CD44v6, c-Met, and HGF. We have now defined the function of the ERM proteins and the step they promote in the signaling cascade. The association of ERM proteins to the coreceptor is absolutely required to mediate the HGF-dependent activation of Ras by the guanine nucleotide exchange factor Sos. The ERM proteins need, in addition, to be linked to the actin cytoskeleton to catalyze the activation of Ras. Thus, we describe here a new function of the cytoskeleton. It is part of a "signalosome" complex that organizes the activation of Ras by Sos. So far the cytoskeleton has mainly been identified as a "responder" to signal transduction. Here, we show now that F-actin acts as an "inducer" that actively organizes the signaling cascade.

Osada, S., M. Kanematsu, et al.; (2008). "Clinical significance of serum HGF and c-Met expression in tumor tissue for evaluation of properties and treatment of hepatocellular carcinoma." Hepatogastroenterology; 55(82-83); 544-549.

                BACKGROUND/AIMS: To develop a prognostic marker for evaluation of intrahepatic metastasis (IM) of hepatocellular carcinoma (HCC), the ligand-stimulated receptor activity of c-Met due to hepatocyte growth factor (HGF) was estimated. METHODOLOGY: For specimens from 30 HCC patients, who were operated on at the Department of Surgical Oncology, Gifu University School of Medicine, for 2 recent years, the induction value of HGF and c-Met were estimated by western blot. RESULTS: Firstly, the serum HGF levels were significantly higher in invasive gross type or in IM-positive tumors. Secondly, the mean expression value of HGF protein in tumors was 0.56 +/- 0.35, which was not different from non-tumor tissue, 0.59 +/- 0.40. And there was no significant differences based on tumor profiles. Thirdly, the value of c-Met in tumor tissue, 1.36 +/- 0.12, was clearly higher than in non-tumor tissue, 1.07 +/- 0.06. The c-Met expressions were significantly higher in the invasive type of HCC as determined by gross type, vessel invasion, IM presence and histological type. Finally, in individual studies about the relationship between the level of serum HGF and c-Met expression in tumor tissue, the presence of IM could be easily detected. Furthermore, the level of serum HGF after hepatectomy was significantly higher than the preoperative value, and individual studies with c-Met expression were associated with early recurrence. CONCLUSIONS: The induction of c-Met might be important to evaluate the progression of HCC, especially to caution for the presence of IM.

Osada, S., M. Kanematsu, et al.; (2005). "Evaluation of extracellular signal regulated kinase expression and its relation to treatment of hepatocellular carcinoma." J Am Coll Surg; 201(3); 405-411.

                BACKGROUND: The aim of this study was to evaluate hepatocellular carcinoma (HCC) using a combination of extracellular signal regulated kinase (ERK) and other markers related to hepatocyte growth factor (HGF) in tumor tissue. STUDY DESIGN: Using specimens from 30 hepatocellular carcinoma patients operated on in our department from 2002 to 2003, we evaluated expression levels of HGF, c-Met, ERK, and cyclin D1 by Western blot. RESULTS: Expression levels of ERK and cyclin D1 proteins were significantly higher in patients with less well-differentiated type tumors by histologic examination or the presence of intrahepatic metastasis (IM). ERK expression in tumor tissue significantly correlated with both tumor size (p=0.0017, R(2)=0.355) and serum levels of HGF (p=0.0247, R(2)=0.218). Nontumor tissue level of cyclin D1 was significantly higher in patients with poor liver function (p=0.047). In patients with higher expression of ERK in tumor tissue compared with nontumor tissue, the histologic finding was more progressed; but a similar tendency was not observed for cyclin D1. In patients with overexpression of HGF and c-Met, the expression level of ERK was significantly higher, but cyclin D1 expression was not. The detected level of cyclin D1 was significantly higher in patients with overexpressed ERK in tumor tissue. Values of ERK and c-Met were correlated, and IM presence was detected more frequently in patients with high expression of ERK and c-Met protein. Even after complete removal of visible IM tumor, recurrence tumors were detected within 6 months in 7 patients with high expressions of both ERK and c-Met protein. CONCLUSIONS: Combination study of tumor expression of ERK might be useful to estimate the properties of hepatocellular carcinoma, especially for the presence of IM.

Osada, S. and K. Yoshida; (2010). "Application of biological study for met expression to cancer therapy." Anticancer Agents Med Chem; 10(1); 58-63.

                Metastasization is an undesirable process in cancer development and may represent the most critical factor in deciding patient prognosis. Organ specificity of the metastasis process suggests the importance of the paracrine factors: one of the most potent paracrine regulators of tumor cell migration is hepatocyte growth factor/scatter factor (HGF/SF). Because the liver-specific growth factor is HGF, its receptor c-Met expression might play a critical role in metastasization to the liver. Activation of HGF/c-Met signaling has been shown to promote cancer cell invasiveness and trigger metastasis though direct involvement of the angiogenic pathway. Given the importance of aberrant HGF/c-Met signaling, several different therapeutic strategies aimed at inhibiting the pathway have been developed and are currently being evaluated in clinical trials. Among these agents, NK4 and AM102 were introduced as HGF inhibitors, and PHA-665752 and Su11274 as c-Met inhibitors and are under study in clinical trials. Further, clinical experience-based study to apply the accumulation of biological knowledge concerning HGF/c-Met to the surgical field is presented.

Ozaki, I., T. Mizuta, et al.; (2003). "Induction of multiple matrix metalloproteinase genes in human hepatocellular carcinoma by hepatocyte growth factor via a transcription factor Ets-1." Hepatol Res; 27(4); 289-301.

                Matrix metalloproteinases (MMPs) have been implicated in progression of hepatocellular carcinoma (HCC), as have hepatocyte growth factor (HGF) and its c-Met receptor. We investigated regulation of MMP gene expression by HGF in human HCC. Expression of mRNAs encoding MMPs, HGF and c-Met receptor was examined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) in human HCC and in five human HCC cell lines. HCC cells were treated with HGF, and mRNA expression for MMPs and Ets-1 which activates transcription of MMPs was investigated. Ets binding activity was determined by gel mobility shift assay. MMP promoter activities were evaluated by reporter gene assay. Effects of Ets-1 antisense oligonucleotides were also examined. At the mRNA level, MMP-1, -3, -7 as well as c-Met were overexpressed in HCC compared with corresponding nonneoplastic liver tissues, although MMP-2, -9 or HGF were not. HGF dose-dependently induced Ets-1 together with an increased Ets binding activity, followed by transcription of MMP-1, -3, and -7. HGF increased MMP promoter activity, as did cotransfection with Ets-1. Ets-1 antisense oligonucleotide transfection down-regulated the MMP expression, and abolished induction by HGF. In conclusion, certain MMPs and c-Met, overexpressed in HCC cells, are induced by HGF via Ets-1. This pathway may contribute to tumor progression.

Paranjpe, S., W. C. Bowen, et al.; (2010). "RNA interference against hepatic epidermal growth factor receptor has suppressive effects on liver regeneration in rats." American Journal of Pathology; 176(6); 2669-2681.

                Liver regeneration after a two-thirds partial hepatectomy (PHx) is a complex process requiring interaction and cooperation of many growth factors and cytokines and cross talk between multiple pathways. Along with hepatocyte growth factor and its receptor MET (HGF-MET), the epidermal growth factor receptor (EGFR) signaling pathway is activated within 60 minutes after PHx. To investigate the role of EGFR in liver regeneration, we used two EGFR-specific short hairpin silencing RNAs to inhibit EGFR expression in regenerating normal rat liver. Suppression of EGFR mRNA and protein was evident in treated rats. There was also a demonstrable decrease but not complete elimination of bromo-deoxyuridine incorporation and mitoses at 24 hours after PHx. In addition, we observed up-regulation of MET and Src as well as activation of the ErbB-3-ErbB-2-PI3K-Akt pathway and down-regulation of STAT 3, cyclin D1, cyclin E1, p21, and C/EBP beta. The decrease in the ratio of C/EBP alpha to C/EBP beta known to occur after PHx was offset in shEGFR-treated rats. Despite suppression of hepatocyte proliferation lasting into day 3 after PHx, liver weight restoration occurred. Interestingly, hepatocytes in shEGFR-treated rats were considerably larger when compared with ScrRNA-treated controls. The data indicate that although the MET and EGFR pathways are similar, the contributions made by MET and EGFR are unique and are not compensated by each other or other cytokines.

Park, B. H., J. H. Lee, et al.; (2005). "Vascular administration of adenoviral vector soaked in absorbable gelatin sponge particles (GSP) prolongs the transgene expression in hepatocytes." Cancer Gene Ther; 12(2); 116-121.

                Transcatheter hepatic arterial chemoembolization using emulsions composed of anticancer agents and gelatin sponges (GS) has been an efficient and safe palliative treatment for inoperable hepatocellular carcinoma (HCC). We employed catheter-mediated left hepatic arterial embolization (CHAE) to increase transduction efficiency of adenoviral vector in canine hepatocytes. The emulsion was prepared by mixing pieces of GSP and adenoviral vectors expressing recombinant beta-galactosidase (Ad.LacZ) or human hepatocyte growth factor (Ad.hHGF). After the left hepatic artery was catheterized under angiography, CHAE with Ad.LacZ or Ad.hHGF was performed. Livers were removed and stained for LacZ activity on day 7. The expression pattern of LacZ staining was either scarce or patchy around the central hilum of the hepatic artery, or was homogeneously distributed in whole lobes, depending on whether large or small pieces of GSP were used. Hematological and serum biochemical changes during CHAE exhibited only a few effects. The chronological measurement of serum HGF concentration showed that the duration of transgene expression was greater after CHAE with Ad.hHGF. A similar pattern of transgene expression was observed in a rat model after hepatic arterial embolization with differential doses of Ad.hHGF soaked in GSP. These results suggest that hepatic arterial embolization by transcatheter mediated infusion with a mixture of adenovirus-GSP could be used for human HCC.

Patil, M. A., S. A. Lee, et al.; (2009). "Role of cyclin D1 as a mediator of c-Met- and beta-catenin-induced hepatocarcinogenesis." Cancer Res; 69(1); 253-261.

                Activation of c-Met signaling and beta-catenin mutations are frequent genetic events observed in liver cancer development. Recently, we demonstrated that activated beta-catenin can cooperate with c-Met to induce liver cancer formation in a mouse model. Cyclin D1 (CCND1) is an important cell cycle regulator that is considered to be a downstream target of beta-catenin. To determine the importance of CCND1 as a mediator of c-Met- and beta-catenin-induced hepatocarcinogenesis, we investigated the genetic interactions between CCND1, beta-catenin, and c-Met in liver cancer development using mouse models. We coexpressed CCND1 with c-Met in mice and found CCND1 to cooperate with c-Met to promote liver cancer formation. Tumors induced by CCND1/c-Met had a longer latency period, formed at a lower frequency, and seemed to be more benign compared with those induced by beta-catenin/c-Met. In addition, when activated beta-catenin and c-Met were coinjected into CCND1-null mice, liver tumors developed despite the absence of CCND1. Intriguingly, we observed a moderate accelerated tumor growth and increased tumor malignancy in these CCND1-null mice. Molecular analysis showed an up-regulation of cyclin D2 (CCND2) expression in CCND1-null tumor samples, indicating that CCND2 may replace CCND1 in hepatic tumorigenesis. Together, our results suggest that CCND1 functions as a mediator of beta-catenin during HCC pathogenesis, although other molecules may be required to fully propagate beta-catenin signaling. Moreover, our data suggest that CCND1 expression is not essential for liver tumor development induced by c-Met and beta-catenin.

Porta, C. and C. Paglino; (2010). "Medical treatment of unresectable hepatocellular carcinoma: Going beyond sorafenib." World J Hepatol; 2(3); 103-113.

