Effects of stimulation with free fatty acids on proliferation and migration of HCC cells in vitro

Introduction: Hepatocellular carcinoma (HCC) is the most common primary liver cancer worldwide. Although new strategies for HCC therapy have evolved perpetually, it still remains a highly aggressive form of cancer with only poor prognosis due to frequently late diagnosis. The majority of patients suffers from chronic liver disease such as non-alcoholic steatohepatitis (NASH), which often develops in obese people. In fact, obesity and its consequences make a larger contribution to overall HCC burden than HBV and HCV infections. Both, NASH and its primary stages NAFLD and steatosis show elevated levels of free fatty acids (FFA). These patients often do have a metablic syndrome as comorbidity, which is a multifaceted disease. Its main characteristics are hyperglycemia, insulin resistance, hypertension and obesity. Today, it gradually becomes a more relevant health problem due to prosperity in some societies. Elevated levels of FFA is a common feature of obesity, metabolic syndrome, NAFLD and HCC. Moreover, elevated levels of FFA not only are important for development of HCC but also for the genuine tumor metabolism. Therefore, the aim of this thesis was to assess the effects of FFA and cellular lipid accumulation, respectively, on proliferation and migration of HCC cells in vitro. Methods and results: No signs of cytotoxicity could be detected in supernatants of four different HCC cell lines, which were stimulated exemplarily with oleate of a chosen dose range. As a marker for cell viability, the amount of lactate dehydrogenase (LDH) is a commonly used parameter and therefore was determined in laboratory analysis. A dose-dependent accumulation of FFA and triglycerides was striking. Oil Red O staining confirmed these results visually. Proliferation was investigated by the use of XTT proliferation assay. In low concentrations of oleate and BSA no effect on proliferation rates could be detected, whereas for higher concentration levels a slightly negative effect of oleate was noticeable. Surprisingly, proliferation rates increased dose-dependently when cells were stimulated with BSA. This effect especially could be shown when using xCELLigence system. This method also revealed a dose-dependent lowered proliferation rate with oleate in culture medium. The second part of this study examined the migratory potential of HCC cells. Both, random and directional cell migration showed no significant changes concerning cell migration. Eventually, a closer look was taken on the relevance of glucose concentration in culture medium. Therefore low glucose medium was used for experimental trials, as well. When compared to each other, a positive effect could be detected for high glucose as culture medium. Discussion: It could be demonstrated that oleate dose-dependently has a slightly inhibiting effect on proliferation of HCC cells. As regards migration HCC cells acted indifferently. The performed in vitro analyses could not prove any protumorigenic influence of oleate exhibited on HCC cells. The striking positive correlation of high levels of FFA in HCC progression and development seems to be mediated by other FFA or at least unlikely by FFA in a direct way