Silymarin inhibited proliferation and induced apoptosis in hepatic cancer cells

Objectives: The aim of this study was to investigate mechanisms involved in the growth inhibitory effect of silymarin, in humanhepatocellular carcinoma. Materials and Methods: The human hepatocellular carcinoma cell line HepG2 was utilized and the MTT assay was performed to study the antiproliferative effect of silymarin. Dual staining was undertaken for ethidium bromide/acridine orange, propidium iodide staining and DNA fragmentation studies were executed to confirm the presence of apoptosis. Cell-cycle analysis was revealed by flow cytometry and mitochondrial transmembrane potential was measured by uptake of the mitochondrial-specific lipophilic cationic dye rhodamine 123. Western blotting analysis for cytochrome c, p53, Bax, Bcl-2, APAF-1, caspase-3, survivin, β-catenin, cyclin D1, c-Myc and PCNA was carried out. Results: Silymarin inhibited population growth of the hepatocellular carcinoma cells in a dose-dependent manner, and the percentage of apoptotic cells was increased after treatment with 50 and 75 g/ml silymarin for 24 h. Silymarin treatment increased the proportion of cells with reduced DNA content (sub-G ₀/G₁ or A₀ peak), indicative of apoptosis with loss of cells in the G₁ phase. Silymarin also decreased mitochondrial transmembrane potential of the cells, thereby increasing levels of cytosolic cytochrome c while up-regulating expression of pro-apoptotic proteins (such as p53, Bax, APAF-1 and caspase-3) with concomitant decrease in anti-apoptotic proteins (Bcl-2 and survivin) and proliferation-associated proteins (β-catenin, cyclin D1, c-Myc and PCNA). Conclusions: Our results demonstrate that silymarin treatment inhibited proliferation and induced apoptosis in the human hepatocellular carcinoma cell line HepG2.