                Even though Sorafenib has radically changed the natural history of those hepatocellular carcinoma patients who are not amenable for curative treatments, further therapeutic improvements are badly needed. As it was for Sorafenib, our increasingly refined understanding of the complex mechanisms underlying HCC carcinogenesis are the starting point for the future development of such treatments. Presently, a number of molecularly targeted agents are in different stages of development for this once orphan cancer. Indeed, several pathways are presently being explored to identify potentially active drugs, including epidermal growth factor receptor, vascular endothelial growth factor/vascular endothelial growth factor receptors, mammalian target of rapamycin, phosphatidyl-inositol-3-kinase/Akt, insulin growth factor, Aurora kinase, Wnt/beta-catenin, retinoic acid receptor and hepatocyte growth factor/C-Met. This review is aimed at addressing the results obtained so far with these newer drugs, also considering the challenges we shall face in the near future, including the issue of response evaluation and identification of predictive/prognostic biomarkers.

Price, J. A., S. J. Kovach, et al.; (2002). "Insulin-like growth factor I is a comitogen for hepatocyte growth factor in a rat model of hepatocellular carcinoma." Hepatology; 36(5); 1089-1097.

                Hepatocyte growth factor-scatter factor (HGF-SF) is a potent hepatic mitogen yet inhibits hepatocellular carcinoma (HCC) cell growth in vitro. Insulin-like growth factor I (IGF-I) is a pleiotropic growth factor shown to be important in cell growth and differentiation in other tumors. We hypothesized that IGF-I may play a role in regulating HGF-SF activity and HCC progression. Using an in vivo model of HCC, we showed elevated IGF-I messenger RNA (mRNA) expression in normal liver from tumor-burdened animals in the absence of changes in circulating IGF-I levels. Analysis of IGF-I receptor (IGF-IR) and HGF-SF (c-met) receptor expression showed significantly higher expression of both receptors in normal liver compared with an HCC specimen. Using cultured HCC cells from this model, we next showed that treatment with IGF-I led to significant increases in mitogen-activated protein kinase (MAPK) activity. Furthermore, we observed significant time-dependent increases in the expression of the c-fos and c-jun proto-oncogenes after addition of IGF-I (n = 5 per group, P

Qin, L. X. and Z. Y. Tang; (2002). "The prognostic molecular markers in hepatocellular carcinoma." World J Gastroenterol; 8(3); 385-392.

                The prognosis of hepatocellular carcinoma (HCC) still remains dismal, although many advances in its clinical study have been made. It is important for tumor control to identify the factors that predispose patients to death. With new discoveries in cancer biology, the pathological and biological prognostic factors of HCC have been studied quite extensively. Analyzing molecular markers (biomarkers) with prognostic significance is a complementary method. A large number of molecular factors have been shown to associate with the invasiveness of HCC, and have potential prognostic significance. One important aspect is the analysis of molecular markers for the cellular malignancy phenotype. These include alterations in DNA ploidy, cellular proliferation markers (PCNA, Ki-67, Mcm2, MIB1, MIA, and CSE1L/CAS protein), nuclear morphology, the p53 gene and its related molecule MD M2, other cell cycle regulators (cyclin A, cyclin D, cyclin E, cdc2, p27, p73), oncogenes and their receptors (such as ras, c-myc, c-fms, HGF, c-met, and erb-B receptor family members), apoptosis related factors (Fas and FasL), as well as telomerase activity. Another important aspect is the analysis of molecular markers involved in the process of cancer invasion and metastasis. Adhesion molecules (E-cadherin, catenins, serum intercellular adhesion molecule-1, CD44 variants), proteinases involved in the degradation of extracellular matrix (MMP-2, MMP-9, uPA, uPAR, PAI), as well as other molecules have been regarded as biomarkers for the malignant phenotype of HCC, and are related to prognosis and therapeutic outcomes. Tumor angiogenesis is critical to both the growth and metastasis of cancers including HCC, and has drawn much attention in recent years. Many angiogenesis-related markers, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF), thrombospondin (TSP), angiogenin, pleiotrophin, and endostatin (ES) levels, as well as intratumor microvessel density (MVD) have been evaluated and found to be of prognostic significance. Body fluid (particularly blood and urinary) testing for biomarkers is easily accessible and useful in clinical patients. The prognostic significance of circulating DNA in plasma or serum, and its genetic alterations in HCC are other important trends. More attention should be paid to these two areas in future. As the progress of the human genome project advances, so does a clearer understanding of tumor biology, and more and more new prognostic markers with high sensitivity and specificity will be found and used in clinical assays. However, the combination of some items, i.e., the pathological features and some biomarkers mentioned above, seems to be more practical for now.

Quiles-Perez, R., J. A. Munoz-Gamez, et al.; (2010). "Inhibition of poly adenosine diphosphate-ribose polymerase decreases hepatocellular carcinoma growth by modulation of tumor-related gene expression." Hepatology; 51(1); 255-266.

                Hepatocellular carcinoma (HCC) is associated with a poor prognosis due to a lack of effective treatment options. In HCC a significant role is played by DNA damage and the inflammatory response. Poly (ADP-ribose) polymerase-1 (PARP-1) is an important protein that regulates both these mechanisms. The objective of this study was to examine the effect of pharmacology PARP-1 inhibition on the reduction of tumor volume of HCC xenograft and on the hepatocarcinogenesis induced by diethyl-nitrosamine (DEN). Pharmacologic PARP-1 inhibition with DPQ greatly reduces tumor xenograft volume with regard to a nontreated xenograft (394 mm(3) versus 2,942 mm(3), P

Radaeva, S., B. Jaruga, et al.; (2002). "Interferon-alpha activates multiple STAT signals and down-regulates c-Met in primary human hepatocytes." Gastroenterology; 122(4); 1020-1034.

                BACKGROUND & AIMS: Interferon (IFN)-alpha therapy is currently the primary choice for viral hepatitis and a promising treatment for hepatocellular carcinoma (HCC). Primary mouse and rat hepatocytes respond poorly to IFN-alpha stimulation. Thus, it is very important to examine the IFN-alpha signal pathway in primary human hepatocytes. METHODS: The IFN-alpha-activated signals and genes in primary human hepatocytes and hepatoma cells were examined by Western blotting and microarray analyses. RESULTS: Primary human hepatocytes respond very well to IFN-alpha stimulation as shown by activation of multiple signal transducer and activator of transcription factor (STAT) 1, 2, 3, 5, and multiple genes. The differential response to IFN-alpha stimulation in primary human and mouse hepatocytes may be caused by expression of predominant functional IFN-alpha receptor 2c (IFNAR2c) in primary human hepatocytes vs. expression of predominant inhibitory IFNAR2a in mouse hepatocytes. Microarray analyses of primary human hepatocytes show that IFN-alpha up-regulates about 44 genes by over 2-fold and down-regulates about 9 genes by 50%. The up-regulated genes include a variety of antiviral and tumor suppressors/proapoptotic genes. The down-regulated genes include c-myc and c-Met, the hepatocyte growth factor (HGF) receptor. Down-regulation of c-Met is caused by IFN-alpha suppression of the c-Met promoter through down-regulation of Sp1 binding and results in attenuation of HGF-induced signals and cell proliferation. CONCLUSIONS: IFN-alpha directly targets human hepatocytes, followed by activation of multiple STATs and regulation of a wide variety of genes, which may contribute to the antiviral and antitumor activities of IFN-alpha in human liver.

Ranganathan, S., X. Tan, et al.; (2005). "beta-Catenin and met deregulation in childhood Hepatoblastomas." Pediatr Dev Pathol; 8(4); 435-447.

                Activation of the Wnt/beta-catenin and hepatocyte growth factor/Met signaling has been implicated in various tumors. Owing to the cross-talk between these pathways and aberrant redistribution of beta-catenin in hepatoblastomas, we examined their status in this tumor. This study examined changes in beta-catenin and Met in paired pretreatment and post-treatment hepatoblastoma tissues in relation to their effects on proliferation and target genes such as c-myc and cyclin-D1. In this study we compared proliferation indices, beta-catenin staining and its known molecular targets, c-myc and cyclin-D1, and Met, a tyrosine kinase receptor for hepatocyte growth factor in pretreatment and post-treatment specimens. Pretreatment and post-treatment sections from 13 children, ages 11 weeks to 9 years, were analyzed for these markers by immunohistochemistry. All tumors (13 of 13) displayed increased proliferation and beta-catenin (cytoplasmic and nuclear) staining in pretreatment biopsies that remained relatively unaffected after treatment. Aberrant Met staining (cytoplasmic) was observed in all pretreatment samples that decreased considerably after treatment in 11 of 13 patients. A significant subset of these tumors showed increased c-myc and cyclin-D1 staining in pretreatment biopsies that decreased after chemotherapy in most cases. beta-Catenin redistribution in tumor cells corresponds to proliferation in hepatoblastomas. However, beta-catenin nuclear localization remains unaffected in viable hepatoblastoma tissue after chemotherapy. In contrast, Met undergoes a prominent decrease after treatment and thus might be important in pathogenesis of hepatoblastoma.

Rao, U. N., S. M. Gollin, et al.; (2001). "Comparative genomic hybridization of hepatocellular carcinoma: correlation with fluorescence in situ hybridization in paraffin-embedded tissue." Molecular Diagnosis; 6(1); 27-37.

                BACKGROUND: Proto-oncogene MYC, mapped to chromosomal band 8q24 and the genes for hepatocyte growth factor (HGF at 7q21) and its receptor, MET, at chromosomal band 7q31, have an important role in the biology and growth of normal and neoplastic liver. Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) studies have reported frequent abnormalities of chromosomes 1 and 8 in hepatocellular carcinomas (HCCs) of various clinical and pathological stages. Chromosome 7 involvement is reported to be less frequent. MATERIALS AND METHODS: Frozen tissue from 17 HCCs was used for CGH analysis and sections of corresponding formalin-fixed, paraffin-embedded HCC tissue were used for dual-color FISH with locus-specific (LSI-cMYC for chromosome 8q24 and LSI D7S486 for chromosome 7q31) and centromeric probes, CEP8 (8p11.1-q11.2) and CEP7 (7p11.1-q11.2) (Vysis, Inc, Downers Grove, IL). This study intended to determine the pattern of chromosomal aberrations in early-stage (incidental) HCC and large surgically resected HCC, and also compared the efficiency and usefulness of the two cytogenetic methods. RESULTS: CGH showed abnormalities on chromosomes 1q, 5q, 7q, 8q, 9, 10, 13q, 15, 16, 17p, 18q, 19, 20, 21, 22, and X. Gains of 8q were noted in 50% of the HCCs, including five cases of incidental HCCs by CGH. Increase in copy numbers of MYC detected by FISH was noted in 25% of tumors that had shown 8q gains by CGH and in five cases with no chromosome abnormalities noted by CGH. Three cases with 7q31 copy number abnormalities were found by FISH in addition to those detected by CGH. CONCLUSION: Combined use of CGH and FISH may provide important information about early and/or primary genetic changes in the development of HCC.

Rogler, C. E. and F. V. Chisari; (1992). "Cellular and molecular mechanisms of hepatocarcinogenesis." Semin Liver Dis; 12(3); 265-278.

                For colorectal carcinomas, the rate of tumor development is proportional to the fourth to sixth power of elapsed time, suggesting that four to six independent events are necessary. Although similar calculations have not been made for HBV-associated HCCs, it is likely that this is also the case for HCCs, since individuals with persistent HBV infection do not usually develop HCC until they are 45 or greater years old. As evidence for specific genetic and epigenetic changes in HCCs accumulate, the important players in multistep hepatocarcinogenesis are becoming clearer. However, even though Myc family oncogenes are clearly implicated in woodchuck HCC, similar integrations have not been found in human HCCs. Therefore, although rodent and human systems have many similarities, we must realize that important differences may also exist. Regarding tumor suppressor genes, the evidence for p53 alterations in HCC is strong. A growing body of evidence suggests further that alterations in the retinoblastoma gene and one or more tumor suppressor genes on chromosome 11 are also involved in HCC. HBV integrations may certainly play a role in the generation of chromosome aberrations leading to loss of tumor suppressor alleles, since chromosomes 11 and 17 are the most common integration sites. Finally, the role of X proteins as participants in malignant transformation has been demonstrated for certain immortalized, nontransformed hepatocytes. Altered autocrine mechanisms of cell growth control, possibly involving IGF-II, are clearly implicated in HCC. Paracrine mechanisms for the control of hepatocyte growth and differentiated functions may also be altered as a result of the synthesis and secretion of a complex array of interleukins, HGF, and basic and acidic FGFs by cells in the inflammatory and cirrhotic lesions of precancerous livers. Whether the order of molecular changes in the hepatocyte is important for malignant progression is presently not clear. What is clear, however, is that hepatocarcinogenesis involves alterations in the concerted action of protooncogenes, growth factor, and tumor suppressor genes. How these factors interact will provide a more complete understanding of the mechanism or mechanisms of hepatic oncogenesis.

