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 Ca ²⁺ 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 Ca ²⁺ influx blockade by Ec is required for cell death.