In the present study, we have shown that PRIMA-1 can sensitize chemoresistant ovarian cancer cells with p53 mutation to CDDP when Akt function is down-regulated. Our data also suggest that the action of PRIMA-1 is associated with p53 phosphorylation and activation. The mechanism by which PRIMA-1 sensitizes mutant p53 and induces apoptosis has not been well elucidated. Whether PRIMA-1 binds directly to mutant p53 or it acts through indirect mechanisms remains an opened question. p53 binds to a specific DNA sequence and transactivates target genes involved in the regulation of cell cycle arrest and apoptosis. Tumor cells containing wt-p53 are usually more chemosensitive than those bearing mutant p53. We have previously shown that while CDDP up-regulates p53 in CDDP-sensitive wt-p53 cells (OV2008), but not its resistant wt-p53 variant (C13*) in vitro, suggesting that regulation of p53 content/function by CDDP may be an important determinant of sensitivity .
PRIMA-1 synergizes with chemotherapeutic drugs to induce tumor cell apoptosis [12, 19–21]. PRIMA-1 restores wild-type confirmation to mutant p53 by binding to the core and induces apoptosis in human tumor cells. Whereas wt-p53 is rapidly degraded by MDM2 in normal cells, the mutant p53 protein fails to undergo degradation in tumor cells and accumulates extensively . Several studies have demonstrated that PRIMA-1 is able to restore the sequence-specific DNA-binding and to transactivate some mutant p53 proteins in vitro and to induce apoptosis in vivo [12, 23–25]. p53 activates many genes involved in cell cycle arrest and apoptosis, mainly through its transcription-dependent activity [26, 27]. It is essential that p53 reactivation in tumor cells trigger apoptosis rather than cell arrest, as the therapeutic goal is to kill the tumor cells. Heat shock protein 90 is a candidate target for p53 mutation reactivation by PRIMA-1 in breast cancer cells . Some data have indicated that treatment with PRIMA-1 leads to upregulation of at least some of p53 target genes; for example, Bax and Noxa but not c-Jun-NH2-kinase (JNK) signaling [29, 30]. On the other hand, Li et al. reported that JNK pathway plays an important role on PRIMA-1-induced apoptosis . Others have shown that PRIMA-1 is capable of inducing apoptosis in a transcription independent manner  or even mutant p53-independent . It has also been reported that PRIMA-1 induces activation of caspase-2, caspase-3 and caspase-9, consistent with induction of apoptosis via the mitochondrial pathway . Microarray analysis revealed that PRIMA-1 induces a limited set of genes in a mutant p53-dependent manner, followed by ER stress .
Akt activation promotes cell survival, suppresses apoptotic death and confers resistance of ovarian cancer cells to CDDP-induced apoptosis [2, 11, 35]. Over-expression/activation of the PI3K-Akt pathway is commonly observed in ovarian cancer [36, 37]. However, precisely how Akt controls p53 activation is still unclear. Activation of Akt promotes the entry of MDM2 into the nucleus and its interaction with the tumor suppressor protein p53. Binding of MDM2 to p53 inhibits the transcriptional activity of p53 and targets it for proteasomal degradation . We previously demonstrated that activated Akt is an important regulator of both X-linked inhibitor of apoptosis protein (Xiap) and p53 levels after CDDP challenge and that p53 mutational status is a determinant of Akt-mediated chemoresistance [2, 11]. Inhibition of Akt activity facilitated the CDDP-induced mitochondrial release and nuclear accumulation of apoptosis-inducing factor (AIF)-dependent, CDDP-induced apoptosis . Activation of Akt confers resistance by blocking p53-mediated transactivation and p53 phosphorylation . In the present study, suppression of Akt sensitized chemoresistant cells to CDDP in a p53-dependent manner, suggesting a functional link between Akt-mediated chemoresistance and p53.
Recent data demonstrated that p53 is essential for CDDP-induced apoptosis in human ovarian cancer cells, and that p53-mediated apoptosis is dependent on the phosphorylation of several N-terminal residues, including Ser15, Ser20 and Ser37 [40–42]. Our previous data suggested that this is mediated, at least in part, through the phosphorylation of p53 on Ser15 and Ser20 . As Ser15 phosphorylation affects p53 stability , phosphorylation of p53 at serine15 by PRIMA-1 seems to be involved in the reactivation of p53 transcriptional function. Mutation of Ser15 to Ala significantly attenuates p53-mediated apoptosis . Our data show that down-regulation of Akt sensitizes the cells to CDDP-induced apoptosis and p-p53 at Ser15 is associated in the PRIMA-1-CDDP interaction, suggesting that Ser15 phosphorylation is needed for its function and apoptosis. This also suggests that CDDP induces p53 phosphorylation on Ser15 residue, which absent in chemoresistant cells and is required for CDDP-induced apoptosis. Akt efficiently blocks this processes, thereby conferring resistance to CDDP-induced apoptosis. In addition, while Akt modulates other p53-dependent cellular events, including the down-regulation of FLIP, our evidence suggests that effective p53 phosphorylation and activation is essential for CDDP-induced apoptosis. Thus, it will be of interest to study the effects of chemotherapy on total and phospho-p53 in human ovarian tumors. Moreover, since Akt attenuates both processes, it is important to study the relationship between activation/overexpression of Akt in ovarian tumors and sensitivity to CDDP.
In summary, inhibition of Akt activity may represent a novel therapeutic approach to the combined treatment of PRIMA-1 and CDDP. Further examination of the role and regulation of Akt in the PRIMA-1-CDDP interaction in the ovarian tumour xenograft might provide novel insights into a possible new therapy for chemoresistant ovarian cancer.