| Backgroud and Objective:prostate cancer is a leading cause of cancer death in men, with an estimated 27,050 deaths in 2007, and 186,320 new cases will occur in the US during 2008. Although death rates have been declining since the early 1990s, it remains the second most common malignancy among American men. With advances in our molecular understanding of normal cell proliferation and differentiation plus the various mechanisms controlling the tumor generation, the manipulation of gene expressioin to treat diseases have moved from the realm of science fiction to a reality.Survivin, a new member of the inhibitor of apoptosis (IAP) protein family found in 1997 that is located on human chromosome 17q25, is expressed in the majority of human tumors as well as in human and mouse embryon tissues, but is barely detectable in terminally differentiated normal cells. This differential pattern of expression makes survivin a useful tool in cancer diagnosis and an attractive target for cancer therapy. Moreover, survivin is an anti-apoptosis gene that overexpressed in the majority of human tumors including prostate cancer. Reactivation and abnormal expression of survivin in prostate cancer decreases cell apoptosis, which is involved in the generation and progression of prostate cancer. Overexpression of survivin in prostate cancer is highly correlated with p53 gene mutation that reduce expression of wide type p53 or create mutant p53 protein..Prostate cancer results from accumulation of certain gene mutantions. For example, p53 mutations are acquired during the progression to cancer in more than half of all human tumors, including prostate cancer. It is now clear that wide type p53 has anti-proliferative and anti-transforming activity. Re-expression of wide type p53 in cancer cells, in which the endogenous p53 gene was deleted or mutated, restore a non-tumorigenic phenotype by suppressing tumor growth. Even though p53 is expressed at 40% of wild type levels in the tumors, the mutation of dysfunction of certain other oncogenes or regulators still result in tumor progression. The cell growth and cell death are usually determined by a balance between oncogenes and tumor suppressor genes. This precarious balance between oncogenes and tumor suppression factors may limit the solitary use of p53 gene-directed therapy. So it is valuable to investigate the anti-tumor effects and mechanism that wide tpye p53 combined with other gene for more insight into anti-tumor activity.RNA interference (RNAi) induced by small interfering RNA(siRNA) has recently emerged as a powerful technique that is capable of suppressing expression of individual genes with high specificity. In previous studies, we applied a DNA vector -based survivin-specific RNAi approach to knockdown survivin expression, which resulted a strong inhibition of prostate cancer cells growth. However, in mammalian cells, RNAi does not completely block gene expression, especially for abnormally high expressing genes. In order to develop a more effective therapeutic regimen, we designed a combination therapeutic stategy, in which two antitumor factors, siRNA-survivin and p53 protein-expressing elements, were constructed to co-express in the same plasmid, and to study the effects and mechanisms of the two factors on androgen independent prostate cancer.Methods:1. Detection expression levels of mutant p53(mt-p53) and survivin proteins by immunohistochemical staining in prostate cancer tissues and normal prostate tissues.2. Construction and identification of a recombinant plasmid which will express siRNA-survivin and p53 two antitumor factors:Amplified the sequence of p53 gene with Kozak sequence using pQE40-p53 plasmid as template by PCR. All designed elements that were confirmed by sequencing analyses. Clone constructed DNA fragment into pcDNA3.1(+) eukaryotic expression vector, designated as plasmid pcDNA3.1-p53. Amplified U6 promoter and the sequence of siRNA-survivin using pGCsiRNA-survivin plasmid as template by PCR, then cloned into pcDNA3.1-p53 vector to construct a plasmid pcDNA3.1-U6 si-survivin/p53, called Psp53, that can express both siRNA-survivin and p53 protein.3. Studies in vitro:The prostate cancer cell lines, PC-3 were transfected 48h or 72h with plasmids p53, si-survivin, or Psp53 to determine the biological behavior of recombinant plasmids. To observe the expression levels of the survivin siRNA and p53 after transfection, the semi-quantitative RT-PCR analysis, and Western blot analysis with samples extracted from transfected and control cells were performed. The cells were also analyzed for cell cycle phase