Diphenyliodonium, an NAD(p)H oxidase inhibitor, also induces apoptosis

Nianyu Li, Kathy Ragheb, Gretchen Lawler, Bartek Rajwa, J. Paul Robinson

Basic Medical Scineces, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907-124 (#6402)

The mitochondrial respiratory chain is the major site of reactive oxygen species (ROS) production under physiological conditions. At the same time, mitochondria play a central role in apoptosis. We have previously reported that IDP (diphenyliodonium) successfully inhibited the production of superoxide by NADPH oxidase, which is the predominate superoxide producing enzyme in neutrophil. However, we also found that IDP greatly increased the fluorescence of HE (an superoxide indicator) on HL-60 cells during incubation. Since these findings suggested the involvement of IDP on mitochondrial respiratory chain ROS production, we thus investigated the effects of IDP on mitochondrial respiratory chain and apoptosis. Our results showed that incubating with 1mM to 2mM of IDP for 6-8 hours could induce DNA fragmentation on HL-60 cells. DNA breakdown was also investigated using Propidium Iodide (PI) by flow cytometry and the results were consistent with DNA ladder observation. Z-VAD, a non-specific caspases inhibitor, could successfully inhibit this process. Another interesting finding is that Cyclosporine A, which is a mitochondrial permeability transition pore (MPT pore) inhibitor, could partly inhibit the IDP induced DNA fragmentation as well as IDP induced superoxide production. Confocal microscopy studies also showed that mitochondria permeability transition happened within one hour, which could be inhibited by cyclosporine A. Other possible contributors of IDP induced apoptosis, such as cytochrome c release, PARP cleavage and mitochondrial membrane depolarization, were investigated as well. Taken together, our observations suggested that IDP, which is a mitochondrial complex I inhibitor, could also induce apoptosis.

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