| Wild-type p53(wtp53) is a major tumor suppressor whose function is critical for protection against cancer. It has been reported that wtp53can induce cell cycle arrest,DNA damage,angiogenesis and metastasis inhibition. Many human tumors carry missense mutations in the p53gene and the affected tumor cells accumulate excessive amounts of the mutant p53(mp53) protein. Various lines of evidence indicate that, in addition to abrogating the tumor suppressor functions of wtp53, the common types of cancer-associated p53mutations also endow the mutant protein with new activities that can contribute actively to various stages of tumor progression and to increased resistance to anticancer treatments. Collectively, these activities are referred to as mp53gain-of-function(mp53GOF). At present, the research on mp53becomes increasingly important as a valid target for inactivation by prospective anticancer therapies. An effective approach to explore mp53GOF, which has become feasible by the advent of siRNA technology, relies on knocking down endogenous mp53in tumor derived cells. It is suitable for monitoring the changes in cell phenotype while excluding dominant-negative effects over wtp53. However, the researches on its distinctive roles in the urogenital cancers are rare.This paper focused on the mp53GOF in prostatic cancer and bladder cancer. Chemotherapy was thought to have minimal clinical efficacy in men with metastatic, hormone-refractory prostate cancer and drug resistant bladder cancer. Therefore, novel therapeutic strategies for the treatment are urgently required. In this study, we knocked down the endogenous mp53in DU145human androgen-independent prostate cancer cell line and T24,5637bladder cancer cells with the siRNA technology, analyzed the outcomes of cells growth, viability, apoptosis and drug resistance, evaluates its therapeutic potential.The main investigations and results are as follows:1. Expressions of mp53mRNA and protein were down-regulated significantly in cell lines DU145,T24and5637after siRNA transfections which were detected by real-time RT-PCR and Western blotting. Knockdown of mp53inhibits cells growth and viability and this phenomenon was concentration and time-dependent demonstrated by MTT assay.2. The impact of silencing mutant p53on cell cycle and apoptosis was examined by flow-cytometric analysis of cells labeled by PI and annexin V, and the related proteins of cell cycle and apoptosis were detected by Western blotting. p53-siRNA induced the arrest of cell proliferation at the both G1and G2phase in DU145cells while induced G2-phase cell cycle arrest in5637and T24cells. The p53-siRNA could also induce remarkable apoptosis in these cell lines and it maybe occurs with the involvement of Bcl-2family pathway.3. Suppression of p53mutants by siRNA resulted in attenuation of PI3K/Akt pathway., which might be one of the mechanisms related to the effects of p53-siRNA.4. Silence of mp53by siRNA increased responsiveness to chemotherapeutic cisplatin in5637cells by MTT assay and flow-cytometric analysis. They exhibited a characteristic of synergic action to reduce the cells viability.In conclution, suppression of p53mutants by siRNA could be an effective and important technology in the treatment of urogenital cancers, especially for treating the hormone-refractory prostate cancer and the drug resistant bladder cancer. Meanwhile, our study also provided evidence and basis for its future clinical application in the therapeutics of other cancer with mutant p53. However, there are still a lot of problems need to be investigated and improved, including the exact mechanisms and in vivo therapeutic experiments. |
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