1. Attenuated Salmonella Typhi Carrying Theplasmid-co-expressed GRIM-19 And Survivin-specific ShRNA For Prostate Cancer Therapy 2. Organic Selenium Inhibiting The Relapse Of Androgen Independence Prostate Cancer

1 Attenuated Salmonella typhi carrying theplasmid-co-expressed GRIM-19 and Survivin-specific shRNA for prostate cancer therapyProstate cancer is a common malignant tumor which is harmful to male of middle and old age. There is no ideal treatment except early prostate cancer could be effectively treated by radical operation. A high degree of malignant prostate cancer tends to micrometastasis earlier. So it is very significant to search the candidate which is no toxic but effective. RNA interference (RNA interference, RNAi) technology was a great discovery and had been used for the treatment of many diseases at the end of the 20th century in academic medicine. The main material of RNAi is the long double strand RNA (dsRNA), when it goes into the cell it is cleavaged into short dsRNAs with 21-23 nucleotides which are called siRNA (small interfering RNA also called short double-stranded RNA), siRNA is a sequence-specific post-transcriptional gene silencing mechanism, which is triggered by double-stranded RNA (dsRNA) and causes degradation of mRNA homologous in sequence to the dsRNA. This new approach has been successfully adopted to inhibit tumorigenicity. RNAi technology is currently being evaluated not only as an extremely powerful instrument for functional genomic analyses, but also as a potentially useful method to develop specific dsRNA based gene-silencing therapeutics.The target gene-specific siRNA preparation method:①Chemical synthesis: this type of siRNA goes into the cell directly and plays a key role but with high price, easily degradated and short half-life;②Preparation of siRNA recombinant plasmid: Endogenous delivery is possible by inserting DNA templates for siRNAs into RNA polymeraseⅢ(polⅢ) transcription units, which are based on the sequences of the natural transcription units of the small nuclear RNA U6 or the human RNase P RNA H1. the approach is available for expressing siRNAs with stability. With the development of the RNAi technology and deeper understanding of this field, a promising new modality of treatment appeared, which can be used in combination with the existing therapies for prostate cancer therapy.We based on the fact that the siRNA-Survivin had significant role in inhibiting the growth of prostate cancer. In order to improve its anti tumor effect we took the cell death regulator GRIM-19 (gene associated with retinoid-IFN-induced mortality19, to construct pGRIM-19-siRNA -Survivin co-expression plasmid and explore its synergistic anti-prostate cancer effect.Recombinant plasmid carrier research: RNAi technology drug is highly efficient, very small amounts of RNAi inducer could make specific target genes silence, reverse the malignant phenotype of tumor cells and induce apoptosis of tumor cells. However, the recombinant plasmid expressing siRNA can not penetrate cell membrane. The delivery of RNAi effector to target cells is one of the key factors determining therapeutic efficacy, because gene silencing is limited to cells reached by RNAi effectors. The biggest challenge is how to solve the carrier. In recent years, studies have demonstrated that attenuated Salmonella typhi has tropic-tumor activity. The preliminary research confirmed that the attenuated Salmonella typhi had gathered in solid tumor. Compared with normal tissue, the reproductive advantage was more than 1,000 times. In addition, it not only grew in severe hypoxia of solid tumors but also in the micrometastasis tumor, and had the effect of tumor lysis. In particular, attenuated Salmonella typhi (Ty21a) as an oral vaccine carrier have been used for 25-year in clinic. Therefore, this article selected attenuated Salmonella typhi as carrier for pGRIM-19-siRNA-Survivin recombinant plasmid for the delivery of anti-prostate cancer research.1.1 Objective:Attenuated Salmonella typhi carrying pGRIM-19-siRNA-Survivin co-expression plasmid as delivery vector for anti-prostate cancer research was applied. The aim is to explore the pGRIM-19-siRNA-Survivin expression plasmid whether or not had synergic effect in the role of anti-prostate cancer and confirm the feasibility of Attenuated Salmonella typhi as delivery for prostate cancer xenografts therapy.1.2 Methods: 1.2.1 pGRIM-19-siRNA-Survivin expression vector constructed successfully: As psi-Survivin plasmid as a template, PCR was applied for amplification of U6 promoter and the psi-Survivin sequence, then the PCR products were cloned into the pGRIM-19 eukaryotic expression vector to construct the co-expression pGRIM-19 -siRNA-Survivin plasmid which at the same time expresses si-Survivin and GRIM-19 gene1.2.2 Studies In vitro:Human androgen-independent DU145 prostate cancer cell lines were used for