ANALYSIS OF THE ANTI-FIBROTIC EFFECTS OF HOP BITTER ACIDS ON HEPATIC STELLATE CELLS AND THEIR ANTI- TUMORIGENIC EFFECTS ON HEPATOCELLULAR CARCINOMA CELLS

Bitter acids (BA) from the hop plant Humulus lupulus L. exhibit multiple beneficial biological properties. However, no studies are available regarding the effects of hop bitter acids on liver cells or chronic liver diseases, respectively. Chronic liver diseases are accompanied by chronic hepatic inflammation which can lead to liver fibrosis. If left untreated, liver fibrosis can progress to cirrhosis, which can cause liver failure. Importantly, cirrhosis is also the main risk factor for the development of hepatocellular carcinoma (HCC). Both cirrhosis and HCC are associated with high morbidity and mortality, and up until now, no effective treatment options are available. The aim of this thesis was to assess the effects of BA on hepatic stellate cells, the central mediators of liver fibrosis, and on hepatocellular carcinoma cells. First, the effects of hop bitter acids on hepatic stellate cells (HSC) were analyzed. HSC were isolated from murine and human liver tissues and were incubated with a standardized hop bitter acid extract. At a concentration of 25 µg/ml hop bitter acids (BA) started to induce lactate dehydrogenase leakage. Already at lower concentrations BA led to a dose dependent inhibition of HSC proliferation and inhibited IkappaB-alpha-phosphorylation and nuclear p65 translocation in a dose dependent way. Accordingly, the same doses of BA inhibited the expression of pro-inflammatory and NFkappaB-regulated genes, but did not affect expression of genes not related to NFkappaB signaling. In addition to these effects on activated HSC, BA inhibited the in vitro activation of non-activated HSC as evidenced by delayed expression of collagen I and alpha-sma mRNA and protein. Together, these findings indicate that BA inhibit NFkappaB activation, and herewith, the activation and development of pro-fibrogenic phenotype of HSC in vitro. Next, we analyzed the effects of BA on hepatocellular carcinoma (HCC) cells. Here, we used two different standardized BA extracts enriched for either alpha-acids or beta-acids to get a first insight whether biological activity vary between these two groups of bitter acids. At a concentration of 25 µg/ml, only the beta-acid rich extract started to induce aspartate aminotransferase (AST) release as marker for cell injury, whereas at a dose of 50 µg/ml both extracts led to a significant increase of AST liberation. Already at lower concentrations both extracts dose-dependently inhibited proliferation and migration of HCC cells. Analysis of different signaling pathways revealed an inhibition of ERK1/2 phosphorylation, down-regulation of AP-1 activity and an alleviation of NFkappaB activity in HCC cells in vitro. Hereby, the beta-acid rich extract showed more pronounced effects. Furthermore, the stability of BA in a rodent chow supplemented with an alpha-acid rich extract was assessed applying high pressure liquid chromatography diode array detection technology. Mimicking feasible storage conditions we observed a very poor recovery, indicative of a strong degradation of BA. This prompted us not to proceed with application of this particular BA supplemented chow in rodent models, since in addition to imprecise BA-concentration also potential ill-defined degradation products would not have allowed a sound interpretation of such studies. In conclusion, hop bitter acids alleviate the pro-fibrogenic phenotype of hepatic stellate cells and inhibit central signaling pathways, which are known to play important roles in tumor development and progression, in hepatocellular carcinoma cells. These data suggest the potential use of hop bitter acids as functional nutrient or therapeutical agent for both prevention and treatment of chronic liver diseases. Further studies are needed to verify the beneficial effects in experimental models of liver injury in vivo. However, analysis of a newly designed BA-supplemented experimental chow for rodents revealed strong degradation processes even under optimal storage conditions. Therefore, special care has to be taken when planning long term BA-application experiments.