Studies On Proanthocyanidin Induces The Human Hepatoma Cancer HepG-2 Cell Apoptosis And Mechanism Through Mitochondria Pathways

This study explores the actions of the inhibition of tumor cells cultured in vitro by proanthocyanidin(PC) and the apoptosis action and mechanism of mitochondria pathway induced by PC, certificate that PC has anti-tumor action in vivo using the transplantation tumor experiment.In vitro studies, it has been found that PC can inhibit the growth of human hepatocarcinoma cells HepG-2. Measurements using mononuclear cell direct cytotoxicity assay(the MTT method) shows that its cytotoxic effect on HepG-2 is strong, with IC50 being 47.67μmol·L-1 respectively.PC can induce tumor cells apoptosis and can induce tumor cells apoptosis via mitochondria pathway(HepG-2).Measurement of action of apoptosis using morphology with Hoechst 33258 stain show that the karyoplasmic ratio of tumor cells diminish, cell nucleus pyconsis,the staining extent become deeper,chromatin prosperous aggregation, fragmentation. The cells breaks up into several apoptotic bodies of different sizes. As PC concentration is increased, these morphological changes under the microscope become more and more clear, indicating that the proportion of cells undergoing apoptosis is gradually increasing. Measurement the tumor cell cycle dealed with PC using flow cytometry shows that PC leads to a decrease in the proportion of cells in the G0/G1 phase, and increase in the proportion of cells in the S phase and G2/M phase, but have no obvious change in the G2/M phase. This shows that PC has the influence in the tumor cell cycle and can block the tumor cells in the S phase. The Kit of Bcl-2 and Bax was used in the HepG-2 cells of Bcl-2 and Bax protein detection.When increasing the doses of PC, Bcl-2 protein expression was decreased and Bax protein expression was increased. Measurement the mitochondrial membrane electric potentiall(Δψm) treated with PC using the flow cytometry with Rodanmin 123 stain shows that PC can obviously cut down theΔψm of the tumor cells with the increasement of the dose. This effect shows dose-dependent. Measurement the content of Ca2+ using confocal laser scanning microscope with Fluo-3/AM stain shows that the PC has influence in the content of Ca2+, the high dose can increase the content of Ca2+ in tumor cells.This effect shows dose-dependent. Measurement the activities of Caspase-3 and Caspase-9 using the kit of Caspase-3,9 shows that the activities of Caspase-3 and Caspase-9 in tumor cells have gradually increase with the dose of PC. This effect shows dose-dependent too.In vivo study, Measurement the best dose of PC using acute toxicity test and dosage bloting method shows that the best dose of PC is 120mg/kg·d,60mg/kg·d,30mg/kg·d.Using the transplantation tumor test in S180 and intragastric administration method shows that all of high,middle and low dose group can inhibit the tumor cells growth. The dose of high and middle group have significance difference,the low dose group has disparity.At the same time,measurement the content of calcium pump using the kit of Ca2+,Mg2+-ATPnase shows that PC can cut down the content of calcium pump in tumor cells comparing with the isotonic Na chloride group. The high group of PC can obviously cut down the content of calcium pump in tumor cells. In a word, PC has the anti-tumor effect in vitro or vivo and PC can induce the tumor cell apoptosis via mitochondria pathways.