An Experimental Study On Apoptosis And Oncosis Of Human Lung Cancer Cell-line A-549 Induced By Arsenic Trioxide

Objective To explore the apoptosis and oncosis inducing effects of arsenic trioxide(As₂O₃) on human lung cancer cell line A-549 and elucidate possible mechanisms and preliminary classification of apoptosis and oncosis.Methods After treatment with As₂O₃, the Cell growth curves of human lung cancer cell line A-549 were described by cell count; cell growth and proliferation inhibition rate were studied by MTT; cell cycle,apoptosis index,oncosis index and cell volume were detected by flowcytometry; the morphology changes were observed after treated with As₂O₃ in different concentrations by inverted phase contrast microscope; and the ultra microstructure changes were observed by transmission electron microscope after treatment,Δψm was detected by flowcytometry with staining both Rh123 and PI. The concentration of intracellar calcium were detected by laser scanning confocal microscope(LSCM) with fluo-3P/AM staining. The expression of Bcl-2,PCNA and AIF was analyzed with immunohistochemistry staining. Porimin was detected by western blotting and Caspase-3,Caspase-9 ,Caspase-8 were detected by flowcytometry.Results The data showed that As₂O₃ significantly inhibited the growth and proliferation of A-549 cells and the inhibiting effects had a combining dose and time dependence. The cell cycle were regulated including that cell cycle progress in G2/M phase was blocked by As₂O₃ (0.5 and 2.0μmol/L, 72h), but ell cycle progress in S phase was blocked by As₂O₃ (0.25 ,1.0 and 4.0μmol/L, 72h). And the influence of apoptosis and oncosis induced by As₂O₃ was as following: the two peak of apoptosis index(13.05% and 16.95) appearanced on the concentration of 0.25μmol/L and 2.0μmol/L. the AI reduced to 12.95% , but OI rised to 18.94% after the cells were treated by As₂O₃ (4.0μmol/L) for 72hours.The oncosis index had a time and dose dependence relation with As₂O₃. Gradually dose and time dependent morphology changes were shot under the light microscope. Typical apoptotic cell (induced by 2.0μmol/L As₂O₃ for 72h), oncotic and necrotic cells (induced by 4.0μmol/L As₂O₃ for 72h) were found through transmission electron microscope. Living cells,apoptotic cells and oncotic cells were observed by laser scanning confocal microscope(LSCM). And the cell rate consistent with apoptosis index and oncosis index in each group. The molecular mechanisms included that the change ofΔψm and concentration intracellar calcium had a time and dose dependence relation with As₂O₃. The expression of Bcl-2 and PCNA of experimental group decreased, the highest inhibitive rate of Bcl-2 was induced by As₂O₃ (4.0μmol/L) for 36 hours, and the highest inhibitive rate induced by As₂O₃ (4.0μmol/L) for 72 hours; The expression of AIF and porimin also had a time and dose dependence relation with As₂O₃; the family of Caspase were activated after treatment with As₂O₃, and the two activation mechanisms (including mitochondrion pathway and death receptor pathway)were had a difference in according to the concentration of As₂O₃.Conclusion: As₂O₃ significantly inhibited the growth and proliferation and regulated cell cycle of A-549 cells, the inhibiting effects include apoptosis and oncosis and the mechanisms of apoptosis and oncosis were that the family of Caspase were activated. The activation pathways (including mitochondrion pathway and death receptor pathway)varied with the time and dose of As₂O₃.We presume the concentration amplitude (0.25～2.0μmol/L) of As₂O₃ is safe and effective in experiment in vivo. We also prognosticate that As₂O₃ is a valuable anti-lung cancer medicine in future.