Acta Pharmacologica Sinica 2008 January; 29 (1): 123-134; doi: 10.1111/j.1745-7254.2008.00732.x

Aim: To evaluate the contribution of an autophagic mechanism to the As₂O₃-induced death of human acute myeloid leukaemia cell line HL60 cells.

Methods: The growth inhibition of HL60 cells induced by As₂O₃ was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. The activation of autophagy was determined with monodansylcadaverine labeling and transmission electron microscope. The role of autophagy in the As₂O₃-induced death of HL60 cells was assessed using autophagic and lysosomal inhibitors. Immunofluorescence, flow cytometry, and Western blot analysis were used to study the apoptotic and autophagic mechanisms.

Results: After treatment with As₂O₃, the proliferation of HL60 cells was significantly inhibited and the formation of autophagosomes increased. The blockade of autophagy maturation with the autophagy-specific inhibitor 3-methyladenine (3-MA) or the lysosome-neutralizing agent NH₄Cl 1 h before As₂O₃ potentiated the As₂O₃-induced death of HL60 cells. In contrast, 3-MA attenuated As₂O₃-induced death when administered 30 min after As₂O₃. 3-MA and NH₄Cl also inhibited As₂O₃-induced upregulation of microtubule-associated protein 1 light chain 3, the protein required for autophagy in mammalian cells. Following As₂O₃, lysosomes were activated as indicated by increased levels of cathepsins B and L. The apoptotic response of HL60 cells to As₂O₃ was suggested by the collapse of mitochondrial membrane potential, release of cytochrome c from mitochondria, and the activation of caspase-3. Pretreatment with 3-MA prior to As₂O₃ amplified these apoptotic signals, while post-treatment with 3-MA 30 min after As₂O₃ attenuated the apoptotic pathways.

Conclusion: Autophagy plays complex roles in the As₂O₃-induced death of HL60 cells; it inhibits As₂O₃-induced apoptosis in the initiation stage, but amplifies the As₂O₃-mediated apoptotic program if it is persistently activated.

Keywords: As₂O₃; autophagy; HL60 cells; 3-methylad-enine; lysosome; mitochondria