Sagmeister, S., M. Eisenbauer, et al.; (2008). "New cellular tools reveal complex epithelial-mesenchymal interactions in hepatocarcinogenesis." Br J Cancer; 99(1); 151-159.

                To enable detailed analyses of cell interactions in tumour development, new epithelial and mesenchymal cell lines were established from human hepatocellular carcinoma by spontaneous outgrowth in culture. We obtained several hepatocarcinoma (HCC)-, B-lymphoblastoid (BLC)-, and myofibroblastoid (MF)-lines from seven cases. In-depth characterisation included cell kinetics, genotype, tumourigenicity, expression of cell-type specific markers, and proteome patterns. Many functions of the cells of origin were found to be preserved. We studied the impact of the mesenchymal lines on hepatocarcinogenesis by in vitro assays. BLC- and MF-supernatants strongly increased the DNA replication of premalignant hepatocytes. The stimulation by MF-lines was mainly attributed to HGF secretion. In HCC-cells, MF-supernatant had only minor effects on cell growth but enhanced migration. MF-lines also stimulated neoangiogenesis through vEGF release. BLC-supernatant dramatically induced death of HCC-cells, which could be largely abrogated by preincubating the supernatant with TNFbeta-antiserum. Thus, the new cell lines reveal stage-specific stimulatory and inhibitory interactions between mesenchymal and epithelial tumour cells. In conclusion, the new cell lines provide unique tools to analyse essential components of the complex interplay between the microenvironment and the developing liver cancer, and to identify factors affecting proliferation, migration and death of tumour cells, neoangiogenesis, and outgrowth of additional malignancy.

Sakaida, I., T. Kimura, et al.; (2005). "Cytochrome c is a possible new marker for fulminant hepatitis in humans." J Gastroenterol; 40(2); 179-185.

                BACKGROUND: Cytochrome c is known as a substance related to apoptosis. We investigated serum cytochrome c levels in patients with fulminant hepatitis (FH) compared with these levels in patients with acute or chronic liver diseases. METHODS: Serum cytochrome c was measured by an electrochemiluminescence immunoassay (ECLIA) method. The numbers of patients were as follows: fulminant hepatitis (FH; n = 15), acute hepatitis (AH; n = 12), chronic hepatitis (CH; n = 30), chronic hepatitis with acute aggravation (CHA; n = 6), liver cirrhosis (LC; n = 30), hepatocellular carcinoma (HCC; n = 30), and healthy volunteers (controls; n = 9). RESULTS: The serum cytochrome c level in FH was 10 686 +/- 7787 pg/ml, with a significant difference (P

Salvi, A., C. Sabelli, et al.; (2009). "MicroRNA-23b mediates urokinase and c-met downmodulation and a decreased migration of human hepatocellular carcinoma cells." FEBS J; 276(11); 2966-2982.

                Urokinase-type plasminogen activator (uPA) and c-met play a major role in cancer invasion and metastasis. Evidence has suggested that uPA and c-met overexpression may be coordinated in human hepatocellular carcinoma (HCC). In the present study, to understand whether the expression of these genes might be coregulated by specific microRNAs (miRs) in human cells, we predicted that Homo sapiens microRNA-23b could recognize two sites in the 3'-UTR of uPA and four sites in the c-met 3'-UTR by the algorithm pictar. The miR-23b expression analysis in human tumor and normal cells revealed an inverse trend with uPA and c-met expression, indicating that uPA and c-met negative regulation might depend on miR-23b expression. Transfection of miR-23b molecules in HCC cells (SKHep1C3) led to inhibition of protein expression of the target genes and caused a decrease in cell migration and proliferation capabilities. Furthermore, anti-miR-23b transfection in human normal AB2 dermal fibroblasts upregulated the expression of endogenous uPA and c-met. Cotransfection experiments in HCC cells of the miR-23b with pGL4.71 Renilla luciferase reporter gene constructs, containing the putative uPA and c-met 3'-UTR target sites, and with the pGL3 firefly luciferase-expressing vector showed a decrease in the relative luciferase activity. This would indicate that miR-23b can recognize target sites in the 3'-UTR of uPA and of c-met mRNAs and translationally repress the expression of uPA and c-met in HCC cells. The evidence obtained shows that overexpression of miR-23b leads to uPA and c-met downregulation and to decreased migration and proliferation abilities of HCC cells.

Selden, C., S. Farnaud, et al.; (1994). "Expression of hepatocyte growth factor mRNA, and c-met mRNA (hepatocyte growth factor receptor) in human liver tumours." Journal of Hepatology; 21(2); 227-234.

                We have quantified mRNA for the hepatocyte growth factor and its putative receptor the c-met proto-oncogene protein product, in a series of human primary and secondary liver tumours and adjacent non-neoplastic liver. In all hepatocellular cancers, hepatocyte growth factor 6 kb mRNA expression was less (mean 23.93% +/- 6.33% S.E.M. n = 7) in the tumours than in the adjacent normal liver. Both relative over- and under-expression of c-met transcripts were found in tumour tissue compared to non-neoplastic liver. Thus hepatocellular cancer tissue does not over-express mRNA for hepatocyte growth factor, though this growth factor might play a role in hyperproliferative states leading to liver cancer.

Shen, Z., O. G. Wong, et al.; (2008). "A novel and effective hepatocyte growth factor kringle 1 domain and p53 cocktail viral gene therapy for the treatment of hepatocellular carcinoma." Cancer Lett; 272(2); 268-276.

                Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, yet effective therapeutic options for advanced HCC are limited. Kringle 1 domain of HGF (HGFK1) has been demonstrated as a potent anti-tumor molecule and p53 is a well established tumor suppressor. Recently we developed AAV transducing HGFK1 (AAV-HGFK1) as a gene therapy for HCC. Here we investigated the possibility of enhancing the effect of AAV-HGFK1 by combining it with Adv transducing p53 (Adv-p53). In vitro expression experiments suggested a small amount of Adv-p53 could increase the expression of AAV transgenes. AAV-HGFK1+Adv-p53 cocktail strongly inhibited the proliferation of microvascular endothelial cell (MEC) and two HCC cell lines, Hepa1-6 and McA-RH7777. In two orthotopic mice and rat HCC models the cocktail gene therapy also significantly reduced the tumor burdens and prolonged the survival time by inhibiting tumor angiogenesis and inducing tumor cell death. Significantly, tumor metastasis was completely prevented. AAV-HGFK1+Adv-p53 viral cocktail may be a promising cancer therapy for the treatment of HCC.

Shen, Z., Z. F. Yang, et al.; (2008). "The kringle 1 domain of hepatocyte growth factor has antiangiogenic and antitumor cell effects on hepatocellular carcinoma." Cancer Res; 68(2); 404-414.

                The kringle 1 domain of human hepatocyte growth factor (HGFK1) was previously shown to inhibit bovine aortic endothelial cell proliferation, suggesting that it might be an antiangiogenic molecule. Here, we evaluated the in vivo efficacy of a recombinant adenoassociated virus carrying HGFK1 (rAAV-HGFK1) for the treatment of hepatocellular carcinoma (HCC) in a rat orthotopic HCC model and explored its molecular mechanisms in vitro in both endothelial and tumor cells. We first showed that rAAV-HGFK1 treatment significantly prolonged the survival time of rats transplanted with tumor cells. Treatment with rAAV-HGFK1 inhibited tumor growth, decreased tumor microvessel density, and completely prevented intrahepatic, lung, and peritoneal metastasis in this in vivo model. In vitro, rAAV-HGFK1 exhibited both antiangiogenic and antitumor cell effects, inhibiting the proliferation of both murine microvascular endothelial cells (MEC) and tumor cells, and inducing apoptosis and G(0)-G(1) phase arrest in these cells. To our surprise, rAAV-HGFK1 did not act through the hepatocyte growth factor/hepatocyte growth factor receptor pathway. Instead, it worked mainly through epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) signaling, with more minor contributions from vascular endothelial growth factor/vascular endothelial growth factor receptor and beta fibroblast growth factor (bFGF)/beta fibroblast growth factor receptor (bFGFR) signaling. In both MECs and tumor cells, rAAV-HGFK1 acted through two pathways downstream of EGFR, namely inhibition of extracellular signal-regulated kinase activation and stimulation of p38 mitogen-activated protein kinase/c-Jun-NH(2)-kinase activation. These results suggest for the first time that HGFK1 exerts both antiangiogenic and antitumor cell activities mainly through EGF/EGFR signaling, and may thus be considered as a novel therapeutic strategy for the treatment of HCC.

Shiota, G. and H. Kawasaki; (1998). "Hepatocyte growth factor in transgenic mice." Int J Exp Pathol; 79(5); 267-277.

                In order to clarify the function of hepatocyte growth factor (HGF) in vivo, we have developed transgenic mice expressing HGF in the liver. The bromodeoxyuridine labelling indices in livers from HGF transgenic mice were doubled, compared to those from wild type mice. Livers of HGF transgenic mice expressed high levels of c-myc, which was the consequence of increased transcription rates through the c-myc promoter. After 70% partial hepatectomy, the livers of HGF transgenic mice recovered in half the time needed for their normal siblings. Since we found that HGF inhibits growth of hepatocellular carcinoma (HCC) cells in vitro, we have made two kinds of double transgenic mice: HGF/TGF alpha and HGF/c-myc mice. The double transgenic mice expressing both HGF and TGF alpha had lower tumour yields, compared to TGF alpha transgenic mice. The HGF/c-myc double transgenic mice had a lower incidence of hepatocellular adenoma (HCA) and HCC in comparison with c-myc transgenic mice. In HGF/c-myc mice, there were more apoptotic cells and less mitotic cells than c-myc transgenic mice. These data indicate that HGF inhibits growth and occurrence of HCC in vivo. We also found that HGF protects liver from D-galactosamine (D-GalN)-induced injury. Hepatic prostaglandin E 2 (PGE2) contents in HGF transgenic mice were much higher than those in wild type mice, and were associated with hepatic HGF contents. An anti-HGF antibody inhibits production of PGE2 in liver after D-GalN administration. These data suggest that HGF protects liver from D-GalN-induced injury through increased liver PGE2 production. The data obtained from HGF transgenic mice suggests the possibility that HGF could be applicable for therapy of human liver diseases in the future.

Shiota, G., H. Kawasaki, et al.; (1994). "Inhibitory effect of hepatocyte growth factor against FaO hepatocellular carcinoma cells may be associated with changes of intracellular signalling pathways mediated by protein kinase C." Res Commun Mol Pathol Pharmacol; 85(3); 271-278.

                Hepatocyte growth factor (HGF) stimulates growth of mature hepatocytes, whereas it inhibits growth of cancer cells including hepatocellular carcinoma (HCC) cells. However, the regulatory mechanisms for this phenomenon remains unclear. An important intermediary in HGF signal transduction in normal hepatocytes, c-myc, was not induced in FaO HCC cells after HGF stimulation, suggesting that intracellular signalling pathways of HGF in FaO HCC cells were different from those in normal hepatocytes. Protein kinase C (PKC) has been reported to be involved in signalling pathways of many growth factors. To study whether PKC is associated with this inhibitory mechanism, we studied the effects of HGF and/or 12-O-tetradecanoyl phorbol-13-acetate (TPA) on the growth of normal hepatocytes and FaO HCC cells. Consequently, HGF or TPA stimulated growth of normal hepatocytes, while equal doses of TPA or HGF inhibited growth of FaO HCC cells, respectively. In addition, TPA reversed the HGF effect in both normal hepatocytes and FaO HCC cells. These data suggest that an inhibitory effect of HGF on FaO HCC cells may be associated with changes of protein kinase C-mediated intracellular signalling pathways.

