2-Phenylethynesulfonamide (PES) uncovers a necrotic process regulated by oxidative stress and p53.

2-Phenylethynesulfonamide (PES) or pifithrin-µ is a promising anticancer agent with preferential toxicity for cancer cells. The type of cell death and the molecular cascades activated by this compound are controversial. Here, we demonstrate PES elicits a caspase- and BAX/BAK-independent non-necroptotic necrotic cell death, since it is not inhibited by necrostatin-1. This process is characterized by an early generation of reactive oxygen species (ROS) resulting in p53 up-regulation. Accordingly, thiolic antioxidants protect cells from PES-induced death. Furthermore, inhibiting the natural sources of glutathione with l-buthionine-sulfoximine (BSO) strongly cooperates with PES in triggering cytotoxicity. Genetically modified p53-null or p53 knocked-down cells show resistance to PES-driven necrosis. The predominant localization of p53 in chromatin-enriched fractions added to the up-regulation of the p53-responsive gene p21, strongly suggest the involvement of a transcription-dependent p53 program. On the other hand, we report an augmented production of ROS in p53-positive cells that, added to the increased p53 content in response to PES-elicited ROS, suggests that p53 and ROS are mutually regulated in response to PES. In sum, p53 up-regulation by ROS triggers a positive feedback loop responsible of further increasing ROS production and reinforcing PES-driven non-necroptotic necrosis.

Cell death induced by 2-phenylethynesulfonamide uncovers a pro-survival function of BAX.

PES (2-phenylethynesulfonamide) was initially identified as an inhibitor of p53 translocation to mitochondria and named Pifithrin-µ. Further studies showed that PES selectively killed tumour cells and was thus a promising anticancer agent. PES-induced cell death was characterised by a non-apoptotic, autophagosome-rich phenotype. We observed this phenotype via electron microscopy in wild type (wt) and double Bax-/- Bak-/- (DKO) mouse embryonic fibroblasts (MEFs) treated with PES. We excluded the involvement of effector caspases, BAX and BAK, in causing PES-triggered cell death. Therefore, apoptosis was ruled out as the lethal mode of action of PES. Surprisingly, MEFs containing BAX were significantly protected from PES treatments. BAX overexpression in Bax-/- MEFs confirmed this pro-survival effect. Moreover, this protective effect required the ability of BAX to localise to mitochondrial membranes. Conversely, mitochondrial fusion induced by treatment with Mdivi-1 conferred increased resistance to MEFs subjected to PES treatment. The involvement of BAX in the regulation of mitochondrial dynamics has been reported. We propose the promotion of mitochondrial fusion by BAX to be the pro-survival function attributed to BAX.