魚藤素誘發人類非小細胞肺癌細胞株 NCI-H460 之凋亡機制

Abstract

Deguelin, is a natural product, isolated from Derris trifoliata Lour. It was reported that deguelin inhibited cell proliferation and caused cell death in human leukemia and breast cancer cells through the induction of apoptosis. Therefore, we investigate deguelin whether or not affect human lung cancer cells in vitro . First, we observed the change of cell morphology and percentage of cell viability after various doses of deguelin treatment, flow cytometric analysis and trypan blue staining were used to detect the cell viability and the cell growth inhibition of NCI-H460 cells. Second, we used annexin V, DAPI staining, comet assay and DNA gel electrophoresis to determined the phenomenon of apoptosis. Finally, we investigated signal transduction by examining the intracellular change of mitochondria membrane potential (Δψm) and Ca2+ levels, western blotting, caspase activity, AKT kinase assay and immunofluoresence were used in NCI-H460 cell line. Based on these results, we found that deguelin greatly suppressed cell proliferation and decreased cell viability in time- and dose-dependent manners. Deguelin induced cell apoptosis and DNA damage including chromatin condensation, the formation of apoptotic bodies, and translocation of phosphatidylserine (PS) of the plasma membrane. Western blotting and immunofluoresence proved that deguelin inhibited p-AKT protein expressions, resulting in activation of Bad and then translocate from cytosol to mitochondria where it binds to Bcl-2 and Bcl-X, followed by promoted the influence of Bcl-2 family proteins. There is a decrease in mitochondria membrane potential (Δψm), causing release of cytochrome c and AIF. Cytochrome c combined with apaf-1 and caspase-9 to an apoptosome , activated caspase-3, leading to apoptosis. Then, AIF translocated into nuclear causing DNA damage. Overall, we suggest that the major mechanism of deguelin-induced apoptosis in NCI-H460 is through AKT and Bad-releated signaling pathway.