Abstract
BackgroundCell death induced by poly(ADP-ribose) (PAR) and mediated by apoptosis-inducing factor (AIF) is well-characterized in models of ischemic tissue injury, but their roles in cancer cell death after chemotherapy are less understood.MethodsHere we investigated the roles of PAR and AIF by RNA interference (RNAi) in MDA-MB-231 and MCF-7 breast adenocarcinoma cells after chemotherapy. Differences in effects were statistically tested by analysis-of-variance and unpaired student’s t-test.ResultsSilencing of AIF by RNAi led to decreased MDA-MB-231 and MCF-7 breast cancer cell death after chemotherapy, which demonstrates a critical role for AIF. RNAi silencing of PAR glycohydrolase (PARG), the primary enzyme that catalyzes the hydrolysis of PAR, led to increased PAR levels but decreased cell death. Further investigation into the possible role of PAR in apoptosis revealed decreased caspase-3/7/8/9 activity in PARG-null cells. Interestingly, the pharmacologic inhibition of caspase activity in PARG-silenced breast cancer cells led to increased cell death after chemotherapy, which indicates that an alternative cell death pathway is activated due to elevated PAR levels and caspase inhibition. AIF silencing in these cells led to profound protection from chemotherapy, which demonstrates that the increased cell death after PARG silencing and caspase inhibition was mediated by AIF.ConclusionsThe results show a role for AIF in breast cancer cell death after chemotherapy, the ability of PAR to regulate caspase activity, and the ability of AIF to substitute as a primary mediator of breast cancer cell death in the absence of caspases. Thus, the induction of cell death by PAR/AIF may represent a novel strategy to optimize the eradication of breast tumors by activating an alternative cell death pathway.
Highlights
Cell death induced by poly(ADP-ribose) (PAR) and mediated by apoptosis-inducing factor (AIF) is well-characterized in models of ischemic tissue injury, but their roles in cancer cell death after chemotherapy are less understood
Nuclear AIF in AIF-silenced cells was significantly decreased. These results demonstrate that RNA interference (RNAi) silencing of AIF in breast adenocarcinoma cells leads to decreased AIF levels and decreased levels of nuclear AIF after treatment with a cytotoxic DNA-damaging agent
Since AIF-mediated necroptosis leads to the dual staining of cells with propidium iodide (PI)/annexin V [5], these results demonstrate that AIF is the primary mediator of cell death in PAR glycohydrolase (PARG)-silenced cells pretreated with Q-VD-OPh. These results demonstrate that AIF can substitute as the primary mediator of cell death in PARG-silenced MDA-MB-231 cells in the absence of caspases, and it potentially identifies a critical role for AIF in the death of breast adenocarcinoma cells after chemotherapy
Summary
Cell death induced by poly(ADP-ribose) (PAR) and mediated by apoptosis-inducing factor (AIF) is well-characterized in models of ischemic tissue injury, but their roles in cancer cell death after chemotherapy are less understood. The ability to induce alternative pathways of cell death in tumors after chemotherapy is potentially an innovative strategy to optimize cancer cell death. Such optimization could lead to the improved eradication of tumors or the successful chemotherapeutic treatment of cancer cells that evade apoptotic cell death. One controversial, yet potentially novel, cell death pathway is poly (ADP-ribose) (PAR)-dependent cell death mediated by apoptosis-inducing factor (AIF). Therapeutic doses of DNA-damaging chemotherapeutic agents activate the synthesis of PAR and leads to the translocation of AIF from the mitochondria to the nucleus [4]. AIF is a pro-cell death mediator for a unique programmed cell death pathway known as “necroptosis” [6]
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