PO-091 Induction of anti-Fas regulates PUMA and MYC: P53-dependent apoptosis pathway in hepatocellular carcinoma cells

Abstract

IntroductionIncreased oxidative stress is a hallmark of cancer cells, which makes them more vulnerable to induction of reactive oxygen species (ROS). P53 plays a crucial role in sensing and removing oxidative damage to DNA, and inactivating mutations in the TP53 gene attenuate this function. In addition, it was shown that mutant p53 is able to suppress the function of major antioxidant factors. Therefore, mutant p53 renders cancer cells even more susceptible to the induction of oxidative stress. Besides p53, the poly (ADP-ribose) polymerase 1 (PARP-1) protein plays an important role in the repair of ROS-induced DNA-damage. This led us to explore the potential of combining oxidative stress induction with the targeted inhibition of the PARP-1 protein to selectively target mutant p53 NSCLC cancer cells.Material and methodsAPR-246 and Auranofin (inhibition glutathione (GSH) and/or thioredoxin reductase 1 (TrxR1)) and Olaparib (PARP-1 inhibitor) were used. The cytotoxicity (SRB-assay) of these compounds was determined in a panel of NSCLC cell lines with different p53 status, including isogenic cell lines (p53 shRNA-knockdown, p53 knock-in). Total GSH content (GSH/GSSG-GloTM) and ROS content (CellROX) were determined. N-acetyl-l-cysteine (NAC) was used as a potent ROS-scavenger. Induction of apoptosis/cell death was determined by the AnnV/PI assay (FC) or the Cytotox Reagent (IncuCyte). DNA-damage was assessed by g-H2AX foci (IF) and the Comet Assay. Synergism was determined using the Additive model.Results and discussionsP53Mut knock-down reduces the cytotoxic effect of APR-246, Auranofin and Olaparib, while p53Mut knock-in sensitised cells for all three compounds. APR-246/Olaparib treatment reduced GSH levels and increased ROS content, resulting in a strong accumulation of DNA-damage and synergistic induction of cell death. Co-treatment with NAC or p53-knockdown significantly reduced this cytotoxic response. Similar synergistic effects were observed for Auranofin/Olaparib treatment in several cell lines with clinically relevant p53 mutations.ConclusionMutant p53 protein expression renders NSCLC cells more susceptible to APR-246, Auranofin and Olaparib treatment. In addition, the combination of oxidative stress induction (APR-246, Auranofin) with PARP-1 inhibition (Olaparib) results in remarkable synergistic effects in the presence of mutant p53. Therefore, this combination strategy could be a promising and selective treatment option for mutant p53 NSCLC patients in which resistance to standard therapies often occurs.