Induction of p53-dependent apoptosis by prostaglandin A₂

Prostaglandin (PG) A₂, one of cyclopentenone PGs, is known to induce activation of apoptosis in various cancer cells. Although PGA₂ has been reported to cause activation of apoptosis by altering the expression of apoptosis-related genes, the role of p53, one of the most critical proapoptotic genes, on PGA₂-induced apoptosis has not been clarified yet. To address this issue, we compared the apoptosis in HCT116 p53 null cells (HCT116 p53-/-) to that in HCT116 cells containing the wild type p53 gene. Cell death induced by PGA₂ was associated with phosphorylation of histone H2A variant H2AX (H2AX), activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase 1 in HCT116 cells. Induction of apoptosis in PGA₂-treated cells was almost completely prevented by pretreatment with a pan-caspase inhibitor, z-VAD-Fmk, or an inhibitor of protein synthesis, cycloheximide. While PGA₂ induced apoptosis in HCT116 cells, phosphorylation of p53 and transcriptional induction of p53-target genes such as p21^WAF1, PUMA, BAX, NOXA, and DR5 occurred. Besides, pretreatment of pifithrin-α (PFT-α), a chemical inhibitor of p53’s transcriptional activity, interfered with the induction of apoptosis in PGA₂-treated HCT116 cells. Pretreatment of NU7441, a small molecule inhibitor of DNA-activated protein kinase (DNA-PK) suppressed PGA₂induced phosphorylation of p53 and apoptosis as well. Moreover, among target genes of p53, knockdown of DR5 expression by RNA interference, suppressed PGA₂-induced apoptosis. In the meanwhile, in HCT116 p53-/-cells, PGA₂ induced apoptosis in delayed time points and with less potency. Delayed apoptosis by PGA₂ in HCT116 p53-/-cells was also associated with phosphorylation of H2AX but was not inhibited by either PFT-ɑ or NU7441. Collectively, these results suggest the following. PGA₂ may induce p53-dependent apoptosis in which DNA-PK activates p53, and DR5, a transcriptional target of p53, plays a pivotal role in HCT116 cells. In contrast to apoptosis in HCT116 cells, PGA₂ may induce apoptosis in a fashion of less potency, which is independent of p53 and DNA-PK in HCT116 p53-/-cells.