Shiota, G., H. Kawasaki, et al.; (1995). "Characterization of double transgenic mice expressing hepatocye growth factor and transforming growth factor alpha." Res Commun Mol Pathol Pharmacol; 90(1); 17-24.

                Hepatocyte growth factor (HGF) and transforming growth factor alpha (TGF alpha) are potent mitogens for mature hepatocytes in primary culture (Nakamura, et al. 1984, Mead et al., 1989). However, these cytokines have completely different effects on tumor cell growth (Jhappan et al., 1990, Shiota et al, 1992). To clarify dual effects of these cytokines in liver, we developed double transgenic mice of HGF and TGF alpha using albumin promoter-HGF cDNA transgenic mice (AlbHGF) and metallothionein promoter-TGF alpha transgenic mice (MthTGF alpha). Double transgenic mice were examined on DNA synthesis, c-myc mRNA and occurrence of hepatocelular carcinoma (HCC). Higher labeling indices using BrDU were observed, in sequence, in AlbHGF mice, AlbHGF/MthTGF alpha, MthTGF alpha and wild type mice. Hepatic expression of c-myc mRNA in AlbHGF mice was elevated, compared to that in AlbHGF/MthTGF alpha or MthTGF alpha mice. After long-term observation, MthTGF alpha mice developed HCC in 6/10 (60%), whereas AlbHGF/MthTGF alpha mice developed HCC in 3/9 (33%). These data suggest that HGF is the potent mitogen, stimulating DNA synthesis and up-regulating c-myc mRNA. In addition, HGF may excert some inhibitory effect on occurrence of HCC by TGF alpha.

Shiota, G., H. Kawasaki, et al.; (1996). "Inhibitory effect of hepatocyte growth factor on metastasis of hepatocellular carcinoma in transgenic mice." Res Commun Mol Pathol Pharmacol; 91(1); 33-39.

                Hepatocyte growth factor (HGF) has different effects on different cell types. Recently, it has been reported that HGF has an anti-proliferative effect against tumor cells including hepatocellular carcinoma (HCC) cells. To clarify whether HGF inhibits the metastasis of HCC cells in vivo, we examined the metastatic potential of HCC cells into liver where HGF was highly expressed in transgenic animals. Three HCC cell lines including HuH7, Hep3B and FaO cells were transplanted into liver through the portal vein in chimeric mice or HGF transgenic mice and athymic nude mice (HGF/nude). The incidence of liver metastasis and number of metastatic tumors were significantly lower in mice expressing HGF (HGF/nude), compared with their siblings expressing no HGF (+/nude) (p

Shiota, G., T. Nakamura, et al.; (1994). "Hepatocyte growth factor regulates transforming growth factor alpha in HepG2 hepatic cells." Biochem Biophys Res Commun; 200(2); 1099-1104.

                Hepatocyte growth factor (HGF) is an important paracrine regulator for liver growth, whereas it inhibits growth of tumor cells including hepatocellular carcinoma. In contrast, transforming growth factor alpha (TGF alpha) is a factor which stimulates hepatocyte growth and causes hepatocellular carcinoma in an autocrine fashion. To determine whether TGF alpha is affected by HGF, we examined the regulation of TGF alpha expression in response to exogenous HGF in HepG2 cells. We first examined TGF alpha mRNA and protein in the presence of HGF and found nearly a 3.6-fold increase in mRNA and a 2.5-fold increase in TGF alpha protein. This induction was dose-dependent and followed delayed kinetics. Nuclear run-on experiments suggested that the mechanism for this induction was an increase in transcription of TGF alpha mRNA. These data suggest that HGF may modulate TGF alpha expression and the interaction of these cytokines may play an important role in liver diseases.

Shiota, G., J. Okano, et al.; (1995). "Serum hepatocyte growth factor levels in liver diseases: clinical implications." Hepatology; 21(1); 106-112.

                Although recent studies have shown that hepatocyte growth factor (HGF) is a potent mitogen in vivo, the significance of serum HGF in liver diseases remains unclear. To clarify clinical significance of serum HGF in liver diseases, serum HGF was measured in 127 patients with liver diseases and in 200 healthy individuals, using a highly sensitive immunoradiometric assay (IRMA). This assay is specific for HGF and is sensitive enough to detect 0.1 ng/mL of HGF. Mean values for serum HGF in acute hepatitis (AH), chronic hepatitis (CH), liver cirrhosis (LC), hepatocellular carcinoma (HCC), primary biliary cirrhosis (PBC), fulminant hepatic failure (FHF), and normal controls were 0.45, 0.40, 1.05, 1.06, 0.44, 16.40, and 0.27 ng/mL, respectively. Serum HGF levels in these diseases were significantly increased compared with those in the controls (P

Shiota, G., D. B. Rhoads, et al.; (1992). "Hepatocyte growth factor inhibits growth of hepatocellular carcinoma cells." Proc Natl Acad Sci U S A; 89(1); 373-377.

                Hepatocyte growth factor (HGF) is a potent mitogen for primary hepatocytes. Therefore, we examined HGF as a possible autocrine growth factor in hepatocellular carcinoma (HCC). We introduced an albumin-HGF expression vector into Fao HCC cells and transgenic mice. Expression of the albumin-HGF vector in Fao HCC cells inhibited their growth in vitro. In vivo, FaoHGF cells produced tumors that averaged 10% of the sizes of G418-resistant controls when transplanted into nude mice. In contrast, hepatocytes from transgenic mice expressing HGF grew more rapidly than did those from normal siblings. Further, growth of eight additional HCC cell lines was inhibited by the addition of recombinant HGF. Finally, of 35 tumor cell lines surveyed, only 6 cell lines expressed HGF mRNA, and no HCC cell line expressed HGF. Although HGF stimulates normal hepatocytes, it is a negative growth regulator for HCC cells.

Shiota, G., K. Umeki, et al.; (1995). "Hepatocyte growth factor and acute phase proteins in patients with chronic liver diseases." J Med; 26(5-6); 295-308.

                Serum hepatocyte growth factor (HGF) levels are increased in patients with liver diseases. HGF has been recently reported to stimulate production of acute phase proteins such as alpha 2-macroglobulin and albumin of hepatocytes in primary culture. To clarify whether serum HGF concentrations have any relation to concentrations of acute phase proteins, we measured serum HGF and acute phase proteins in chronic liver diseases where the synthesis of many plasma proteins is decreased with the decline of liver function. Eighty three patients with chronic liver diseases and 20 normal individuals were examined for serum HGF, albumin, C-reactive protein (CRP), alpha-fetoprotein (AFP), alpha 1-acid glycoprotein (alpha 1-AG) and alpha 2-macroglobulin (alpha 2-MG). Mean values for serum HGF in chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) were 0.37, 0.79 and 0.66 ng/mL, which were significantly higher than those in controls (p

Singh, R. K., R. Tsan, et al.; (1997). "Influence of the host microenvironment on the clonal selection of human colon carcinoma cells during primary tumor growth and metastasis." Clin Exp Metastasis; 15(2); 140-150.

                The purpose of this study was to determine the subpopulation dynamics of human colon carcinoma (HCC) cells growing at orthotopic (cecum, liver) or ectopic (subcutis, kidney, spleen) sites in nude mice and to correlate any outgrowth of distinct clones with the differential expression of metastasis-related genes. Low metastatic KM12C HCC cells were genetically tagged with a retrovirus harboring the neomycin-resistance (Neo(R)) gene. Southern blot analyses demonstrated only minor resolution of the Neo(R) hybridization pattern in DNA isolated from primary tumors growing orthotopically or ectopically, suggesting a polyclonal outgrowth. In contrast, a major resolution of the Neo(R) hybridization pattern was observed in liver-specific metastases, demonstrating the outgrowth of single dominant clones. Expression of epidermal growth factor receptor (EGR-R) increased 20-60% in the liver metastases vs spleen tumors and the KM12C Neo(R) cells. Transforming growth factor alpha (TGF-alpha), amphiregulin (AR), and c-met showed only modest differences in mRNA expression. A 20-80% increase in type IV collagenase mRNA levels was also observed in all tumor specimens. Furthermore, expression of the multi-drug resistance gene PGY-1 and the carcinoembryonic antigen (CEA) gene were elevated in the liver metastases compared with the spleen tumors and cultured cells. Transcript levels of the angiogenic factors interleukin-8 and basic fibroblast growth factor did not correlate with clonal outgrowth. These data demonstrate a correlation between EGF-R, type IV collagenase, CEA, and PGY-1 gene expression and the production of liver metastases. Our results suggest that distinct HCC clones differentially expressing specific mRNA transcripts for metastasis-related genes are the forerunners of the experimental liver metastatic lesions.

Skopkova, M., A. Penesova, et al.; (2007). "Protein array reveals differentially expressed proteins in subcutaneous adipose tissue in obesity." Obesity (Silver Spring); 15(10); 2396-2406.

                OBJECTIVE: Many adipokines, inflammatory cytokines, and other proteins produced by adipose tissue have been shown to be involved in the development of obesity-related insulin resistance. Nevertheless, new factors that play an important role in these processes are still emerging. Therefore, we screened the level of 120 different proteins in biopsies of subcutaneous adipose tissue (ScAT) of lean and obese subjects. RESEARCH METHODS AND PROCEDURES: All studied volunteers (12 obese with BMI >30 and 6 lean with BMI

Sripa, B., S. Leungwattanawanit, et al.; (2005). "Establishment and characterization of an opisthorchiasis-associated cholangiocarcinoma cell line (KKU-100)." World J Gastroenterol; 11(22); 3392-3397.

                AIM: To establish and characterize a new cholangiocarcinoma cell line from a patient living in the Opisthorchis viverrini (O. viverrini) endemic area of Northeast Thailand. METHODS: Fresh liver biopsy and bile specimens were obtained from a 65-year-old Thai woman with cholangiocarcinoma of the porta hepatis. After digestion, the cells were cultured in Ham's F12 media. The established cell line was then characterized for growth kinetics, cell morphology, imm-unocytochemistry and cytogenetics. Tumorigenicity of the cell line was determined by heterotransplanting in nude mice. RESULTS: The primary tumor was a poorly differentiated tubular adenocarcinoma. Examination of the bile revealed malignant cells with O. viverrini eggs. The cholangioc-arcinoma cell line KKU-100 was established 4 mo after the primary culture-population doubling time was 72 h. KKU-100 possesses compact and polygonal-shaped epithelial cells. Immunocytochemically, this cell line exhibited cytokeratin, EMA, CEA, and CA125, but not alpha-fetoprotein (AFP), CA19-9, desmin, c-met, or p53. Such protein expressions parallel those of the primary tumor. Cytogenetic analysis identified aneuploidy karyotypes with a modal chromosome number of 78 and marked chromosomal structural changes. Inoculation of KKU-100 cells into nude mice produced a transplantable, poorly differentiated aden-ocarcinoma, similar to the original tumor. CONCLUSION: KKU-100 is the first egg-proven, Opisthorchis-associated cholangiocarcinoma cell line, which should prove useful for further investigations of the tumor biology of this cancer.

Sun, H., J. Y. Wan, et al.; (2008). "[Screening and identification of human anti-c-Met Fab from a phage antibody library]." Zhonghua Gan Zang Bing Za Zhi; 16(7); 505-508.

                OBJECTIVE: To screen anti-c-Met Fab from a phage antibody library and identify its binding activity. METHODS: The expression of c-Met of HCC lines was identified by Western blot and immunofluorescence. Antibodies against c-Met were screened with immobilized antigen. After five rounds of panning, 30 randomly selected clones were identified by phage ELISA to select specific clones with high affinity. The positive clones were selected for Fab soluble expression in TOP10F and the binding activities were analysed in HCC lines. RESULTS: c-Met expressed in HCC membrane was confirmed by Western blot and immunofluorescence. A Fab fragment named AM2-26 with fine activity to c-Met was selected. AM2-26 binding specificity was confirmed by IP, FACS and immunofluorescence. CONCLUSION: The anti-c-Met Fab binding to c-Met in HCC provides a promising candidate for the biotherapy of hepatoma.

Suzuki, A., M. Hayashida, et al.; (2000). "Hepatocyte growth factor promotes cell survival from fas-mediated cell death in hepatocellular carcinoma cells via Akt activation and Fas-death-inducing signaling complex suppression." Hepatology; 32(4 Pt 1); 796-802.

