| Prostate cancer (PCa) is a global health problem and has become the second most common non-cutaneous cancer in men, accounting for10%of male cancers. In recent years,5-year survival rate of patients with PCa has been ranked at the third highest of all cancers. An efficient therapy for PCa remains an urgent need. Results from two recent randomized clinical trials indicated that docetaxel-based chemotherapy improved the survival of inpatients with hormone-refractory prostate cancer (HRPC). Taxane-based therapy was also a promising approach to treat PCa with and without radiation therapy. In addition, adding carboplatin to docetaxel was found to be well tolerated, resulting in additional therapeutic effect in men with HRPC who developed progressive disease during or shortly after treatment with docetaxel. These studies provided further evidence that platinum salts may have a role in the treatment of this disease.However, cisplatin (DDP)-based regimens often causes severe toxic side-effects such as myelosuppression, asthenia and gastrointestinal disorder, as well as long-term cardiac, renal and neurological consequences. These adverse events usually cause drug discontinuation and limit therapeutic efficacy. One promising strategy to reduce such toxicity of DDP, but maintain its therapeutic effect, is the combination of low-dose conventional chemotherapeutic drugs such as DDP with other approaches that are likely to increase cellular chemo-sensitivity.Cancer gene therapy is one of these approaches and offers numerous unique opportunities. It is well-known that cell growth and death are determined usually by a balance between oncogenes and tumor suppressor genes. P53plays a regulatory role in the development and homeostasis of cells and tissues. Inactivation of P53caused by its negative regulators, such as Murine Double Minute2(mdm2), can contribute to the development of a large number of human cancers. mdm2is an E3ubiquitin ligase that directly binds to p53for its ubiquitylation. Tumors with over expression of mdm2were resistant to p53gene therapy. Reportedly PCa with mdm2knockdown were sensitive to radiation therapy both in vitro and in vivo. Therefore, the anti-tumor strategy to interfere with the p53-mdm2feedback loop holds a promise to restore p53tumor suppressing pathway. Therefore, to directly down-regulate mdm2expression by its specific siRNA in the tumor cells with forcing-expression of exogenous p53gene may be an effective approach to treatment of PCa. In our previous experiments, we demonstrated that knockdown of MDM2expression by its specific siRNA with the overexpression of wild-type (WT) p53gene offered a synergistic inhibition of PCa cell growth in vitro and in vivo.Choosing an efficient gene delivery system has been a major concern for gene therapy. The delivery system could selectively target tumors, deliver the tumor suppressor genes into tumors without damage to normal tissue. Attenuated Salmonella typhimurium, a facultative anaerobe, offers a particular promise as an anticancer vector over other vectors currently used. Salmonella typhimurium can preferentially amplify and reside within solid tumors (1,000fold greater in tumors than in normal tissues) with a minimal toxicity to normal tissues. Recently, these bacteria have been engineered to express numerous genes, including siRNA, including multiple drug resistance1(MDR1) and STAT3siRNAs and interleukin-2and interleukin-12. For instance, we have demonstrated that the attenuated S. typhimurium carrying STAT3-specific siRNA provided a significantly suppressive effect on PCa cell growth. In previous study, we have co-transfected mdm2-siRNA to directly down-regulate MDM2expression and human WT p53gene in PCa cells in vitro and in vivo; For the in vivo study, we have used S. typhimurium to carry various plasmids predominantly transfected into PCa cells in a nude mouse model of xenografts.In our previous studies, we have demonstrated that knockdown of mdm2expression by its specific siRNA with an overexpression of WT p53gene offered a synergistic inhibition of PCa cell growth in vitro and in vivo without any treatment. Here, we have further explored the use of the attenuated S. typhimurium to deliver the plasmid co-expressing mdm2siRNA and WT p53gene with and without low-dose DDP therapy to examine their antitumor effects in a PCa mouse model of xenografts. Our results showed that DDP combined with the attenuated Salmonella carrying plasmid co-expressing mdm2siRNA and WT p53have the optimally therapeutic effect on the PCa as compared to DDP alone in nude model of xenografts. Mechanistic studies showed that the optimally therapeutic effect was associated with the optimal induction of apoptotic cell death in the tumors through increasing expression of cleaved caspase3, caspase9, and bax/bcl2ratio in the combination of DDP with mdm2siRNA/WT p53gene. More importantly there was no apparent toxic side-effect in the animals treated with the combination.Objective:To observe the low-dose cisplatin combined with attenuated Salmonella typhimurium co-expression plasmid mdm2-siRNA and p53effect of combination therapy of prostate carcinoma transplanted subcutaneously in nude mice, explore cisplatin synergy and common expression plasmid and anti-tumor mechanism for prostate cancer clinical treatment provided new experimental evidence.Methods:1. Planking method detected Salmonella in the tumor and organ distribution.2. HE staining of:detecting the morphology of tumor cells in each group of tumor tissue sections. 3. In Vivo studies:Copy subcutaneous xenograft model of prostate cancer in nude mice were divided into six groups, attenuated Salmonella nude mice injected into the tail vein injection, intraperitoneal injection, cisplatin observed antitumor effect. Immunohistochemical detection of mdm2, PCNA protein expression; RT-PCR and Western blot analysis of the expression of tumor related genes and proteins (p53, mdm2, bax, bcl2, caspase3, caspase9).4. TUNEL assay of tumor cell apoptosis.Results:1.72hours and10days after the intravenous injection of attenuated Salmonella, At72h, many bacterial distribution in the tumor tissue and the liver, spleen, lung, kidney. At10days, many colonial distributed only in the tumor tissue, we almostly cannot see the colony growth in liver, spleen and other organs. This shows that Salmonella tumor targeting.2. Compared with the control group, the Pmp53group, DDP group, DDP+si-scramble group and DDP Pmp53group showed varying degrees of necrosis, apoptosis, and the extent of the most significant the DDP+Pmp53group, and we don’t find abviously side effects.3. Cisplatin combined the total expression plasmid Pmp53significantly inhibit the growth of the tumor, the tumor weight and volume of combined treatment group significantly reducing compared to the control group; Immunohistochemical detection of tumor tissue the mdm2, of PCNA of combined treatment group significantly reduce expression; by RT-PCR and Western blot test results, cisplatin plus Pmp53enhanced the expression of p53, bax, caspase3, caspase9and reduced the expression of mdm2and bcl2.4. TUNEL assay showed that combined treatment group increased significantly compared with the control in apoptotic cells.Conclusions:1. Attenuated Salmonella can carry the gene co-expression plasmid successfully reach the tumor cells.2. Cisplatin combined with attenuated Salmonella carrying co-expression plasmid p53and siRNA-mdm2inhibition of mdm2gene expression and an increase expression of p53in tumors in nude mice.3. Compared with monotherapy group, the combined treatment of cisplatin and Pmp53shows a stronger role in inhibiting the growth of prostate cancer, and we don’t find abviously side effects.4. The inhibitory mechanism of the combination therapy may be due to the synergistic effect of both in vivo and show a good therapeutic effect on prostate cancer, one of the mechanisms may be provided with p53to activate the expression of bax, bcl2expression is suppressed, lead bcl2/bax ratio decreased final activation caspase3, and promote apoptosis of tumor cells.These results suggest that cisplatin for p53/mdm2axis may be a new strategy for treatment of cancer. |
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