Inhibition of Ca2+ influx is required for mitochondrial reactive oxygen species-induced endoplasmic reticulum Ca2+ depletion and cell death in leukemia cells.
Disturbances of endoplasmic reticulum (ER) Ca2+ homeostasis or protein processing can lead to ER stress-induced cell death. Increasing evidence suggests that oxidative stress (OS) plays an important role in a variety of cell death mechanisms. To investigate the role of OS in ER stress, we measured OS in response to three ER stress agents: econazole (Ec), which stimulates ER Ca2+ release and blocks Ca2+ influx; thapsigargin (Tg), a sarco(endo)plasmic reticulum Ca2+ ATPase inhibitor that releases ER Ca2+ and stimulates Ca2+ influx; and tunicamycin (Tu), a glycosylation inhibitor that causes protein accumulation in the ER. Ec, but not Tg or Tu, caused a rapid increase in OS. Reactive oxygen species (ROS) generation was observed within mitochondria immediately after exposure to Ec. Furthermore, Ec hyperpolarized the mitochondrial membrane and inhibited adenine nucleotide transport in cell-free mitochondria, suggesting a mitochondrial target. Antimycin A, an inhibitor of complex III in electron transport, reversed mitochondrial hyperpolarization, OS generation, ER Ca2+ depletion, and cell death by Ec, suggesting complex III dependence for these effects. Antioxidants butylated hydroxytoluene and N-Acetyl-L-cysteine prevented ER Ca2+ depletion and cell death by Ec. However, inhibition of Ca2+ influx by Ec was unaffected by either antimycin A or the antioxidants, suggesting that this target is distinct from the mitochondrial target of Ec. Atractyloside, an adenine nucleotide transport inhibitor, generated ROS and stimulated ER Ca2+ release, but it did not block Ca2+ influx, deplete the ER or induce cell death. Taken together, these results demonstrate that combined mitochondrial ROS generation and Ca2+ influx blockade by Ec is required for cell death.
Zhang
Yicheng
Y
Arthritis and Immune Disorder Research Centre, University Health Network and Department of Immunology, University of Toronto, Medical Discovery Tower, 8th Floor, Rm 8-356, 101 College St., Toronto, ON, Canada M5G 1L7.
Soboloff
Jonathan
J
Zhu
Ziping
Z
Berger
Stuart A
SA
D000227:Adenine Nucleotides / Q000378:metabolism; D000968:Antimycin A / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D001278:Atractyloside / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004464:Econazole / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism* / Q000502:physiology; D008954:Models, Biological; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism*
D000227:Adenine Nucleotides; D000975:Antioxidants; D017382:Reactive Oxygen Species; D001278:Atractyloside; D000968:Antimycin A; D004464:Econazole; D020169:Caspases; D002118:Calcium