                The Akt/PI-3 kinase pathway is a system essential for cell survival. In the current study, we showed that hepatocyte growth factor (HGF) activates the Akt/PI-3 kinase pathway to suppress Fas-mediated cell death in human hepatocellular carcinoma (HCC; 3 lines; SK-Hep1, HLE, and Chang Liver cell lines), hepatoblastoma (1 line; HepG2), and embryonic hepatocyte (1 line; WRL). Five tested cell lines showed the resistance to Fas-mediated cell death by the pretreatment of HGF. This HGF-induced cell survival was suppressed by wortmannin (Akt/PI-3 kinase pathway inhibitor), suggesting an involvement of Akt. When cells were pretreated with HGF, Fas-mediated cell death was suppressed, followed by Akt phosphorylation at Ser473. Fas-death-inducing signaling complex (DISC) formation, especially FADD and caspase 8 interaction, was suppressed by HGF and the suppression of the Akt/PI-3 kinase pathway by transient expression of PTEN, resulting in acquisition of Fas-DISC formation and Fas-mediated cell death in HGF-treated cells. We suggest that HGF promotes cell survival in hepatocyte-derived cell lines (HCC, hepatoblastoma, and embryonic hepatocyte) from Fas-mediated cell death via Fas-DISC suppression as a result of Akt activation.

Suzuki, H., M. Mori, et al.; (1999). "Serum vascular endothelial growth factor in the course of transcatheter arterial embolization of hepatocellular carcinoma." Int J Oncol; 14(6); 1087-1090.

                We previously reported that in vitro hypoxic condition enhanced VEGF level and its receptor expression in hepatic cancer cell line, HepG2. Transcatheter hepatic arterial embolization (TAE) therapy is one of the vasculo-occlusive and hypoxic challenges to hepatocellular carcinoma (HCC). Therefore, we examined the level of VEGF in sera of patients with HCC who underwent TAE during the course of the treatment. Thirty-eight patients with HCC and hepatitis C virus-positive cirrhosis were studied. Peripheral blood samples were taken before and 1, 3 and 7 days after TAE with informed consent. The serum levels of VEGF as well as hepatocyte growth factor (HGF), another hepatic remodeling factor, were measured. The molar ratio (BTR) of serum branched chain amino acid (BCAA) to tyrosine (Tyr), the serum levels of AST, ALT and LDH were also examined. Although the level of AST, ALT and LDH reached the peak value within 1 day after TAE, VEGF level increased significantly 7 days later. On the other hand, there were no significant alterations in the levels of HGF and BTR during the course of TAE. Although the level of HGF was significantly correlated with the level of VEGF before TAE, this correlation was no more observed after TAE. These data collectively suggest that VEGF may be secreted in response to clinical hypoxic intervention, TAE, independent of HGF or altered amino acid metabolism. VEGF may play a role as a sensitive marker for tumor ischemia.

Tameda, Y. and Y. Horiguchi; (1994). "[Development of diagnostic method for liver disease]." Rinsho Byori; 42(10); 1001-1002.

                Advance of current methods for management of liver diseases was discussed by eight discussers in this symposium. About three theme such as; 1. what laboratory parameters were helpful for prediction of effect of interferon (IFN) for HCV positive chronic hepatitis, 2. what tests were available for early diagnosis and prediction of prognosis in fulminant hepatitis, and 3. what laboratory parameters were useful for early diagnosis and prediction of development of hepatocellular carcinoma (HCC), hot discussion was performed. It was presented that time course of titration of HCV core antibody was available for prediction of effect of interferon in some cases. In patients with low concentration of HCV-RNA and genotype III, good effect of IFN was obtained. The possibility that the measurement of HCV serotype would become more common instead of that of HCV genotype because of its simplicity in methodology was presented. The measurements of plasma amino acids and human hepatocyte growth factor (h-HGF) were useful in early diagnosis of fulminant hepatitis. As current topics in management of HCC, the utility of determination of lectin affinity alpha-fetoprotein in early diagnosis of HCC, the importance of making definite diagnosis by target biopsy of HCC in early phase and prediction of development of HCC by abdominal sonogram were reported.

Tanimizu, N., M. Nishikawa, et al.; (2003). "Isolation of hepatoblasts based on the expression of Dlk/Pref-1." J Cell Sci; 116(Pt 9); 1775-1786.

                Hepatoblasts are common progenitors for hepatocytes and biliary epithelial cells, although their nature remains largely unknown. In order to isolate and to characterize hepatoblasts, we searched for cell surface antigens expressed in mouse fetal hepatic cells by the signal sequence trap method and found that Dlk, also known as Pref-1, was strongly expressed in fetal liver. Immunohistochemical as well as northern analysis indicated that Dlk was highly expressed in the E10.5 liver bud. The strong expression continued until the E16.5 stage and was significantly downregulated thereafter. Using a monoclonal antibody against Dlk, we isolated Dlk+ cells either by a fluorescence-activated cell sorter or by an automatic magnetic cell sorter. Dlk+ cells isolated from fetal livers expressed albumin and formed colonies when cultured at low density with HGF and EGF for 5 days. Over 60% of colonies derived from E14.5 Dlk+ cells contained both albumin+ and cytokeratin 19+ cells, indicating that a majority of colony-forming Dlk+ cells are able to differentiate into both hepatocyte and biliary epithelial cell lineages. In addition, numerous microvilli were observed by electronmicroscopic analysis in most of those cultured cells, also indicating differentiation of Dlk+ cells under this condition. Furthermore, 7% of the colony-forming Dlk+ cells were not only bipotential but also highly proliferative, forming a large colony containing more than 100 cells during 5 days of culture. By transplantation of Dlk+ cells into the spleen, donor-derived hepatocytes were found in the recipient liver, indicating that Dlk+ cells differentiated into hepatocytes in vivo. These results indicate that Dlk+ cells are hepatoblasts and that Dlk is a useful marker to enrich highly proliferative hepatoblasts from fetal liver.

Tavian, D., G. De Petro, et al.; (2000). "u-PA and c-MET mRNA expression is co-ordinately enhanced while hepatocyte growth factor mRNA is down-regulated in human hepatocellular carcinoma." International Journal of Cancer; 87(5); 644-649.

                Hepatocyte growth factor/scatter factor (HGF/SF) is one of the most important humoral mediators of liver regeneration. It is potentially related to molecular mechanisms of hepatocarcinogenesis via a paracrine system involving its cellular receptor, c-met. In this study, the expression patterns of HGF and c-met were evidenced by multiplex RT-PCR in different specimens of human hepatic tissues (n = 71). A significant increase of c-met mRNA expression was detected in hepatitis (P = 0.001), cirrhosis (P = 0.006), and hepatocellular carcinoma (HCC) tissue (P = 0.003) compared with normal parenchyma and steatosis. HGF mRNA expression was significantly higher only in hepatitis (P = 0.01). Over-expression of c-met mRNA and under-expression of HGF mRNA were detected in the HCCs compared with the corresponding peri-tumoral tissues. Neither HGF nor c-met expression was related to age, sex, tumor size, grading, presence of pseudocapsula, and proliferative activity of the malignant hepatocytes. A significant inverse correlation was found between c-met mRNA expression level and survival (in months) of patients (P = 0.007), as previously shown for urokinase-type plasminogen activator (u-PA) mRNA (P = 0.027). In addition, c-met mRNA expression was strictly associated with u-PA mRNA level in HCC samples (P = 0.001). These data show that a loss of balance concerning HGF, c-met, and u-PA mRNA expression occurs during hepatocarcinogenesis. Particularly, up-regulation of c-met and u-PA mRNA transcription appears to be coordinately regulated, and their levels of expression are inversely correlated with survival; they must therefore play an important role in the development and progression of human HCC and may also be relevant prognostic markers.

Terada, T., Y. Nakanuma, et al.; (1998). "Immunohistochemical demonstration of MET overexpression in human intrahepatic cholangiocarcinoma and in hepatolithiasis." Hum Pathol; 29(2); 175-180.

                Expression of MET, the c-met-encoded receptor for hepatocyte growth factor, has not been investigated in proliferative biliary diseases of human liver, including hepatolithiasis and cholangiocarcinoma. Comparatively, we analyzed by immunohistochemistry the expression of MET in normal adult human livers (n = 20), normal postnatal preadult livers (n = 21), fetal livers (n = 36), hepatolithiatic livers (n = 32), and intrahepatic cholangiocarcinomas (n = 26). In normal adult livers, obvious MET immunoreactivity was not found in any cell types. In fetal liver, MET was weakly expressed in primitive biliary cells (ductal plate and immature bile ducts) and immature hepatocytes during 8 to 30 gestational weeks but was essentially negative thereafter. In hepatolithiasis, a condition of risk for cholangiocarcinoma development, MET was overexpressed in proliferated biliary cells in 26 of 32 cases (81%). In this nonneoplastic proliferative biliary condition, MET immunoreactivity was observed to be most prominent in the hyperplastic septal and large bile ducts of liver, and in the proliferated peribiliary glands associated with intrahepatic large bile ducts. In intrahepatic cholangiocarcinoma, MET overexpression in neoplastic biliary epithelium was observed in 15 of 26 cases (58%) and correlated with the degree of tumor differentiation, being highest in well-differentiated tumors and relatively low in poorly differentiated tumors. These data show for the first time that overexpression of MET is a common feature of hyperplastic and neoplastic biliary epithelial cells in human liver and suggest that MET/hepatocyte growth factor may be playing an important role in human biliary hyperplasia and in cholangiocarcinogenesis in vivo.

Tomiya, T., S. Hayashi, et al.; (1997). "Serum transforming growth factor-alpha level can be a parameter for evaluating liver regeneration after partial hepatectomy in patients with liver cancer." Semin Oncol; 24(2 Suppl 6); S6-14-S16-17.

                Sensitive and reliable laboratory parameters are necessary to evaluate the degree of liver regeneration serially in patients after partial hepatectomy for liver cancer. We evaluated the serum levels of transforming growth factor-alpha (TGF-alpha) and hepatocyte growth factor (HGF), both of which are potent mitogens for hepatocytes, in 22 hepatectomized patients with liver cancer: 10 patients with hepatocellular carcinoma and 12 patients with metastatic liver tumors. Ten patients who underwent laparotomy for nonhepatic surgery were also studied as surgical controls. The serum TGF-alpha and HGF levels were measured by sandwich enzyme-linked immunosorbent assay techniques. Both the serum TGF-alpha and HGF levels increased after partial hepatectomy. However, there was no correlation between the levels of TGF-alpha and HGF. The maximal level of TGF-alpha achieved in each case correlated significantly with the resected liver volume and the increased volume of the remaining liver. Hepatocyte growth factor showed no such correlations. After nonhepatic surgery, the HGF level also increased significantly, while the TGF-alpha level did not. These results suggest that the serum TGF-alpha level varies depending on the regenerative stimulus to the liver, and that its increase corresponds with the degree of liver regeneration that occurs in patients after partial hepatectomy for liver cancer. In contrast, it is unlikely that the serum HGF level reflects liver regeneration. In conclusion, the serum TGF-alpha level can be used as a parameter for evaluating liver regeneration in patients who have undergone partial hepatectomy.

Trusolino, L., A. Bertotti, et al.; (2010). "MET signalling: principles and functions in development, organ regeneration and cancer." Nat Rev Mol Cell Biol; 11(12); 834-848.

                The MET tyrosine kinase receptor (also known as the HGF receptor) promotes tissue remodelling, which underlies developmental morphogenesis, wound repair, organ homeostasis and cancer metastasis, by integrating growth, survival and migration cues in response to environmental stimuli or cell-autonomous perturbations. The versatility of MET-mediated biological responses is sustained by qualitative and quantitative signal modulation. Qualitative mechanisms include the engagement of dedicated signal transducers and the subcellular compartmentalization of MET signalling pathways, whereas quantitative regulation involves MET partnering with adaptor amplifiers or being degraded through the shedding of its extracellular domain or through intracellular ubiquitylation. Controlled activation of MET signalling can be exploited in regenerative medicine, whereas MET inhibition might slow down tumour progression.