the project, recombinant plasmids were transfected into cells with lipofection. Cell proliferation was detected by MTT at different time; cell cycle and apoptosis were detected through flow cytometry (FCM), Annexin V-FITC kit, acridine orange staining and Tunel method. Survivin protein distribution with immunofluorescent staining ; the target gene and protein (Survivin, GRIM-19) and associated genes and protins (Stat3, c-Myc, CyclinD1, BcL-xL, caspase3 and VEGF) expressions by RT– PCR and Western blot1.2.3 Studies in vivo:In order to observe the anti-prostate cancer effect of the recombinant plasmids, we constructed nude prostate cancer xenografts hypodermically and injected Attenuated Salmonella typhi carrying different plasmids into peritoneal cavity to the tumor. We measured tumor volumes, compared tumor growth rates and tumor weights between groups at the same time. RT-PCR, Western blot and immunohistochemical staining were used to detect the target genes and proteins and associated genes and proteins (Stat3, c-Myc, cyclinD1, BcL-xL, caspase3 and VEGF); HE and TUNEL staining of tumor tissue for were used to detect the changes in morphology and apoptosis. Through the assays we evaluated the anti-prostate cancer effects and revealed the related mechanisms of the co-expression plasmid.1.3 Results:1.3.1 RT-PCR, immunohistochemistry and Western blot results showed that: gene and protein expressions of Survivin in prostate cancer and DU145 cells were significantly higher than that of normal prostate tissue, while the expressions of GRIM-19 significantly lower than normal prostate tissue.1.3.2 We successfully constructed the co-expression pGRIM-19-siRNA-Survivin plasmid identified by restriction enzyme digestion and sequencing which has two promoters and expresses si-RNA-Survivin and GRIM-19 at the same time.1.3.3 Results In vitro: the Survivin expression was dowenregulated in gene and protein level after transfection of psi-Survivin, pGRIM-19 and pGRIM-19- siRNA- Survivin plasmids, but GRIM-19 gene and protein levels in pGRIM-19 and pGRIM -19-siRNA-Survivin groups were upgegulated. proliferation inhibiting experiments showed that co-expression plasmid pGRIM-19-siRNA-Survivin inhibited cell growth better than single gene therapy group; FCM and Annexin V-FITC Detection: In psi-Survivin, pGRIM-19 and pGRIM-19-siRNA-Survivin groups, G1 phase arrest and apoptosis cells were observed, in co-expression pGRIM-19-siRNA-Survivin plasmid group there was more obvious G1 phase arrest cells and apoptosis cells; acridine orange and TUNEL staining found: co-expression plasmid could promote apoptosis significantly. immunofluorescence results found Survivin gathered in nucleus and the cytoplasm around in the transfected psi-Survivin, PGRIM-19 and pGRIM-19-siRNA-Survivin groups; RT-PCR and Western methods further revealed the pGRIM-19-si-Survivin expression plasmid could downgegulated Stat3, c-Myc, cyclinD1, BcL-xL expression while increased of the expression of caspase3.1.3.4 Results in vivo: In order to observe the anti-prostate cancer effects of pGRIM-19-si-Survivin recombinant plasmid in vivo, the subcutaneous xenografts were constructed in nude mice. The recombinant bacteria carrying various plasmids were injected into intraperitoneal cavity into the tumor. The results once again proved that si-Survivin and GRIM-19 could inhibit the prostate cancer xenografts growth with synergism. Compared with the two single gene therapy groups,the mean tumor weight and mean tumor volume were lower, the tumor cells occurred apoptosis obviously through HE and TUNEL staining in Attenuated Salmonella typhi carrying pGRIM-19-si- Survivin plasmid group. The total RNA and proteins were extracted and the related genes and proteins were detected by semi-quantitative RT-PCR and Western blot. The results found that the co-expression plasmid inhibited prostate xenografts growth by downregulating Stat3, c-myc, cyclin D1,BcL-xL and VEGF but upregulating caspase31.4 Conclusion:1.4.1 The co-expression pGRIM-19-si-Survivin plasmid showed synergic anti tumor effect not only for hormone-independent DU145 prostate cancer cells in vitro but also for the subcutaneous xenografrs of prostate cancer constructed with DU145 cells in vitro.1.4.2 The main anti-tumor mechanism of the co-expression pGRIM-19-si-Survivin plasmid was to promote prostate cancer cells apoptosis.1.4.3 The main barrier of gene therapy is that recombinant plasmid could not penetrate cell membrane by itself. Application of attenuated Salmonella typhi as the delivery of co-expression plasmid gene therapy vector has solved the problem.RNAi technology is a revolutionary event at the end of the 20th century. We applied Attenuated Salmonella typhi usually used with oral vaccine as carrier for co-expression pGRIM-19-si-Survivin plasmid for prostate cancer xenografts therapy and found that the co-expression had strong anti tumor