Galangin induces apoptosis of hepatocellular carcinoma cells via the mitochondrial pathway

Abstract

Aim: To investigate the mechanism by which galangin, a polyphenolic compound derived from medicinal herbs, induces apoptosis of hepatocellular carcinoma (HCC) cells.

Methods: The 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was used to measure cell viability. Apoptosis was evaluated by in situ uptake of propidium iodide and Hoechst 33258 and was then detected by fluorescence microscopy. Protein expressions were detected by Western blotting. To confirm the apoptotic pathway mediated by galangin, cells were transfected by bcl-2 gene to overexpress Bcl-2 or siRNA to down-regulate Bcl-2 expression.

Results: Galangin (46.25-370.0 micromol/L) exerted an anti-proliferative effect, induced apoptosis, and decreased mitochondrial membrane potential in a dose and time-dependent manner. Treatment with galangin induced apoptosis by translocating the pro-apoptotic protein Bax to the mitochondria, which released apoptosis-inducing factor and cytochrome c into the cytosol. Overexpression of Bcl-2 attenuated galangin-induced HepG2 cell apoptosis, while decreasing Bcl-2 expression enhanced galangin-induced cell apoptosis.

Conclusion: Our data suggests that galangin mediates apoptosis through a mitochondrial pathway, and may be a potential chemotherapeutic drug for the treatment of HCC.

Figure 1

Effects of galangin on cell viability of three hepatocellular carcinoma cell lines. A: Three hepatocellular carcinoma (HCC) cell lines were treated with 46.25, 92.5, 185, and 370 μmol/L galangin for 24 h. The IC₅₀ of galangin to HepG2, Hep3B, and PLC/PRF/5 cells were 134.0, 87.3 and 79.8 μmol/L, respectively; B: Three HCC cell lines were treated with 92.5 μmol/L galangin for 24, 48 and 72 h. mean ± SD. n = 4. ^aP < 0.05 vs HepG2 cell/dimethyl sulfoxide (DMSO)-treatment group; ^cP < 0.05 vs PLC/PRF/5 cell/DMSO-treatment group; ^eP < 0.05 vs Hep3B/DMSO cell/DMSO-treatment group.

Figure 2

Hepatocellular carcinoma cells apoptosis induced by galangin. A: Morphology of apoptotic cells, pictures were taken under a 20 × objective; B: Cells were treated with different concentrations of galangin for 24 h and stained with Hoechst 33258/PI to measure apoptosis. mean ± SD. n = 4. ^aP < 0.05 vs HepG2 cell/dimethyl sulfoxide (DMSO)-treatment group; ^cP < 0.05 vs PLC/PRF/5 cell/DMSO-treatment group; ^eP < 0.05 vs Hep3B/DMSO cell/DMSO-treatment group.

Figure 3

Effect of galangin on the mitochondrial membrane potential of hepatocellular carcinoma cell lines. A: Three hepatocellular carcinoma (HCC) lines were treated with 46.25, 92.5, 185, and 370 μmol/L galangin for 24 h; B: Three HCC were treated with 92.5 μmol/L galangin for 24, 48 and 72 h. mean ± SD. n = 4. ^aP < 0.05 vs HepG2 cell/dimethyl sulfoxide (DMSO)-treatment group; ^cP < 0.05 vs PLC/PRF/5 cell/DMSO-treatment group; ^eP < 0.05 vs Hep3B/DMSO-treatment group.

Figure 4

Effects of galangin-treated hepatocellular carcinoma cells on protein expression involved in the mitochondria pathway. A: The changes of Bax in the mitochondria; B: The changes of cytochrome c and apoptosis-inducing factor (AIF) in the cytosol; C: Caspase-3 and caspase-9 activation, and Poly(ADP-ribose) polymerase (PARP) cleavage induced by galangin. 1: Blank control group; 2: Dimethyl sulfoxide-treatment groups; 3: Cells were treated with 46.25 μmol/L galangin for 24 h; 4: Cells were treated with 92.5 μmol/L galangin for 24 h; 5: Cells were treated with 185 μmol/L galangin for 24 h; 6: Cells were treated with 370 μmol/L galangin for 24 h.

Figure 5

Effects of Bcl-2 on galangin-induced HepG2 cell apoptosis. A: Bcl-2 protein level was measured in HepG2 cells by Western blotting after cells were transfected; B: Morphology of apoptotic cells. Cells were treated with 185 μmol/L galangin for 24 h and stained with Hoechst 33258/PI to measure apoptosis. Pictures were taken under a 20 × objective. 1: Control vector group; 2: pcDNA3.1-bcl-2-transfected group; 3: Control siRNA group; 4: Bcl-2 siRNA-transfected group; C: Bcl-2 level affect apoptotic index of HepG2 cell induced by galangin. mean ± SD. n = 4. ^aP < 0.05 vs pcDNA3.1 group; ^cP < 0.05 vs control siRNA group; D: The effect of Bcl-2 level on cell viability induced by galangin. mean ± SD. n = 4. ^aP < 0.05 vs HepG2 cell/dimethyl sulfoxide-treatment group; ^cP < 0.05 vs pcDNA3.1-transfected group; ^eP < 0.05 vs control siRNA group.