Tung, E. K., C. M. Wong, et al.; (2009). "HAI-2 is epigenetically downregulated in human hepatocellular carcinoma, and its Kunitz domain type 1 is critical for anti-invasive functions." International Journal of Cancer; 124(8); 1811-1819.

                Pharmacological demethylation-based gene expression profile analysis is a useful tool to identify epigenetically silenced tumour suppressor genes. HGF activator inhibitor 2 (HAI-2), a serine protease inhibitor, has been identified as one of the candidate tumour suppressor genes in human hepatocellular carcinoma (HCC) with this technique. In this study, we aimed to characterise the epigenetic status and tumour suppressive function of HAI-2 in HCC. We validated that HAI-2 expression was either absent or low in most of the HCC cell lines tested, and 5-Aza-2'-deoxycytidine treatment significantly restored its expression in 9 (75%) of these 12 cell lines. HAI-2 was found to be frequently underexpressed in human HCCs (p

Tward, A. D., K. D. Jones, et al.; (2007). "Distinct pathways of genomic progression to benign and malignant tumors of the liver." Proc Natl Acad Sci U S A; 104(37); 14771-14776.

                We used several of the genetic lesions commonly associated with human liver tumors to reconstruct genetic progression to hepatocellular carcinoma and adenoma in mouse models. We initiated tumorigenesis with a transgene of the protooncogene MET or by hydrodynamic transfection of MET in combination with other genes into the livers of adult animals. Hepatocellular carcinoma in both instances arose from cooperation between MET and constitutively active versions of beta-catenin. In contrast, adenomas were produced by cooperation between MET and defective signaling through the transcription factor HNF1alpha. Prompted by these findings, we uncovered a coincidence between activation of the protein-tyrosine kinase encoded by MET and activating mutations of beta-catenin in a subset of human hepatocellular carcinomas. Inactivation of MET transgenes led to regression of hepatocellular carcinomas despite the persistence of activated beta-catenin. The tumors eventually recurred in the absence of MET expression, however, presumably after the occurrence of one or more events that cooperated with activated beta-catenin in lieu of MET. These results offer insight into hepatic tumorigenesis, provide mouse models that should be useful in the further study of hepatic tumorigenesis and for preclinical testing, and identify a subset of human hepatocellular carcinomas that may be susceptible to combination therapy directed against Met and the Wnt signaling pathway.

Ueki, T., J. Fujimoto, et al.; (1997). "Expression of hepatocyte growth factor and its receptor c-met proto-oncogene in hepatocellular carcinoma." Hepatology; 25(4); 862-866.

                The c-met proto-oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor (HGF), a potent mitogen and motogen for epithelial cells. Because of its profound effects on cell growth and motility, HGF may be important in the development of cancer metastases in hepatocellular carcinoma (HCC). In this study, we examined HGF concentration and expression of the c-met-proto-oncogene product (c-met) in 62 patients with HCC to determine the relationship between the level of expression and clinicopathological features, and patient outcome following hepatectomy. Western blotting was used to examine the c-met expression, and HGF concentration in tumors was measured using an enzyme-linked immunosorbent assay. c-met was found to be overexpressed in HCC compared with nontumorous liver tissue (P

Ueki, T., J. Fujimoto, et al.; (1997). "Expression of hepatocyte growth factor and its receptor, the c-met proto-oncogene, in hepatocellular carcinoma." Hepatology; 25(3); 619-623.

                The c-met proto-oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor (HGF), a potent mitogen and motogen for epithelial cells. Because of its profound effects on cell growth and motility, HGF may be important in the development of cancer metastases in hepatocellular carcinoma (HCC). In this study, we examined HGF concentration and expression of the c-met proto-oncogene product (c-Met) in 62 patients with HCC to determine the relationship between the level of expression and clinicopathological features, and patient outcome following hepatectomy. Western blotting was used to examine the c-Met expression, and HGF concentration in tumors was measured using an enzyme-linked immunosorbent assay. c-Met was found to be overexpressed in HCC compared with nontumorous liver tissue (P

Ueno, Y., H. Sakurai, et al.; (2005). "Selective inhibition of TNF-alpha-induced activation of mitogen-activated protein kinases and metastatic activities by gefitinib." Br J Cancer; 92(9); 1690-1695.

                We have reported that the selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib ('Iressa', ZD1839), suppressed intrahepatic metastasis of hepatocellular carcinoma CBO140C12 cells. In this study, we focused on the tumour necrosis factor-alpha (TNF-alpha) signalling pathways. Real-time reverse transcription-polymerase chain reaction showed that TNF-alpha mRNA was expressed in large quantities in the implanted tumour. Gefitinib inhibited EGF- but not hepatocyte growth factor (HGF)-induced activation of mitogen-activated protein kinase (MAPK) cascades, suggesting selectivity of the inhibitor. However, gefitinib inhibited the TNF-alpha-induced activation of MAPKs and Akt. In addition, TNF-alpha-induced metastatic properties including adhesion to fibronectin, mRNA expression of integrin alpha v, production of matrix metalloproteinase-9 and invasion were inhibited by gefitinib without affecting cell proliferation. Furthermore, the TNF-alpha-induced responses except for NF-kappaB activation were blocked by metalloprotease inhibitors, suggesting that gefitinib inhibited the transactivation of EGFR induced by TNF-alpha. These results suggest that the TNF-alpha signalling pathway is a possible target of gefitinib in suppressing the intrahepatic metastasis of hepatocellular carcinoma.

Utsunomiya, I., A. Iemura, et al.; (1999). "Establishment and characterization of a new human hepatocellular carcinoma cell line, HAK-3, and its response to growth factors." Int J Oncol; 15(4); 669-675.

                A new human hepatocellular (HCC) cell line, HAK-3, was established from a resected HCC of a Japanese, female patient. HAK-3 retains morphologic features of the original HCC, and proliferates in a monolayered sheet (doubling time: 26 h). HAK-3 is a single aneuploid cell population with a DNA index of 2.42, the karyotype is human, chromosomes are 80-85 (mode: 83), and secretes fibronectin and tissue polypeptide antigen. Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) dose-dependently accelerated the cell proliferation, while deletion-type hepatocyte growth factor (dHGF) tended to suppress the proliferation, and transforming growth factor (TGF)-alpha showed almost no influence. dHGF induced the decrease of cell adhesiveness, changed the cell morphology to spindle-shaped cells, increased cell movement, and showed chemotactic effects with the increase of its concentration gradient in cultures. HAK-3 would be useful in studies on the acceleration mechanisms of cancer cell proliferation by growth factors and of chemotaxis by dHGF.

Varnholt, H., Y. Asayama, et al.; (2002). "C-met and hepatocyte growth factor expression in combined hepatocellular and cholangiocarcinoma." Oncol Rep; 9(1); 35-41.

                The membranous tyrosine kinase receptor c-met and its natural ligand hepatocyte growth factor are prominent mitogens, motogens and morphogens for hepatocytes and many other cell types in vitro as well as in vivo. To clarify the significance of the c-met/hepatocyte growth factor system in the development and spread of combined hepatocellular and cholangiocarcinoma, surgical specimen from 30 patients, consisting of 4 double cancers, 20 combined types and 6 mixed types, were examined immunohistochemically. Immunoreactivity for HGF was significantly correlated with the differentiation degree of cholangiocellular components, being highest in well and moderately differentiated and lowest in poorly differentiated components (p=0.0119). No significant association was observed between expression of c-met or HGF and the presence of liver cirrhosis, vascular invasion, perineural invasion, lymphatic permeation, intrahepatic metastasis or lymph node metastasis. HGF may have an important impact on the differentiation of certain cHCC-CC. Other clinicopathologic factors related to tumor development and spread may not be influenced by c-met or HGF, at least not on the protein level.

Vejchapipat, P., P. Tangkijvanich, et al.; (2004). "Association between serum hepatocyte growth factor and survival in untreated hepatocellular carcinoma." J Gastroenterol; 39(12); 1182-1188.

                BACKGROUND: Hepatocellular carcinoma (HCC) is a common hepatic malignancy worldwide. Its nature of rapid growth results in a grave prognosis. Hepatocyte growth factor (HGF) is a mitogen for hepatocytes, responsible for their proliferation. The aim of the present study was to investigate the prognostic roles of serum HGF in untreated HCC patients. METHODS: Fifty-five patients with inoperable HCC were studied. The diagnosis of HCC was based on either liver histopathology or imaging evidence of a liver mass, together with elevated serum alpha-fetoprotein. Serum HGF levels of the patients, at the time of diagnosis, were compared to those of 28 healthy controls. All patients received only palliative treatments and were followed up until they died. Comparison of survival curves between patients with a serum HGF level of 1.0 ng/ml or more and those with lower serum HGF was performed, using the log-rank test. Data values are expressed as means and SD. RESULTS: Fifty-one men and four women with inoperable HCC were recruited. The mean age was 54.15+/-15.34 years. The serum HGF levels in the inoperable HCC patients were significantly higher than those in the controls (0.58+/-0.43 vs 0.14+/-0.04 ng/ml; P

Wang, Y. C., G. L. Xu, et al.; (2011). "Estrogen Suppresses Metastasis in Rat Hepatocellular Carcinoma through Decreasing Interleukin-6 and Hepatocyte Growth Factor Expression." Inflammation.

                Metastasis remains one of the major challenges before hepatocellular carcinoma (HCC) is finally conquered. Estrogen has recently emerged as a protective factor in the development and progression of HCC, but whether and how it reduces metastasis of HCC remain to be elucidated. We conducted an in vivo highly metastatic rat HCC model in female Sprague-Dawley rats induced by diethylnitrosamine and N-nitrosomorpholine to examine the effects of estrogen on HCC metastasis. Moreover, female rats were randomly distributed into four groups: ovariectomy (OVX), sham operation, ovariectomy followed by 30 mug/kg body weight/day 17alpha-ethynylestradiol supplementation, and sexually intact control groups. Here, we show that, 60% lung metastasis was observed in the rats of OVX group, whereas 17-25% lung metastasis was found in rats of the other three groups. Furthermore, physiological doses of estrogen, no matter endogenous or exogenous, can suppress metastasis of HCC through decreasing interleukin-6 (IL-6) and hepatocyte growth factor (HGF) expression in the tumor microenvironment. In conclusion, the present study demonstrated that estrogen has the potential to inhibit lung metastasis from rat HCCs in vivo. Its mechanism of action may involve modulation of inflammatory tumor microenvironment by suppression of HGF and IL-6 production.

Whittaker, S., R. Marais, et al.; (2010). "The role of signaling pathways in the development and treatment of hepatocellular carcinoma." Oncogene; 29(36); 4989-5005.

                Hepatocellular carcinoma (HCC) is a highly prevalent, treatment-resistant malignancy with a multifaceted molecular pathogenesis. Current evidence indicates that during hepatocarcinogenesis, two main pathogenic mechanisms prevail: (1) cirrhosis associated with hepatic regeneration after tissue damage caused by hepatitis infection, toxins (for example, alcohol or aflatoxin) or metabolic influences, and (2) mutations occurring in single or multiple oncogenes or tumor suppressor genes. Both mechanisms have been linked with alterations in several important cellular signaling pathways. These pathways are of interest from a therapeutic perspective, because targeting them may help to reverse, delay or prevent tumorigenesis. In this review, we explore some of the major pathways implicated in HCC. These include the RAF/MEK/ERK pathway, phosphatidylinositol-3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, WNT/beta-catenin pathway, insulin-like growth factor pathway, hepatocyte growth factor/c-MET pathway and growth factor-regulated angiogenic signaling. We focus on the role of these pathways in hepatocarcinogenesis, how they are altered, and the consequences of these abnormalities. In addition, we also review the latest preclinical and clinical data on the rationally designed targeted agents that are now being directed against these pathways, with early evidence of success.

Wu, F. S., S. S. Zheng, et al.; (2006). "[Study on the prognostic value of hepatocyte growth factor and c-met for patients with hepatocellular carcinoma]." Zhonghua Wai Ke Za Zhi; 44(9); 603-608.

