Recently, we have shown that down-regulation of methylthioadenosine phosphorylase (MTAP) in hepatocellular carcinoma (HCC) cells enhances the invasive potential and the resistance against cytokines. Here, we aimed at investigating the molecular mechanism underlying this tumor-promoting effect and expanded the analysis to a large series of human HCC tissues. Liquid chromatography tandem mass spectrometry revealed that reduced MTAP expression resulted in higher intra- and extracellular concentrations of 5'-deoxy-5'-methylthioadenosine (MTA) in cultivated HCC cells and, concordantly, higher levels of MTA in HCC tissue. MTA induced matrix metalloproteinase (MMP) and interleukin-8 transcription in HCC cells in vitro, accompanied by enhanced proliferation and activation of the transcription factor NFκB. In addition, MTA secreted by HCC cells induced expression of fibroblast growth factor-2 and MMP1 in stromal myofibroblasts. In human HCC tissues, MTAP mRNA correlated inversely with MTA levels, and immunohistochemical analysis of a tissue microarray of 140 human HCCs revealed that low MTAP protein expression correlated with advanced tumor stages. In conclusion, MTAP deficiency results in accumulation of MTA, which is associated with increased tumorigenicity. These data further indicate MTAP as a tumor suppressor in HCC, and MTA as a potential biomarker for HCC progression.

In human cancers, giant cadherin FAT1 may function both, as an oncogene and a tumor suppressor. Here, we investigated the expression and function of FAT1 in hepatocellular carcinoma (HCC). FAT1 expression was increased in human HCC cell lines and tissues compared with primary human hepatocytes and non-tumorous liver tissue as assessed by quantitative PCR and western blot analysis. Combined immunohistochemical and tissue microarray analysis showed a significant correlation of FAT1 expression with tumor stage and proliferation. Suppression of FAT1 expression by short hairpin RNA impaired proliferation and migration as well as apoptosis resistance of HCC cells in vitro. In nude mice, tumors formed by FAT1-suppressed HCC cells showed a delayed onset and more apoptosis compared with tumors of control cells. Both hepatocyte growth factor and hypoxia-mediated hypoxia-inducible factor 1 alpha activation were identified as strong inducers of FAT1 in HCC. Moreover, demethylating agents induced FAT1 expression in HCC cells. Hypoxia lead to reduced levels of the methyl group donor S-adenosyl-L-methionine (SAM) and hypoxia-induced FAT1 expression was inhibited by SAM supplementation in HCC cells. Together, these findings indicate that FAT1 expression in HCC is regulated via promotor methylation. FAT1 appears as relevant mediator of hypoxia and growth receptor signaling to critical tumorigenic pathways in HCC. This knowledge may facilitate the rational design of novel therapeutics against this highly aggressive malignancy.

Background: Silymarin derived from the milk thistle plant "Silybum marianum" is composed of four major flavonolignans. Clinical as well as experimental studies indicate hepatoprotective effects of silymarin. However, the underlying mechanisms are only incompletely understood. The aim of this study was to assess the effect of oral administration of a defined silymarin extract in the model of acute carbon tetrachloride (CCl 4 ) induced liver injury. Methods: A single dose of a silymarin extract (SE; 20 or 100 mg/kg body weight) was given to rats by oral gavage. Subsequently, rats were injected with a single dose of CCl 4 (2 ml/kg body weight). Results: After 24h, analysis of liver to body weight ratio, serum levels of transaminases and histological analysis revealed a marked liver damage which was inhibited by SE in a dose dependent manner. CCl 4 -induced expressions of pro-inflammatory and pro-fibrogenic genes were significantly reduced in SE treated rats. Molecular analysis revealed that SE reduced the expression of the pro-inflammatory chemokine MCP-1, the pro-fibrogenic cytokine TGF-beta as well as collagen I in isolated human hepatic stellate cells (HSC), which are the key effector cells of hepatic fibrosis. Conclusion: Oral administration of the tested silymarin extract inhibited hepatocellular damage in a model of acute liver injury. Moreover, we newly found that the silymarin extract had direct effects on pro-inflammatory and pro-fibrogenic gene expression in HSCs in vitro. This indicates that direct effects on HSC also contribute to the in vivo hepatoprotective effects of silymarin, and further promote its potential as anti-fibrogenic agent also in chronic liver disease.

Bitter acids (BAs) from the hop plant Humulus lupulus L. exhibit multiple beneficial biological properties with promising effects in cancer therapy and prevention, but information regarding the effects on hepatocellular carcinoma (HCC) is missing. Here, we used two different hop bitter acid extracts enriched for either α-acids or β-acids to obtain insight into whether biological activity varies between these two groups of BAs. At a concentration of 25 µg/ml, only the β-acid rich started to induce aspartate transaminase (AST) release, and a significant increase was detected with 50 µg/ml of both extracts. Already at lower concentrations both extracts led to a dose-dependent inhibition of proliferation, and migration was suppressed at a concentration as low as 5 µg/ml in HCC cells. The focus on different signaling pathways revealed an inhibition of ERK1/2 phosphorylation, downregulation of AP-1 activity and an alleviation of nuclear factor κB (NFκB) activity in HepG2 cells incubated with 5 µg/ml of both extracts, whereby the β-acid rich extract showed more pronounced effects. In conclusion, we identified ERK1/2, AP-1 and NFκB, which are important factors in tumor development and progression, as targets of hop BAs. Thus, these data suggest the potential use of BAs as functional nutrients for both prevention and treatment of HCC.

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