Evaluation of Silibinin Effects on the Viability of HepG2 (Human hepatocellular liver carcinoma) and HUVEC (Human Umbilical Vein Endothelial) Cell Lines

Abstract

Human hepatocellular carcinoma is one of the most common recurrent malignancies since there is no effective therapy for it. Silibinin, a widely used drug and supplement for various liver disorders, demonstrated anti-cancer effects on human hepatocellular carcinoma, human prostate adenocarcinoma cells, human breast carcinoma cells, human ectocervical carcinoma cells, and human colon cancer cells. Considering the anti-hepatotoxic activity of silibinin and its strong preventive and anti-cancer efficacy against various epithelial cancers, we investigated the efficacy of silibinin against human HCC and HUVEC cell lines. Silibinin effects on the growth and mode of cell death of these two cell lines are presented in this paper. HepG2 and HUVEC cells were incubated with different doses of silibinin (12.5, 25, 50, 100, 150 and 200 μg/mL) at 24, 48, and 72 h. Cytotoxicity was assessed using MTT and Trypan blue assays. Mode of cell death induced by silibinin was investigated using LDH assay and acridine orange/PI double dye staining. The results showed that silibinin has dose-dependent inhibitory effect on the viability of HepG2 and HUVEC cells. However, Silibinin causes a more continuous dose-dependent cytotoxicity in HepG2 cells compared to the HUVEC cells in which some degrees of resistance is apparent at the beginning. The mode of cell death looks also different in these two cell lines with HepG2 cells being more in favor of apoptosis while necrosis is more evident for the HUVEC cells.

Keywords: HUVEC; HepG2; Human hepatocellular carcinoma; Silibinin.

Figure 1

MTT assay on A) human hepatocarcinoma cell line and B) human umbilical vein endothelial cell line exposed to different concentrations of Silibinin for 24 (dotted), 48 (dash) and 72 h (line). Cell survivals are significantly different from each other in all concentrations with a p <0.05

Figure 2

Trypan Blue die exclusion assay on A) human hepatocarcinoma cell line and B) human umbilical vein endothelial cell line exposed to different concentrations of Silibinin for 24, 48 and 72 h. Cell survivals are significantly different from each other in all concentrations with a p<0.05

Figure 3

LDH release assay on human hepatocarcinoma cell lineexposed to different concentrations of Silibinin for 24. Cell survivals are significantly different from each other in all concentrations with a p <0.05

Figure 4

Fluorescence spectroscopy examination onhuman hepatocellular carcinoma (A) andhuman umbilical vein endothelial (B) cells after 24 h exposure to 100 µg/mL of Silibinin. Cells express more of apoptotic cell death (A; green dotted cells) at the beginning followed by secondary necrosis (B; red dotted and homogeny cells) afterthe exposure to Silibinin