                OBJECTIVE: To analyze the prognostic value of hepatocyte growth factor (HGF) and c-met for patients with hepatocellular carcinoma (HCC) after hepatectomy. METHODS: Twenty-five patients undergoing partial hepatectomy for HCC were studied. Serum HGF level was determined using ELISA kit before and after operation respectively. c-met protein and mRNA expression in cancerous and paracancerous tissues were detected by immunohistochemical and RT-PCR methods respectively. The correlations of clinical-pathologic parameters with the HGF level in serum and c-met expression in cancerous tissue were analyzed respectively. RESULTS: HCC patients had a significantly higher concentration of serum HGF than normal controls and chronic hepatitis B respectively [(1.03 +/- 0.09) ng/ml vs (0.69 +/- 0.02) ng/ml and (0.74 +/- 0.09) ng/ml]. No significant difference in serum HGF was observed between HCC and cirrhosis patients with Child-Pugh score B/C [(1.03 +/- 0.09) ng/ml vs (1.04 +/- 0.11) ng/ml]. Serum HGF concentrations were positively correlated with tumor size (> 5 cm), node cirrhosis, portal vein tumor thrombi (PVTT) and preoperative alpha-fetoprotein (AFP) level (> or = 400 microg/L). After the resection of tumor, serum HGF concentration had a peak on the third postoperative day (POD), and then declined, but did not return to normal level on the tenth POD. From preoperative day to third POD, HGF concentration had a higher elevation in patients with major resection than with local resection. Moderately or strongly positive expression of c-met protein was observed in 21 cancerous regions (21/25), and only in 5 paracancerous regions. The intensive expression of c-met mRNA was 100% (25/25) detectable in the cancerous tissues, but only 24% (6/25) in the paracancerous tissues. The expression extent of c-met protein was correlated with portal vein tumor thrombi (PVTT). In paracancerous tissues, the expression of c-met protein was more intense in patients with cirrhosis than those without cirrhosis. The patients with recurrence or metastases after operation had a higher level of serum HGF and more intensive expression of c-met than other patients. No significant association was observed between HGF in serum and c-met expression in cancerous tissue. CONCLUSIONS: The over-expression of HGF and its receptor c-met indicate an adverse prognosis for HCC patients. The sustained high level of serum HGF after hepatectomy may be a factor related to early tumor recurrence and metastasis.

Xie, B., C. Tang, et al.; (2009). "[Hepatitis B virus X protein regulates c-met promoter via the ERKs singal pathway in HepG2 cells]." Zhonghua Gan Zang Bing Za Zhi; 17(7); 531-534.

                OBJECTIVE: To explore the signal pathway mediating the regulatory effect of Hepatitis B virus X protein (HBX) on c-met gene promoter in HepG2 cells. METHODS: The expression of c-met in HBX-transfected HepG2 cells treated with different signal pathway inhibitors was detected by western blot, the invasion capability of cells was determined by Matrigel invasion assay. RESULTS: ERK inhibitor U0126 inhibited the expression of the c-Met in HBx-transfected HepG2 cells. However, both p38MAPK inhibitor SB203580 and PI-3K inhibitor wortmanin had no effect on expression of the c-Met in HBx-transfected HepG2 cells. Furthermore, the ERK inhibitor U0126 also inhibited the invasiveness of HBX-transfected HepG2 cells. CONCLUSION: HBx induces invasion of HCC via activation of ERK pathway.

Xie, B., R. Xing, et al.; (2010). "Down-regulation of c-Met expression inhibits human HCC cells growth and invasion by RNA interference." Journal of Surgical Research; 162(2); 231-238.

                BACKGROUND: Cell migration is a basis for invasion and metastasis of malignant tumors. Receptor tyrosine kinases are recognized as important therapeutic targets in antineoplastic strategies. C-met is a receptor tyrosine kinase highly expressed in human hepatocellular carcinoma (HCC) cell line MHCC97-H. Higher expression of c-met in tumor tissue can lead to scattering, angiogenesis, proliferation, enhanced cell motility, invasion, and eventually, metastasis. To explore the roles of c-met in modulating the motility of cell, we silenced c-met expression in the HCC line MHCC97-H by RNA interference (RNAi). MATERIALS AND METHODS: For transient expression, c-met-siRNA 1,2 recombinant plasmids were transfected into phoenix A cells. The MHCC97-H cells were cultured in Dulbecco's modified Eagles's medium (DMEM) with 10% fetal bovine serum (FBS) to establish MHCC97-H HCC cells stably expressing c-met-siRNA. MHCC97-H cells were treated with the recombinant virus for assay of c-met mRNA and protein, evaluation of growth and invasion of MHCC97-H cells, and identification of hepatitis B virus X (HBX) protein correlation with c-met. RESULTS: After transfection of c-met-siRNA for 48 h, the expression of c-met decreased markedly in MHCC97-H cells; the most effective site of the siRNA target sequence is at the 537 upstream, far from the transcription start. In addition, the proliferation, motility, and invasive ability of MHCC97-H cells were significantly inhibited. Furthermore, we showed that hepatitis B virus (HBV) X protein (HBX) potentiated the activities of the extracellular signal-regulated kinase 1/2 (ERK1/2) in MHCC97-H cells. Treatment with extracellular signal-regulated kinase (ERK) inhibitor (U0126), but not P38 MAPK inhibitor (SB203580) or phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin), markedly suppressed the expression of c-met protein in MHCC97-H cells. CONCLUSION: These results indicate that the over-expression of c-met protein plays an important role in the cell invasion of MHCC97-H, and HBX protein may promote the expression of c-met by ERKs pathway.

Xie, Q., K. D. Liu, et al.; (2001). "SF/HGF-c-Met autocrine and paracrine promote metastasis of hepatocellular carcinoma." World J Gastroenterol; 7(6); 816-820.

                AIM: To explore the role of SF/HGF-Met autocrine and paracrine in metastasis of hepatocellular carcinoma (HCC). METHODS: SF/HGF and c-met transcription and protein expression in HCC were examined by RT-PCR and Western Blot in 4 HCC cell lines, including HepG2, Hep3B, SMMC7721 and MHCC-1, the last cell line had a higher potential of metastasis. sf/hgf cDNA was transfected by the method of Lipofectin into SMMC7721. SF/HGF and c-met antibody were used to stimulate and block SF/HGF-c-met signal transduction. Cell morphology, mobility, and proliferation were respectively compared by microscopic observation, wound healing assay and cell growth curve. RESULTS: HCC malignancy appeared to be relative to its met-SF/HGF expression. In MHCC-1, c-met expression was much stronger than that in other cell lines with lower potential of metastasis and only SF/HGF autocrine existed in MHCC-1. After sf/hgf cDNA transfection or conditioned medium of MHCC-1 stimulation, SMMC7721 changed into elongated morphology, and the abilities of proliferation (P

Yada, K.; (1994). "Effect of intracellular pH and two growth factors, epidermal growth factor and human hepatocyte growth factor, on DNA synthesis in non-regenerating and regenerating hepatocytes and hepatoma cells." Osaka City Med J; 40(2); 53-69.

                I examined the effects of intracellular pH (pHi) and the growth factors, epidermal growth factor (EGF) and human hepatocyte growth factor (HGF), on DNA synthesis in non-regenerating hepatocytes, regenerating hepatocytes and hepatoma cells. Non-regenerating and regenerating hepatocytes were isolated from the livers of intact adult rat and of the adult rat 24 hours after 70% hepatectomy, respectively. Hep G2 cells, a human hepatoma cell line, was employed as hepatoma cells. Regenerating hepatocytes and Hep G2 cells, but not non-regenerating hepatocytes displayed increased DNA synthesis with increasing pHi in the absence of EGF and HGF. However, non-regenerating hepatocytes displayed little increase, regenerating hepatocytes displayed substantial increase in DNA synthesis with increasing pHi in the presence of EGF or HGF. In contrast, Hep G2 cells displayed decreased DNA synthesis in the presence of HGF but not EGF. These findings indicate that pHi influences the fashion of proliferation in hepatocytes and cancer cells. EGF and HGF stimulate DNA synthesis in hepatocyte and inhibit that in cancer cell, suggesting that increasing pHi and administration of these growth factors may be one of the effective treatment for hepatoma.

Yamagami, H., M. Moriyama, et al.; (2001). "Detection of serum and intrahepatic human hepatocyte growth factor in patients with type C liver diseases." Intervirology; 44(1); 36-42.

                We determined hepatocyte growth factor (HGF) levels in the serum and liver of patients with hepatitis C and assessed the relationship to histological findings of the liver and hepatitis C virus-related markers in the serum in patients with type C liver diseases. The subjects were 108 patients with chronic hepatitis C (CH), 70 patients with liver cirrhosis C (LC), 38 patients with hepatocellular carcinoma (HCC) and 20 patients with acute hepatitis (AH). As normal controls 20 subjects were studied. The serum HGF levels were measured using an enzyme-linked immunosorbent assay kit. Intrahepatic HGF was investigated by immunoperoxidase staining using monoclonal HGF antibody. The serum HGF level was highest in patients with AH. The serum HGF levels tended to be higher in patients with LC and HCC than those with CH. Further, the serum HGF level was related to the degree of intrahepatic inflammatory cell infiltration and fibrosis, and intrahepatic HGF was noted primarily in the cell membrane of mesenchymal cells in focal necrosis. The degree of intrahepatic HGF expression tended to be higher in patients with high serum HGF levels. In patients with HCC, however, HGF showed little localization in cancer cells, but was noted in infiltrating mesenchymal cells in both cancerous and noncancerous regions. In conclusion, the measurement of serum HGF levels may be useful for estimating the degree of intrahepatic inflammatory reaction and fibrosis. Although further study is necessary, the high serum level of HGF revealed high carcinogenic states in chronic hepatitis and liver cirrhosis type C.

Yamagamim, H., M. Moriyama, et al.; (2002). "Serum concentrations of human hepatocyte growth factor is a useful indicator for predicting the occurrence of hepatocellular carcinomas in C-viral chronic liver diseases." Cancer; 95(4); 824-834.

                BACKGROUND: Numerous reports have examined the relationship between hepatocyte growth factor (HGF) and either the facilitation or suppression of the occurrence of hepatocellular carcinoma (HCC). METHODS: In this study, we measured serum HGF concentrations of blood samples and conducted prospective studies to examine the long-term outcome of C-viral chronic hepatitis (CH) and cirrhosis in patients. The subjects examined in this study include 99 patients with C-viral CH, cirrhosis, and HCC. The serum HGF level was measured in blood samples within 48 hours of collection using enzyme-linked immunosorbent assay kits. RESULTS: The serum concentrations of HGF were significantly higher in patients with HCC than in patients with CH or cirrhosis. The detection rate of HGF and its mean serum level were significantly higher in patients with a low platelet count than in patients with a high platelet count. All of the patients with serum HGF concentrations of more than 0.6 ng/mL had HCC, irrespective of the levels of alpha-fetoprotein, vitamin K absence, or antagonist-II in the blood. Serum HGF concentrations increased concomitantly with increases in areas occupied by HCC. The cumulative incidence of occurrence of HCC was significantly higher in patients with high HGF concentrations than in patients with low HGF concentrations. Multivariate analysis revealed that the elevation in serum HGF level is the most important risk factor for the occurrence of HCC. CONCLUSIONS: The serum level of HGF represents the degree of the carcinogenic state in the liver of patients with C-viral CH and cirrhosis. Therefore, the determination of serum HGF concentrations may be useful as a third tumor marker of HCC in detection as well as follow-up therapy.

Yamaguchi, K., M. A. Nalesnik, et al.; (1996). "Expression of HGF mRNA in human rejecting kidney as evidenced by in situ hybridization." Urological Research; 24(6); 349-354.

