Proliferation Inhibition, Cell Cycle Regulation And Apoptosis Induction Of NDPK-A And Its Cys Mutants On A549 Cells

Objective:In order to investigate the effects of NDPK-A and its Cys mutants in proliferation, cell cycle and apopotsis, we transiently transfected recombitant plamids pEGFP-NDPK-A^ pEGFP-NDPK-A C4S,pEGFP-NDPK-A C4/109/145S into A549 cells to compare the diverse bioactivities of them. Our study was designed to investigate the affects of Adriamycin and 17-AAG for the expression of nm23-H1 on A549 cells. We investigated the cell cycle of vero cells to compare the unlikeness between vero cells and A549 cells.Method:Recombitant plasmids pEGFP-NDPK-A,pEGFP-NDPK-A C4S,pEGFP-NDPK-A C4/109/145S were transiently transfected into A549 cells by LTX reagents to study the highest transfected efficiency. The expression parts of NDPK-A and its Cys mutants were investigated by western blot and laser confocal microsopy. The effects of NDPK-A and its Cys mutants in cell proliferation were researched through MTT method in 96 well plate after A549 cells being transfected; In addition, pEGFP-NDPK-A, pEGFP-NDPK-A C4S, pEGFP-NDPK-A C4/109/145S and pEGFP-C1 were transiently transfected into A549 cells and vero cells which were markered with green fluroence were sorted using flow cytometry and dyed with PI and Hoechst plus PI respectively to dectect the cell cycle and apoptosis. The Adriamycin and 17-AAG effects on expression of nm23-H1 in A549 cells were studied by western blot.Results:EGFP-NDPK-A, EGFP-NDPK-A C4S and EGFP-NDPK-A C4/109/145S could express in the cytoplasm of A549 cells as fusion proteins with green fluroence, and their molecular weights were 50kD or so. Wild NDPK-A, C4S and C4/109/145S mutants all played proliferation suppression and the most strongest activity of them was C4S mutant. Cell cycle was regulated by NDPK-A and C4S, C4/109/145S mutant by delaying the S phrase, and NDPK-A behaved more accelerated apoptosis activity than C4S and C4/109/145S mutant after Hoechst and PI double dyed. The Adriamycin and 17-AAG affected the expression rate of nm23-H1 in A549 cells by concentration phenomenon. NDPK-A regulated the cell cycle of vero cells comparing with C4S mutant.Conclusions:The investigation for NDPK-A and its Cys mutants on A549 cells were carried out by fusion proteins EGFP-NDPK-A and mutants, and all the proteins were existed in cytoplasm. C4S mutant exhibited higher bioactivity in cell proliferation while wild NDPK-A exhibited higher accelerated apoptosis activity. Furthurmore, NDPK-A, C4S and C4/109/145S mutants contacted closly with cell cycle that provided better outlook on further study on the correlation between structure and bioactivities. The correction between Adriamycin and 17-AAG with nm23-H1 offered prior meaning for antineoplastic drugs research.