Effects Of Autophagy On Arsenic Trioxide-induced Death Of Human Leukemia Cell Line HL60 Cells

AIM To study the effects and the mechanisms of autophagy on As₂O₃-induced death of HL60 cells.METHODS After treatment with As₂O₃ , the growth inhibition of HL60 cells was assessed by MTT colorimetric assay. MDC staining and Transmission Electron Microscope (TEM) were used to examine autophagy induced by As₂O₃ in HL60 cells. To examine the involvement of autophagy in As₂O₃-induced death of HL60 cells, the autophagy specific inhibitor 3-methyadenine (3-MA) was added with As₂O₃ and the cytotoxicity of As₂O₃ was measured by lactose dehydrogenase (LDH) leakage. Immunohistochemistry, flow cytometry and Western blot analysis were used to study the apoptotic and autophagic mechanisms involved in death of HL60 cells.RESULTS The proliferation of HL60 cells was significantly inhibited in a dose- and time-dependent manner after As₂O₃ treatment. Autophagy was induced in HL60 cells as detected by both MDC staining and TEM; The autophagy specific inhibitor 3-MA exerted opposite effects on As₂O₃-induced death of HL60 cells: 3-MA potentiated As₂O₃'s cytotoxicitiy in HL60 cells when administered 1h before As₂O₃; while it attenuated As₂O₃-induced death of HL60 cells when administered 30 min after As₂O₃. What's more, once autophagy was induced by As₂O₃, LC3, the autophagic protein in mammalian cells was also activated. The expression of cathepsin B, D and Bid increased, mitochondrial membrane potential collapsed, cytochrome c release was induced and caspase-3 activation accelerated during death of HL60 cells following treatment with As₂O₃. Moreover, pretreatment with 3-MA prior to As₂O₃ amplified above-mentioned apoptotic signaling pathways, but it inhibited these signaling pathways when 3-MA was administered post- As₂O₃ insult.