                In situ hybridization was performed to demonstrate hepatocyte growth factor (HGF) mRNA in two patients with normal kidney and in 23 patients with allograft nephrectomy. In situ hybridization was combined with immunohistochemistry to identify HGF-producing cells. In the two patients with normal kidney, no HGF mRNA was obtainable. In 15 of the 23 allograft patients, signals of HGF mRNA were detectable. In six of these 15 patients, the signals were present mainly at the medullocortex junction, and in the other nine patients at the cortex and/or medulla. Strong and frequent signals were present in gland-like structures in 15 cases. Some scattered signals were also present in the fibrosed glomeruli in five cases, in the thickened intimas of large arteries in three cases, and in the arterial muscle coats of two cases. Combined immunohistochemistry and in situ hybridization showed that HGF mRNA-positive cells in gland-like arrangements were also positive for cytokeratin and negative for factor VIII. Cells with HGF mRNA signal and located in the arterial media were also positive for actin. These findings suggest that HGF mRNA is transcribed both in the urinary tubular epithelium and in the mesenchymal cells (fibroblasts, and smooth muscle cells in chronic vascular rejection and endothelial cells and/or mesangial cells in transplant glomerulopathy) in human rejecting kidney.

Yamaguchi, K., M. A. Nalesnik, et al.; (1996). "Hepatocyte growth factor mRNA in human liver cirrhosis as evidenced by in situ hybridization." Scand J Gastroenterol; 31(9); 921-927.

                BACKGROUND: Hepatocyte growth factor (HGF) is a strong mitogen of hepatocytes, and HGF-producing cells have been reported to be Ito cells or endothelial cells in the sinusoid of the liver. No reports have been published about the localization of HGF mRNA in human liver cirrhosis. METHODS: In situ hybridization (ISH) for HGF mRNA was performed in 5 normal liver and 16 human liver cirrhosis specimens, using 1 RNA probe and 3 oligonucleotide probes labeled with 35S. RESULTS: A positive signal was obtained in 15 of these cases. In five normal liver specimens, signals of HGF mRNA were not obtainable. In 13 of the 15 cases of liver cirrhosis, HGF mRNA was present in the periphery of the regenerative nodules. This peripheral pattern was seen in regenerative nodules with irregular nodule to septal interfaces. Combined immunohistochemistry and ISH showed that vimentin and CD 68-positive cells consistent with macrophages expressed HGF mRNA in such cases. In three specimens with diffuse signal for HGF mRNA in the hepatic nodules, signals localized to the sinusoidal spaces. HGF mRNA-positive cells were spindled and polygonal in shape, suggesting endothelial, Kupffer, and/or Ito cells of origin. In the diffuse pattern the peripheral margins of the regenerative nodules appeared well-defined. In one case regenerative nodules with both diffuse and peripheral signal patterns were present in the same section. There was no relationship among HGF mRNA, etiology, and macroscopic appearance of liver cirrhosis. CONCLUSIONS: HGF gene transcription in human liver cirrhosis nodules may be heterogeneous, probably related to the degree of activity of the regenerative nodules. HGF appears to be produced by the mesenchymal cells, including Ito cells, macrophages (Kupffer cells), and endothelial cells in human liver cirrhosis.

Yan, H. X., H. Y. Wang, et al.; (2004). "Negative regulation of hepatocellular carcinoma cell growth by signal regulatory protein alpha1." Hepatology; 40(3); 618-628.

                Signal regulatory protein (SIRP) alpha1 is a member of the SIRP family that undergoes tyrosine phosphorylation and binds SHP-2 tyrosine phosphatase in response to various mitogens. The expression levels of SIRPalpha1 were decreased in HCC tissues, compared with the matched normal tissues. Exogenous expression of wild type SIRPalpha1, but not of a mutant SIRPalpha1 lacking the tyrosine phosphorylation sites, in SIRPalpha1-negative Huh7 human HCC cells resulted in suppression of tumor cell growth both in vitro and in vivo. Treatment of Huh7 transfectants with EGF or HGF induced tyrosine phosphorylation of SIRPalpha1 and its association with SHP-2, which were accompanied by reduced ERK1 activation. Expression of SIRPalpha1 significantly suppressed activation of NF-kappaB and also sensitized Huh7 cells to TNFalpha or cisplatin-induced cell death. In addition, SIRPalpha1-transfected Huh7 cells displayed reduced cell migration and cell spreading in a fashion that was dependent on SIRPalpha1/SHP-2 complex formation. In conclusion, a negative regulatory effect of SIRPalpha1 on hepatocarcinogenesis is exerted, at least in part, through inhibition of ERK and NF-kappaB pathways.

Yang, H., N. Magilnick, et al.; (2007). "Effect of hepatocyte growth factor on methionine adenosyltransferase genes and growth is cell density-dependent in HepG2 cells." J Cell Physiol; 210(3); 766-773.

                Hepatocyte growth factor (HGF) is a potent hepatocyte mitogen but its effect in liver cancer is conflicting. Methionine adenosyltransferase (MAT) is an essential enzyme encoded by two genes (MAT1A and MAT2A), while a third gene (MAT2beta) encodes for a subunit that regulates the MAT2A-encoded isoenzyme. MAT1A is silenced while MAT2A and MAT2beta are induced in hepatocellular carcinoma (HCC). The current work examined expression of HGF/c-met in HCC and whether HGF regulates MAT genes and growth in HepG2 cells. We found the mRNA levels of HGF and c-met are markedly increased in HCC. To study the influence of cell density, HepG2 cells were plated under high-density (HD) or low-density (LD) and treated with HGF (10 ng/ml). Cell density had a dramatic effect on MAT1A expression, being nearly undetectable at LD to a ninefold induction under HD. Cell density also determined the effect of HGF. At HD, HGF increased the mRNA levels of p21 and p27, while lowering the levels of MAT genes, cyclin A, and c-met. At LD, HGF increased the mRNA levels of cyclin A, MAT2A, MAT2beta, and c-met. Consistently, HGF inhibits growth under HD but stimulates growth under LD. HGF induced sustained high ERK activation under HD as compared to LD. In summary, HGF induces genes favoring growth and is mitogenic when HepG2 cells are plated under LD; however, the opposite occurs under HD. This involves cell density-dependent differences in HGF-induced ERK activation. This may explain why HGF is mitogenic only when there is loss of cell-cell contact in vivo.

Yoshida, Y., T. Hirano, et al.; (2007). "Allogeneic bone marrow transplantation for hepatocellular carcinoma: hepatocyte growth factor suppresses graft-vs.-host disease." Am J Physiol Gastrointest Liver Physiol; 293(6); G1114-1123.

                Allogeneic bone-marrow transplantation (BMT) can induce a powerful graft-vs.-tumor (GVT) effect not only on hematological malignancies but also on solid tumors. However, graft-vs.-host disease (GVHD) is a major complication of allogeneic BMT. We assessed GVT effect on hepatocellular carcinoma (HCC) and the effects of hepatocyte growth factor (HGF) gene transduction on GVHD in HCC transplanted mice. (C57BL/6 x C3H/HeJ)F(1)(B6C3F1, H-2(bxk)) mice were used as recipients and C3H/HeJ(H-2(k)) mice were used as donors. Hepa1-a (a C57L mouse-derived hepatoma cell, H-2(b)) was subcutaneously injected into the recipient mice. Tumor bearing mice were treated in the following ways: group 1, no treatment; group 2, total body irradiation (TBI); group 3, TBI and BMT; group 4, TBI and BMT with empty vector; group 5, TBI and BMT with HGF gene transduction; group 6, TBI and BMT with administration of FK506, a representative immunosuppressive agent. Acute GVHD was assessed by histological examination of the liver, small intestines, and large intestines. Tumor growth was markedly suppressed in mice that received an allogeneic BMT. Donor-derived CD8(+) T cells had infiltrated into the tumor, and cytotoxic CD8(+) T cells against HCC were present. However, among the four groups that received a BMT, this suppressive effect was weaker in group 6 compared with the other three groups (groups 3, 4, and 5). HGF gene transduction improved GVHD while preserving the GVT effects. Allogeneic BMT markedly suppresses the growth of HCC. Simultaneous HGF gene transfer can suppress GVHD while preserving the GVT effect.

Zender, L. and S. Kubicka; (2008). "Molecular pathogenesis and targeted therapy of hepatocellular carcinoma." Onkologie; 31(10); 550-555.

                Hepatocellular carcinoma (HCC) constitutes the 5th most frequent cancer worldwide, and due to a lack of treatment options, HCC represents the 3rd most lethal cancer worldwide. The incidence of HCC is continuously rising in Europe and Northern America, which can be explained by spreading of hepatitis C virus infections. Systemic chemotherapy is not an option for most patients with HCC. The most promising strategy for systemic treatment of HCC is targeted therapy. Successful targeted therapy has to inhibit pathways which are necessary for tumor growth, even in the late stages of carcinogenesis. The p16/Rb, p53, and IGF2R checkpoints as well as oncogenic alterations of telomerase, c-myc, Wnt/beta-catenin, PI3K/Akt, hedgehog, and c-met/HGF are most frequently involved in human hepatocarcinogenesis. However, currently, the most attractive target for molecular therapy of HCC appears to be the vascular endothelial growth factor (VEGF). Phase I/II studies showed high progression-free survival rates with antibodies or small molecules targeting the VEGF receptor pathway. Recently, a randomized placebo-controlled phase III study showed that the multikinase inhibitor sorafenib, which inhibits VEGF and Raf, significantly improves survival of patients with advanced HCC and Child A cirrhosis. As a consequence of this study, sorafenib is now the first available drug for effective systemic treatment of patients with advanced HCC.

Zhao, Q. T., S. Q. Yue, et al.; (2007). "Potential involvement of the cyclooxygenase-2 pathway in hepatocellular carcinoma-associated angiogenesis." Life Sci; 80(5); 484-492.

                Angiogenesis plays a crucial role in tumor development and growth. The present study was carried out to investigate the potential involvement of the cyclooxygenase-2 (Cox-2) pathway in the regulation of angiogenesis in hepatocellular carcinoma (HCC). We inhibited Cox-2 expression in HCC cell line HuH-7 by selective Cox-2 inhibitor (SC-58635) or Cox-2 siRNA. Conditioned media (CMs) from HuH-7 cells were used in angiogenic assays in vitro and in vivo. Compared with CMs from untreated and negative siRNA treated HuH-7 cells, CMs from SC-58635 and Cox-2 siRNA treated HuH-7 dramatically suppressed the proliferation, migration, and differentiation of human umbilical vein endothelial cells (HUVECs) in vitro and neovascularization in vivo. These inhibitory effects could be partially reversed by the addition of exogenous PGE2 to CMs. Furthermore, Cox-2 inhibition by SC-58635 resulted in PGE2 reduction accompanied by the down-regulation of four PGE2 receptor (EP receptor) subtypes. Treatment with SC-58635 led to the down-expression of proangiogenic factors such as VEGF, HGF, FGF2, ANGPT1 and ANGPT2 in HCC. An approximately 78% reduction of VEGF level has been found in the CM from SC-58635 treated HuH-7. Our results suggest an involvement of Cox-2 in the control of HCC-associated angiogenesis. PGE2 as a vital angiogenic factor may act directly on endothelial cells to promote HuH-7-stimulated angiogenic process. Moreover, Cox-2/PGE2/EP/VEGF pathway possibly also contributes to tumor angiogenesis in HCC. This study provides the rationale for clinical studies of Cox-2 inhibitors on the treatment or chemoprevention of HCC.

Zhuang, P. Y., J. B. Zhang, et al.; (2010). "Long-term interferon-alpha treatment suppresses tumor growth but promotes metastasis capacity in hepatocellular carcinoma." J Cancer Res Clin Oncol; 136(12); 1891-1900.

                PURPOSE: To elucidate the effect of IFN-alpha treatment on tumor growth and metastasis of hepatocellular carcinoma (HCC). METHODS: IFN-alpha administration was conducted in nude mice using an orthotopic implantation model of human HCC, and the key molecular markers in the IFN-alpha treatment was detected by immunohistochemistry staining and PCR array. RESULTS: Up to 12 weeks of IFN-alpha treatment significantly suppressed tumor growth of HCC, but relatively increased the number of circulating tumor cells, which might be due to the enhanced tumor hypoxia as well as up-regulation of metastasis-related genes, such as HIF-1alpha, c-met, u-PA, PDGF-A, and IL-8. However, IFN-alpha had no direct effect on migration and invasion of HCC cells. CONCLUSIONS: IFN-alpha has janus face of consistently suppressing HCC growth, however, promoting tumor metastasis capacity, which is of clinical indication for the scientific administration of IFN-alpha and the similar antiangiogenesis drugs for their dual effect on tumor growth and metastasis.

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