effect whether in vitro or in vivo. The above results showed the advanced, feasible and original research work. 2. Organic selenium inhibiting the relapse of androgen independence prostate cancerProstate cancer is a leading cause of cancer-related deaths in the Western countries, and in China, the incidence and mortality rates of prostate cancer have increased markedly because of aging population, lifestyle and dietary changing. Men currently diagnosed at the early stages of prostate cancer can, in many cases, be effectively treated by radiation, chemotherapy and endocrine therapy. However, in half of the patients, the disease will recur and ultimately develop into hormone-refractory prostate cancer and metastatic prostate cancers that are essentially incurable. New treatment of tumor therapy is thought highly of increasingly.Selenium (Se) is an essential trace element with important biological functions. Studies have shown that selenium compounds inhibit the growth of prostate cancer cells, including the androgen-dependent LNCaP line and the androgen-independent PC-3 and DU145 lines in vitro. The growth inhibition is mediated by apoptosis induction. Selenium was able to significantly down regulate prostate-specific antigen (PSA) transcript and protein expression in the androgen-responsive LNCaP cells. Selenium also suppressed the binding of AR to the androgen responsive element site. Selenium intervention strategy aimed at toning down the amplitude of androgen signaling could be helpful in controlling morbidity or delaying the genesis of androgen independent prostate cancer.2.1 Objective:To investigate the effects and relative mechanisms that organic seleum postponing androgen inednpence prostatecancer.2.2 Methods:2.2.1. study In vitroThe prostate cancer cell lines LNCaP was treated with different doses of MSA, to determine the expression levels of PSA and AR, the semi-quantitative RT-PCR and Western blot analysis with the samples extracted from treated and control cells were performed. The detection for the inhibition of cell proliferation by SRB; the LNCaP cells were analyzed for apoptosis rate and cellular cycles by flow cytometry, the effect of MSA on Stat3 by immunocytochemistry and immunofluorescence.2.2.2. study In vivo:To study the effects of selenium treatment on prostate tumor with castration in vivo, nude mice were imbeded with androgen-dependence prostate cancer LNCaP mass to establish prostate cancer xenografts subcutaneously. Once the tumors reached adequate volumes the mice were operated with castration. At the same time a dose of 0 or 100μg/20g/day by oral gavage for 16 weeks. Tumor measurements were taken once a week. The growth rate and final weight of the tumors were recorded and compared between groups. In addition, the morphology changing and apoptotic cells were determined by HE and TUNEL assays. Immunohistochemical staining for AR, Stat3 and Hif-1αwere performed to determine the distribution and levels. In addition, RT-PCR and Western blot analysis were used to detect the expression levels of related genes and proteins. PSA concentration in serum was detected through ELASIA assay.2.3 Results:2.3.1 Results in vitro: The results from semi-quantitative RT-PCR and Western blot analyses for the LNCaP cell samples treated with different doses of MSA could specifically reduce AR and PSA expressions. The results of SRB demonstrated that cell proliferation was inhibited with time and dose dependence. MSA could promote LNCaP cells apoptosis by FCM analysis, MSA also could doweregulate Stat3 expression by immunocytochemistry and immunofluorescence significantly.2.3.2 Results vivo: We copied the prostate cancer xenograft model with relapse after castration in vivo the results showed that in MSC group the average weights and volumes of the tumors were lower than controlled group. The tumor cells from MSC group had more significant apoptotic cells and lower proliferation rate. The expression of PSA, AR, Stat3 and Hif-1αwere down regulated by RT-PCR, Western and immunohistochemisty. ELASIA assay results showed that MSC could decrease the level of PSA in serum.2.4 Conclusions:MSA could inhibit androgen dependence prostate cancer LACaP cell proliferation and promote the cell apoptosis in vitro, mechanism might be related with downregulated AR expression. We constructed the relapse model of prostate cancer after castration in nude mouse successuly and found that organic seleum could postpone the relapse of androgen indepence prostate cancer induced by castration, the mechanism was related with that seleum inhibiting angiogenesis, reducing AR expression, inhibiting AR signal transduction and PSA secreation and downregulating HIF-1αand Stat3 expression.100μg/20g MSC didn't manifest obvious side effects through oral administration. The technology roadmap and experimental results have not been reported till now at home and abroad.