Study On Apoptosis Of Human Hepatoma Cell Line HepG-2 Induced By Malanin

Objective: Research malanin induces cancer cells HepG-2 cell proliferation and apoptosis inducing effect, laying the foundation for the development and utilization malanin.Method:melanin was isolated from malanin oleifera seeds by smash,PBS collect,ammonium sulfate precipitation and hydrophobic interaction chromatography(HIC);MTT assay was used to determine HepG-2 cell proliferation with malanin;HepG-2 cell were detected with morphologic observation; DNA agarose electrophoresis were applied to analysis the appearance of DNA-Ladder with different concentrations and time of malanin treating HepG-2 cells;Flow cytometry was used to test different time of malanin in the role of apoptosis in HepG-2 cells and peaks; Flow cytometry was used to test mitochondrial membrane potential;caspase-3,8,9activity was determined by spectrophotometry; MTT assay was used to determine HepG-2 cell proliferation with broad-spectrum caspase inhibitor and caspase-3,8,9inhibitors and malanin. Results: malanin inhibits the proliferation of HepG-2cells,and in a time-dose relationship, HepG-2 cells in time of 24 h,48h,72 h IC50 were respectively 0.2480μg/mL,0.1516μg/mL,0.0739μg/mL; Optical microscopy revealed cell rounding,shrinkage,smaller size,cell gap increases,there is the formation of apoptotic bodies,and fluorescence microscopy revealed nuclear fragmentation,lumps,the fluorescence intensity was significantly higher;DNA agarose electrophoresis analysis showed that after treatment with different time of HepG-2 cell DNA fragmentation turns into a different fragment begin 24 h and disappear at 48h;The mitochondrial membrane potential began to decline at 12 h,and as the delivery time,the mitochondrial membrane potential is more and more low; caspase-3,8,9 activity with the extension of the delivery time increases,36 h reached the maximum;after joining a broad-spectrum caspase inhibitor,caspase-3,8,9 inhibitors the cell inhibition rate was significantly lower.Conclusion:malanin inhibits the proliferation of HepG-2cells,and in a time-dose relationship;morphology,DNA fragmentation results show:apoptosis induced by malanin in human hepatoma cell HepG-2;malanin can enhanced mitochondrial membrane potential depolarization and decreased mitochondrialfunction and induced HepG-2 cells apoptosis;after administration caspase-3,8,9activity were significantly increased,36 h reached the maximum, joining a broad-spectrum caspase inhibitor,caspase-3,8,9 inhibitors the cell inhibition rate was significantly lower,that Description caspase family members mediate apoptosis induced by melanin in human hepatoma cell HepG-2.