Silencing of Apoptosis-Inducing factor and poly (ADP-ribose) glycohydrolase reveals novel roles in breast cancer cell death after chemotherapy

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.

Inhibition of poly(ADP-ribose) polymerase-1 or poly(ADP-ribose) glycohydrolase individually, but not in combination, leads to improved chemotherapeutic efficacy in HeLa cells

“…We previously demonstrated that the RNAi knockdown of PARG surprisingly led to the increased survival of MCF-7 breast adenocarcinoma cells in response to chemo-therapeutic treatments (45). The results here demonstrate the opposite (and desired effect) in HeLa cells, where we show that the RNAi silencing of PARG leads to increased HeLa cell death after chemotherapy.…”

“…The results here demonstrate the opposite (and desired effect) in HeLa cells, where we show that the RNAi silencing of PARG leads to increased HeLa cell death after chemotherapy. These differences in effects between the two cancer cell lines may reflect the ability of PARG silencing to induce alternative pathways of cell death in HeLa cells, since we previously demonstrated that the absence of PARG leads to decreased caspase activation and the activation of caspase-independent cell death (43,45). The differences may also be due to differential outcomes caused by the prolonged presence of PAR synthesized by other PARPs.…”

Poly(ADP-Ribosyl) Glycohydrolase Prevents the Accumulation of Unusual Replication Structures during Unperturbed S Phase

“…PARG has 4 different isoforms in the cells: 99-kDa and 102-kDa isoforms, which localize to the cytoplasm, a 110-kDa isoform, which localizes to the nucleus, and a 60-kDa isoform, which localizes to the mitochondria (15). PARG activity has been previously associated with the control of various cellular processes, including response to oxidative stress and apoptosis (16,17). Depleting all isoforms of PARG in mice results in embryonic lethality (18).…”

The Sound of Silence: RNAi in Poly (ADP-Ribose) Research

“…The delayed degradation of PAR molecules had a protective effect in particular when cells were challenged with the oxidant hydrogen peroxide (H 2 O 2 ). Recently, Feng et al confirmed this finding in human breast cancer cells treated with alkylating agents [164]. Interestingly, the investigation of PARG silencing in mammalian cells after genotoxic challenge has resulted in a hypersensitivity phenotype, as well [111,165,166].…”