Molecular mechanisms of tumour suppression by the P53-stabilizing compound CP-31398

The p53 tumour suppressor protein is considered the guardian of the genome and has a number of biological functions including cell cycle arrest, DNA repair, and apoptosis in response to various stresses, such as ultraviolet irradiation, serum deprivation, and exposure to anticancer drugs. The p53 gene is the most commonly mutated gene in human malignancies. Mutation in p53 leads to loss of tumour suppressive functions or gain of oncogenic functions, and ultimately cancer development. Many strategies are being developed to restore wild-type p53 function in cells with altered p53. CP-31398, a synthetic compound, was recently found to stabilize wildtype p53. Furthermore, CP-31398 rescued mutant p53 to enhance its transcriptional activity in vitro and in vivo and suppressed tumour growth in mice. The objective of this study is to elucidate the molecular mechanisms mediated by CP-31398 under normal and stressed conditions. We first sought to determine the possible molecular mechanisms involved in CP-31398 suppression of tumour growth. Using two isogenic colon carcinoma cell lines with differing p53 status, we first confirmed the p53 stabilizing affect of CP-31398. We demonstrated that CP-31398 induced p53-dependent apoptosis and G2 arrest, as well as p53-independent Gl arrest. We found that CP-31398 upregulated transcription of the p53 downstream target gene, p21[sup Waf1/Cip1]. Furthermore, CP-31398 induced apoptosis through the mitochondrial/caspase-9 pathway. CP-31398 upregulated Bax and Bak mRNA and protein levels, altered mitochondrial membrane potential leading to the release of cytochrome c and activation of caspases-9 and -3. Since ultraviolet-B irradiation in the sunlight is the primary environmental cause for malignant melanoma and p53 plays a crucial role in apoptosis, we then set out to determine if CP-31398 can enhance ultraviolet-B-induced apoptosis. We found that CP-31398 enhances ultraviolet-B-induced apoptosis in a wild-type p53 melanoma cell line, MMRU, by stabilizing p53. The mitochondrial/caspase-9 pathway was found to be activated in CP-31398 enhancement of ultraviolet-B-induced apoptosis, via the upregulation of Bax, change in mitochondrial membrane potential, release of cytochrome c, and activation of caspase-9. Moreover, we found that CP-31398 enhanced cell death induced by ultraviolet-B in other wild-type (RPEP) and mutant p53 (PMWK) melanoma cell lines, but did not enhance cell death in the mutant melanoma cell line, SK-mel-110. Melanoma is a chemoresistant cancer and studies have shown that p53 mutational status is a determinant for melanoma chemosensitivity. Since CP-31398 was shown to be able to rescue mutant p53 functions, we treated melanoma cells with CP-31398 together with the topoisomerase I inhibitor camptothecin which was shown to induce p53-dependent apoptosis, to determine if CP-31398 would enhance melanoma chemosensitivity. We found that CP-31398 did not enhance cell death induced by chemotherapeutic drugs in a wild-type (MMRU) and a mutant (SK-mel-110) p53 melanoma cell line. Pre-treatment of melanoma cells with CP-31398 also failed to enhance cell death induced by camptothecin. The inability of CP-31398 to enhance camptothecin-induced cell death could be explained by the fact that camptothecin-induced p53. accumulation is not enhanced after CP-31398 and camptothecin combination treatment. Furthermore, we demonstrated that CP-31398 did not enhance the sensitivity of MMRU cells to other chemotherapeutic drugs, such as vincristine and cisplatin. In summary, we demonstrate that the p53-stabilizing compound CP-31398 is able to induce apoptosis via activating the mitochondrial/caspase-9 pathway. We hope that a better understanding of the molecular mechanisms of tumour suppression mediated by CP-31398 may lead to the establishment of this compound as a potential anticancer agent or the discovery of other similar synthetic compounds for cancer treatment.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat