Investigation Of Mechanisms Underlying VB1Induces Growth Inhibition And Apoptosis In Human Hepatocellular Carcinoma Hep G2Cells

Part Ⅰ Effects of VB1on the growth and inducing angiogenesis of human hepatocellular carcinoma Hep G2cellsObjectiveIn this study we aimed to determine the effects of purified vitexin compound1(VB1, a neolignan from the seed of Chinese herb Vitex Negundo) on the growth and inducing angiogenesis of human hepatocellular carcinoma cellsMethodsHuman hepatocellular carcinoma cell lines Hep3B, Huh-7, HepG2, and human embryo liver L-02cells, human umbilical vein endothelial (HUVE-12) cells were cultured in different concentrations of VB1. The effects of VB1on the proliferation of HCC cell lines HepG2, Hep3B, Huh-7and human embryo liver L-02cells were investigated using the (MTT) assay. Growth inhibition was assessed by clonogenic assay and cell cycle arrest was investigated using flow cytometry in Hep G2cells. Anti-angiogenesis was evaluated using a matrigel in vitro HUVEC tube formation assay. ResultsAfter treatment HCC cells with different concentrations of VB1(0,2.5,5.0,10.0μM).The result shows that VB1inhibited cell viability in a concentration-dependent manner. The HepG2cell line was most sensitive, the Hep3B cell line was moderate sensitive, and the Huh-7cell line was least sensitive. VB1had little effect on human embryo liver L-02cell line and its inhibitory effect is superior to the conventional chemotherapy drugs5-FU(p<0.05).We examined the effects of VB1on colony formation of the HCC cell line HepG2on plates and showed that VB1inhibited anchorage-dependent growth in a dose dependent manner. We further showed that VB1inhibited colony formation in a dose-dependent manner in a soft agar assay.FCM analysis shows that after the treatment with different concentrations of VB1resulted in an accumulation of cells in the G1/G0phase(42.2%versus63.4%) and a decrease in the number of S-phase cells(46%versus24.3%). Furthermore, the condition media of VB1-treated cells significantly reduced tube formation of HUVECs after6h incubation compared with the media from control cells (p<0.05). ConclusionPurified vitexin compound1(VB1) possesses the actions of inhibition of growth and inducing angiogenesis of hepatocellular carcinoma Hep G2cells. Part Ⅱ Effects of VB1on Apoptosis of human hepatocellular carcinoma Hep G2cellsObjectiveThe objective of this study was to examine the effects of the VB1on Apoptosis of human hepatocellular carcinoma Hep G2cells.MethodsApoptotic cell death of HepG2cells was examined using an enzyme linked immunosorbent assay (ELISA) detection kit, flow cytometry (FCM) after propidium iodide (PI) staining, and by DNA agarose gel electrophoresis. Caspase activity was measured using ELISA.ResultsWe investigated apoptosis using flow cytometric analysis to detect increases in hypodiploid cell populations.The result shows that VB1increased the percentage of the sub-Gl population of cells in a concentration-dependent manner(2.4%versus30.2%)(P<0.05). We also showed that the histone/DNA fragment of HepG2cells increased in concentration-dependent manner following exposure to VB1. DNA fragmentation analysis by agarose gel electrophoresis showed a typical ladder pattern of inter-nucleosomal DNA fragments in cells exposed to VB1(5or10μM) for24h. Active caspase-3,-8and-9levels increased following exposure of Hep G2cells to VB1, and zVAD-fmk abrogated apoptosis induced by VB1, and that zDEVD-fmk zIETD-fmk and zLEHD-fmk attenuated VB1-induced apoptosis. Conclusion Purified vitexin compound1(VB1) induces apoptotic cell death of human hepatocellular carcinoma cell line Hep G2, in a concentration-dependent manner, which is depended on activation of caspase cascade. Part Ⅲ Investigation of the mechanisms underlying VB1induces growth inhibition and apoptosis of hepatocellular carcinoma Hep G2cellsObjectiveThe purpose of the present study was to explore the molecular mechanism by which VB1induces inhibition of growth and inducing angiogenesis and apoptosis of hepatocellular carcinoma Hep G2cells.MethodsPI3K/AKT and MAPK pathways were analyzed using Western blot. Administration of PD98059, a specific MEK antagonist, transfection of a specific AKT siRNA or FOXO3a siRNA were used to explore the molecular mechanisms of VB1-induced inhibition of proliferation and apoptosis in Hep G2cell line.ResultsResults by western blot analysis showed that exposure of HepG2cells to various concentrations of VB1resulted in a decrease in the expression levels of PI3K, AKT and ERK phosphorylated protein, and an increase in JNK phosphorylation, resulting in growth inhibition and apoptotic death. Treatment of Hep G2cells with VBl activated FOXO3a transcription factor. VB1induced the expression of its downstream effector Bim, TRAIL, DR4and DR5proteins and inhibited cyclin D1and survivn expression in Hep G2cells. VB1also reduced the secretion of vascular endothelial growth factor (VEGF), resulting in the inhibition of endothelial tube formation. Knockdown of AKT1by small interfering RNA (siRNA) enhanced growth inhibition, and silencing FOXO3a by siRNA attenuated this action. Knockdown of AKT1by siRNA and the MEK1/2inhibitor, PD98059, enhanced VB1-induced apoptosis and FOXO3a transcriptional activity. Suppression of FOXO3a expression by siRNA effectively attenuated VBl-induced apoptosis.Conclusion:Activation of FOXO3a transcription factor through blockage of the PI3K/AKT and MAPK signal pathways is involved in inhibition of growth and inducing angiogenesis of hepatocellular carcinoma Hep G2cells and pro-apoptotic effects by VB1.