Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: Anti-tumor effect of oleic acid in hepatocellular carcinoma cell lines via autophagy reduction

Oleic acid (OA), as other monounsaturated fatty acids, is a component of the olive oil. Beneficial health effects of olive oil are well-known, such as protection against liver steatosis and against some cancer types. In the present study we focused on OA effects in hepatocellular carcinoma (HCC), investigating responses to OA treatment (50-300 μM) in HCC cell lines (Hep3B and Huh7.5) and in a healthy liver-derived human cell line (THLE-2). Upon OA administration higher lipid accumulation was observed in HCC cells as compared to healthy cells. This observation paralleled the autophagy reduction found in HCC cell lines but not in healthy controls. An inverse relationship between perilipin-2 and autophagy levels has been previously described in liver, we therefore analysed perilipin-2 protein expression and observed a significant perilipin-2 increase in HCC cell lines but not in healthy cells upon increasing OA dosage. We then investigated the effects of OA on viability and proliferation. OA in the presence of 10% FBS significantly reduced viability of HCC cell lines by about 40% at 300µM through Alamar Blue staining evaluation and reduced cyclin D1 expression in a dose-dependent manner while it was ineffective on healthy hepatocytes. In addition, cell cycle analysis by flow cytometry using propidium iodide staining of OA-treated HCC cell lines revealed mild reduction of the S-phase and mild increase in G0/G1 phase in both HCC cell lines, thus supporting the reduction of the proliferation induced by OA. Furthermore, OA increased cell death by about 30% (through Trypan blue-stained-cell counts), inducing apoptosis and necrosis in HCC cells but not in healthy hepatocytes at 300µM dosage. Moreover, by senescence β-galactosidase staining, increase in Hep3B at 300µM OA was observed. Remarkably, 300µM OA significantly inhibited the antiapoptotic proteins c-FLIP and Bcl-2 in HCC cells but not in healthy hepatocytes. All these results led us to conclude that different cell death processes occur in these two HCC cell lines upon OA treatment. Furthermore, to better analyze the role of OA in cell death and proliferation, we investigated the expression level of p44/p42 MAPK (ERK1/2). Indeed, P-ERK inactivation in Thr202/Tyr204 is a marker of reduced proliferation and increased cell death. Western blot analyses revealed that increasing OA concentrations led to a dose-dependent P-ERK reduction in both HCC cell lines but not in the healthy controls. Moreover, to evaluate the effects of OA on cellular aggressiveness, we performed both cell migration (scratch test) and invasion assays. We observed that 300 µM OA significantly reduced the migration and invasion of both HCC cell lines, while it has no effects on healthy cells. Finally, we investigated autophagy role in OA-dependent effects by using the autophagy inducer torin-1. We performed combined OA/torin-1 treatment and observed reduced lipid accumulation as well as reduced cell death as compared to single OA treatment. We therefore concluded that OA effects in HCC cells lines are at least in part dependent on OA-induced autophagy reduction.