distribution by flow cytometry, and the early apoptosis rates were observed by Annexin V-FITC assay and DAPI staining. MTT assay was used to detect the efficacy for inhibition of cell proliferation by different constructs. The distribution and level of survivin protein in cells were analyzed by immunofluresence.4. Studies in vivo:To study the effects of p53, si-survivin and Psp53 recombinant plasmids on prostate tumor growth in vivo, nude mice were injected with PC-3 cells to establish prostate cancer xenografts mouse model. When the tumor growth reached about 5 mm in diameter, the different constructs containing si-scramle,p53, si-survivin and Psp53, respectively, were injected into the tumor tissues directly. Penetration of the materials was enhanced by subsequent electroporation. The growth and final weight of the tumors will be measured and recorded. HE staining and TUNEL assay were used to detect the apoptosis of tumor cells. To study the expression levels of certain genes and proteins which could be involved (e.g. GRIM19, p14ARF, c-Myc, CDK4, E2F-1, eIF2-α), RT-PCR and Western blot analyses were performed.Results:1. Overexpressin of mt-p53 and survivin proteins in prostate cancer tissues.16 samples from normal prostate tissues and 20 samples from prostate cancer tissues were determined by immunohistochemical analyses. The results showed higher expression levels of mutant p53 and increased levels of survivin proteins in prostate cancer tissues compared with normal prostate tissues. With Spearman analysis,the expression of survivin was correlated with p53 protein(rs=0.514).2. The Psp53 recombinant plasmids containing both siRNA-survivin and p53 were successfully constructed, and confirmed by restriction enzyme digest and DNA sequence analysis. The method we used represents a significant improvement from the conventional method.3. The combinational treatment strategy demonstrated that knockdown of survivin gene function by specific siRNA with restoration of the wide type p53 treatment showed a strong efficacy for suppression of PC-3 tumor cells in vitro.MTT assay demonstrated that the Psp53 treatment group showed the most remarkable suppression effect on PC-3 cell growth, indicating a synergistic effect from the two antitumor factors. RT-PCR and Western blot analyses demonstrated that co-expressed plasmid could not only specifecally reduce the survivin expression level but also can increase wt-p53 expression. The data also showed that the expression levels of c-Myc and CDK4 were significantly depressed and GRIM19 and p14ARF levels were obviously enhanced due to the synergistic effect of Psp53. FCM analysis showed that a high percentage of cells were blocked at the checkpoint G1 in p53,si-survivin and Psp53 groups, and this were significant decrease in S phase cell from the Psp53 group. DAPI staining and Annexin V-FITC assay showed early apoptosis were happened in all the treated cells excepted two control groups. Immunofluresence staining showed that survivin protein expression level was significant higher in the nucleus than that in cytoplasm indicating the survivin protein might possibly be translocated from cytoplasm to nucleus.4. Examination of prostate cancer xenograft in nude mouse model showed a synergistic effect of Psp53 coinciding with the in vitro results.In the group treated with co-expressed p53 and siRNA survivin, both the average weights and volumes of the tumors were lower than those of other groups. Compared to two control groups, the tumor tissue cells from p53, si- survivin and Psp53 groups were induced apoptosis by using TUNEL staining, especially in Psp53 group. The expression levels of survivin, CDK4 ,E2F-1 proteins were significant decreased and GRIM-19, p14ARF were upregulated in co-expressed groups, all which displayed supressed growth of prostate cancer by phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2-α). The results indicated a synergistic effect for suppression of tumor growth by the wt-p53 and siRNA-survivin combination.Conclusion:The combinational treatment with a survivin-specific siRNA and p53 co-expressing plasmid showed a synergistic effect on suppression of prostate cancer cell growth in vitro and in vivo. The mechanism of the synergistic effect could be mediated through the key phosphorylation pathway of eIF2-α, which affected by c-Myc, CDK4, E2F-1, GRIM19, p14ARF and increases apoptosis, suppressing cell cycling and tumor growth. This study provides a feasible strategy that could improved cancer therappy efficacy by co-expressing two unique antitumor factors. |
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