Alpinetin down-regulating Bcl-2 promotes apoptosis of human hepatic cancer Hep3B cells

Abstract

Objective To study the effects of alpinetin on apoptosis of Hep3B cells and explore the related mechanism.Methods Hep3B cells were cultured in vitro,treated with alpinetin; RT-PCR and Western blot was used to detect the mRNA and protein levels of Bcl-2; MTT assay was used to detect the cellular growth inhibitory rate; Annexin V-FITC/PI double staining was used to detect the apoptosis rate of cells; Mitochondrial membrane potential was analyzed by flow cytometry; Western blot was used to detect protein expression of Caspase-3,9 and Cytochrome C ; the experiment was carried out in four groups:control group,high dosage of alpinetin group,middle dosage of alpinetin group and low dosage of alpinetin group.Results The expression of Bcl-2 in Hep3B cells were decreased by alpinetin.After treated with different dosages of alpinetin (40,80,120 μmol/L),the apoptotic inhibitory rate detected by MTT were 6.38％ ± 1.32％,21.58％ ± 1.97％ and 43.18％ ± 3.89％,significantly higher than those in control group (tlowdose =13.01,tmiddle dose =15.12,thighdose =14.79,average P ＜ 0.01) ; the expression of mitochondrial membrane potential green fluorescence protein (GFP) were 18.93％ ± 2.3％,31.11％ ± 2.67％ and46.06％ ± 2.95％,significantly higher than those in control group (tlow dose =16.70,tmiddle dose =31.38,thigh dose =48.15,average P ＜ 0.01).Western blot analysis showed that the expression of Caspase-3,9 andCytochrome C in cytoplasm significantly was higher than those in control group(Caspase-3:llow dose =11.94,tmiddle dose =10.18,thigh dose =18.82,average P ＜0.01; Caspase-9:tlow dose =15.11,tmiddle dose =20.41,thish dose =21.25,average P ＜0.01; Cytochrome C:tlow dose =15.11,tmiddle dose =28.47,thigh dose =16.01,average P ＜ 0.01).while that Cytochrome C in mitochondria significantly lower than those in control group (tlow dose =16.70,tmiddle dose =12.00,thighdose =27.61,average P ＜ 0.01).Conclusions Alpinetin promotes apoptosis of human hepatic cancer cells Hep3B by down-regulating Bcl-2,probably through mitochondrial pathway. Key words: Liver neoplasms ; Galangin; Apoptosis ; Gene, Bcl-2 ; Mitochondria, liver