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


CONCLUSIONS
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.
Yang
Ya-ping
YP
Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Medicine, Suzhou, China.
Liang
Zhong-qin
ZQ
Gao
Bo
B
Jia
Yan-li
YL
Qin
Zheng-hong
ZH
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology*; D001343:Autophagy / Q000187:drug effects*; D002103:Cadaverine / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D018922:HL-60 Cells; D006801:Humans; D051336:Mitochondrial Membranes / Q000187:drug effects; D010087:Oxides / Q000494:pharmacology*; D013347:Subcellular Fractions / Q000187:drug effects
D001152:Arsenicals; D010087:Oxides; D000643:Ammonium Chloride; C025946:3-methyladenine; D000225:Adenine; D002103:Cadaverine; D000077237:Arsenic Trioxide