Attenuated Salmonella Carrying The Co-expressed SiRNA-VEGF And P53 Plasmid For Prostate Cancer Therapy In Vitro And In Vivo

Prostate cancer is harmful to the health of the elderly male ,it's the common malignant tumor. Incident rate is first, mortality is after lung cancer. In China , With the aging of population and the change of life style,the incidence rate of prostate cancer has fastly increasing.Although men had diagnosed at the early stages of prostate cancer, it is often detected in later period or after metastasis. The cancer will reaccur and finally transformate to hormone-refractory prostate cancer. Gene therapy as a novel treatment of cancer is being more and more attention.p53 gene is the most systematic study of tumor suppressor gene, which played an important role in regulating cell cycle and growth inducing apoptosis. p53 gene mutations is one of the important reason for cancer. Studies have shown that 50-60% of tumors with p53 gene mutation. Mt-p53 not only showed the role of loss control on the normal tumor suppressor, but also impetused normal cells to malignant .p53 mutation is closely related with prostate cancer metastasis, invasionand prognosis.Vascular Endothelial Growth Factor (VEGF) is an important factor to promote angiogenesis.It is play a very important role in the solid tumor formation of blood vessels.VEGF binds to receptors on endothelial cell membrane and through the relevant signal transduction and play functions, including angiogenesis, promoting endothelial cell proliferation, increase vascular permeability and anti-apoptosis. Many studies have confirmed that VEGF is closely related with tumor angiogenesis, tumor metastasis .Salmonella is an infectious pathogen. attenuated Salmonella typhimurium maintain invasion force while lower pathogenicity by genetic engineering .Salmonella can gather priority in tumor tissues.it can not live in larger and hypoxic tumor but also in smaller and oxygen tumor. With Attenuated Salmonella genetic engineering techniques to improve and perfect, as well as dosage and safety issues resolved, it will certainly become a good prospects for cancer gene therapy transporter. Objective:Observe treatment effect of siRNA-VEGF and p53 plasmid on prostate cancer cell line PC-3 in the combined and to attenuated Salmonella-carrier carrying siRNA-VEGF and p53 plasmid in gene therapy of prostate cancer xenografts in nude mice .the effect of high expression of p53 gene and VEGF gene silencing and mechanism of the synergy, provide new experimental evidence in gene therapy for prostate cancer.Methods:1. Immunohistochemical staining: to detect the expression of mt-p53 protein and VEGF in normal prostate tissue and prostate cancer tissue by using the immunohistochemical staining2. Construction and indentification of co-expressing p53 and siRNA-VEGF plasmid: according to human VEGF gene sequences from GenBank, synthesized oligonucleotides that encoding siRNA- VEGF,selected target site, then ligated into pGCsilencer siRNA expression vector ,finally construct pGCsiRNA-VEGF plasmid; PCR is used to amplified the sequence of siRNA-VEGF include U6 promoter ,Amplified the sequence of p53 gene from using pQE40-p53 plasmid by PCR, then cloned into pcDNA3.1vector to construct a plasmid pcDNA3.1-U6-si-VEGF-p53 (Pvp53) that co-expressed both p53 and siRNA- VEGF.3. Studies in vitro:The prostate cancer cell line PC-3, were transfected with recombinate plasmids siRNA-scramble,p53,siRNA-VEGFand Pvp53. MTT assay were used to detect the efficacy for inhibition of cell proliferation by different constructs after treated 24h,48h and 72h. Using AO/EB staining and Annexin V-FITC to detect apoptosis,using PI staining to detect cell cycle phase and apoptosis.Scrath test and Transwell test to detect migration and invasion. the RT-PCR analysis and Western blot analysis extracted from transfected and control cells were to observe the expression levels of related genes and proteins(p53,VEGF,Bax,B cl-2,Caspase3,HIF-1α,MMP2,MMP9).4. Studies in vivo: We copyed a nude mouse tumor xenograft model. Mice were via.s.c. with PC-3 cells into the right flank. S. typhimurium carried siRNA-scramble,siRNA-VEGF,p53and Pvp53 were via i.v. into the mice. Planking to detect Salmonella survival time, Using immunohistochemical staining to observe Caspase3,HIF1α. RT-PCR analysis and Western blot analysis were used to observe the expression levels of related genes and proteins(p53,VEGF,Bax,Bcl-2,Caspase3,HIF-1α,MMP2,MMP9).Results:1. Immunohistochemistry showed that the expression of mt-p53 and VEGF protein in prostate cancer was significantly higher than in normal prostate tissue.2.The Pvp53 co-expression plasmids containing both p53 and siRNA-VEGF,it was successfully constructed, and confirmed by restriction enzyme digestion and DNA sequencing .3. The combinational treatment method certificate that knockdown of VEGF gene function by siRNA and restoration of the wt- p53 treatment showed a strong effect for inhibiting proliferation , promoting apoptosis and inhibiting migration and invasion of PC-3 tumor cells in vitro experiment. MTT assay showed that the Pvp53 group showed the most remarkable suppression action on PC-3 cell growth, AO/EB staining ,PI staining and Annexin V-FITC showed Pvp53 promote apoptosis of PC-3 cells . Scrath test and Transwell test showed Pvp53 inhibited capacity of migration and invasion.RT-PCR and Western blot analysis showed that recombination plasmid not only specifically increase expression level of Bax and Caspase3 but also can reduce the expression of Bcl-2, MMP2,MMP9,HIF-1α4. Pvp53 plasmid carried by attenuated Salmonella inhibited tumor growth of prostate cancer in nude mice. the group which treated with Pvp53, both the average weights and volumes of the tumors were lower and smaller than those of other groups. Ceiling method to confirm the attenuated Salmonella can carrying the plasmid into the tumor cells .expression of HIF-1αdecreased and the expression of Caspase3 increased were found in the co-expression Pvp53 plasmid group by immunohistochemical staining; RT-PCR and Western blot results demonstrated that co-expression Pvp53 plasmid increased the expression of Bax and Caspase3 and inhibited Bcl-2, MMP2,MMP9,HIF-1αexpression by varying groups.Conclusion:1. The Pvp53 recombinant plasmids containing both p53 and siRNA-VEGF were successfully constructed.2. Co-expression plasmid Pvp53 showed a strong efficacy for inhibiting proliferation , promoting apoptosis and reduced prostate cancer cell migration and invasion in vitro and in vivo. experiments in vivo and in vitro have demonstrated p53 and si-VEGF expression were playing very good synergy.3.While the mechanism may be related to the exression of 53 activated Bax, suressed the exression of Bcl-2, resulting in Bcl-2/Bax ratio decreased, the final activation of Casase3, romote aotosis of tumor cells, on the other hand may be the exression of p53 inhibited HIF-1α, thereby inhibiting the transcrition of VEGF, then inhibit the exression of MMP2 and MMP9 , inhibited tumor cell migration and invasion .4. Salmonella can be used as transport plasmid vector, and Salmonella have good security. Injected through the tail vein of deep tumor gene therapy provides a feasible approach.