Mutations in the TP53 gene, encoding the p53 tumor suppressor, are arguably the most common type of gene-specific alterations in human cancer. This emphasizes the pivotal role of p53 as a major mainstay of the body’s built-in anticancer defense mechanisms. As the field of p53 research evolves, it is increasingly evident that many p53 mutants not only lose their own tumor-suppressive functions and acquire dominant-negative activities over the remaining wild-type allele, but also gain new oncogenic properties that are not present in wild-type p53; this phenomenon is termed the ‘gain-of-function hypothesis’. For instance, overexpression of p53 mutants in cultured cells was shown to interfere with apoptosis, enhance proliferation, and increase resistance of the cells to chemotherapy [22–24]. Moreover, transfection of mutant p53 into TP53-null cells was shown to enhance their ability to form tumors in mice . Therefore, whereas wild-type p53 is a potent tumor suppressor, cancer-associated p53 mutants possess the attributes of oncogenes, suggesting that knockdown of mutant p53 may restrain or reverse the process of oncogenesis.
Recently, therapeutics based on RNA interference (RNAi) have become powerful and useful methods for the treatment of many diseases, including cancer, because of the high specificity, high efficacy and low toxicity of the RNAi trigger, small dsRNA [26, 27]. Stable or conditional knockdown of endogenous mutant p53 by siRNAs in various human cancer cell lines, such as lung, breast, and colon cancer cells, has been reported to reduce their growth rate and chemoresistance in vitro, and their ability to form tumors in nude mice [13, 14, 28]. A recent study also obtained consistent results showing that siRNA targeting mutant p53 could induce cell cycle arrest and apoptosis in human prostate cancer cells . In the current study, we used siRNAs that targeted p53 mutants to transfect human bladder cancer cell lines expressing only p53 mutants endogenously, and found that the transfection resulted in suppressed cell growth and viability via induction of cell cycle arrest and apoptotic cell death.
In numerous cancer types, mutations in TP53 are strongly associated with high expression levels of the proliferation-associated gene cluster, comprised mainly of the genes that participate in the core processes of the cell cycle . Moreover, several p53 mutants were recently shown to repress wild-type p53 target genes, which encode pivotal cell cycle inhibitors (such as p21 and GADD45α (growth arrest and DNA-damage-inducible 45α)), leading to alteration of cyclins/CDKs and an increased proliferation rate . Whereas overcoming the G2/M checkpoint to initiate mitosis requires cyclin B/CDK1 to be activated, cyclin A seems to be required for both S-phase and M-phase . Cyclin A/CDK2 drives G2-phase cells into mitosis, and is a rate-limiting component required for entry into mitosis . Our analysis of cell cycle-related proteins showed that in the siP53-treated cells, there was significantly decreased expression of both cyclin B1 and cyclin A and reduced phosphorylation of CDK1, supporting the induction of G2 phase arrest detected by flow cytometry. Meanwhile, our study also showed activation of caspase-9 and caspase-3 and proteolytic cleavage of PARP in these cells, which play central roles during cell apoptosis [34, 35].
Over the past two decades, cisplatin-based combination chemotherapy regimens, such as CMV (cisplatin, methotrexate, and vinblastin) or M-VAC (methotrexate, vinblastin, doxorubicin, and cisplatin) have been mainly used for patients with advanced bladder cancer [36–39]. However, because of their severe systemic toxicity and the poor overall prognosis of patients, novel therapeutic schemes containing different drug cocktails have been developed, with cisplatin occupying a central position in theseregimen (for example the GC (gemcitabine + cisplatin) regimen) . It is generally accepted that DNA is the preferential and cytotoxic target for cisplatin [41–43]. Cisplatin-mediated damage of genomic DNA causes severe cell cycle perturbation and arrest at certain checkpoints, and in the absence of adequate repair, the affected cells undergo cell apoptosis. There has been controversy about the influence of mutant p53 on the responsiveness to the cisplatin-based systemic chemotherapy in bladder cancer . Several studies have reported that patients with p53-altered bladder cancer benefited from adjuvant chemotherapy , whereas wild-type p53 was related to a poor response to systemic cisplatin-based chemotherapy , which might be related to the protection of cells from DNA damage by wild-type p53. However, other studies showed that mutations in TP53 were associated with drug resistance in several malignancies and cell lines [22, 46], which might be partially attributable to transcriptional activation of the multi-drug resistance 1 (MDR1) gene [47, 48] and interference with apoptosis  by mutant p53. Therefore, targeting mutant p53 may sensitize the cancer cells to chemotherapy in at least some bladder cancers. In the current study, we found that knockdown of mutant p53 by siRNA in 5637 and T24 bladder cancer cells could co-operate with cisplatin and enhance its anticancer effects additively via increased cell apoptosis.