Dynamic effects of autophagy on arsenic trioxide-induced death of human leukemia cell line HL60 cells
Abstract
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 posttreatment 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.
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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 posttreatment 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.