Antitumor Effect Of CXCR4Promoter Mediated Conditionally Replicative Adenovirus To Prostate Cancer

Prostate cancer is most frequently diagnosed in male in Western countries. The incidence of prostate cancer in our country is lower than in Western countries, but with the improvement of living standard and the changes of daily habit, the incidence of prostate cancer showed a rising trend, it has been becoming a major disease which affects the living quality and life expectancy of Chinese aged men. Despite the success of chemical or surgical castration for treatment of prostate cancer, androgen independence and resistance to existing treatments is a common feature of metastatic disease. There is currently no optimal treatment for decreasing disease progression in patients with metastatic disease. In recent years, with the progress of the molecular biology, gene therapy is a potentially viable approach for treating advanced prostate cancer. Now, more and more attention has been paid to how to make cancer management by specifically targeted therapy is not only effective but it can also avoid damage to healthy tissues, and still effective for advanced tumors and metastases.Recently, Conditional proliferation by tumor-specific and break tumor cells by oncolytic adenovirus cause great concern, there is big progress in oncolytic adenovirus for cancer therapy. Oncolytic virus not only replicate in tumor cells leading to cell break and death, but also by the infected cells to release more virus particles, resulting in a cascade amplification effect, until the tumor cells to be cleared. Now, we can use the advanced genetic engineering to give the oncolytic adenovirus effect more specificity and power. Oncolytic adenovirus can be driven by tumor-specific promoter for different tumor tissue, the oncolytic viruses will keep high activity only in specific tumor tissue. The chemokine receptor CXCR4, a member of the super family of G-protein coupled receptors, which encodes352amino acids. CXCR4expression is found indifferent tumors, but is undetectable in normal tissue. CXCR4promoter has been identified in renal cancer and glioma. Thus, the over expression of CXCR4gene indicate that they might be used as tumor-specific promoter in prostate cancer. The aim of our research work is to evaluate and compare the antitumor effect of CXCR4promoter mediated conditionally replicative adenovirus to prostate cancer cell line.Part1The different expression of CXCR4in prostate cancer cells and prostate epithelial cellsObjective:To study the expression of chemokine receptor CXCR4in prostate cancer cell line PC-3, LNCaP and prostate epithelial cell line RWPE-1, evaluate the different expression of CXCR4in prostate cancer cells and prostate epithelial cells.Methods:Cell line LNCaP, PC-3, RWPE-1was cultured regμlarly. Transcriptional expression of CXCR4in the LNCaP、PC-3, RWPE-1was examined by RT-PCR and post-transcriptional expression of CXCR4was examined by Western-blot.Results:Our results confirmed that prostate cancer cell line LNCaP and PC-3express cell-surface CXCR4highly compared with RWPE-1(P<0.01). In prostate cancer cells, LNCaP has highly expression level of CXCR4than PC-3cell line (P<0.05).Conclusion:There is strong expression of CXCR4in prostate cancer cells, indicating that CXCR4might be a tumor-specific expression protein.Part2Evaluation of CXCR4Promoter Transcriptional Activitie in Prostate Cancer CellsObjective:To identify and evaluate the transcriptional activities o f CXCR4promoter in prostate cancer cells.Methods:CXCR4gene promoter were amplified by PCR method and cloned into the plasmid pDsRed2-1and pGL4.17. Prostate cancer cell line LNCaP, PC-3 and prostate epithelial cell line RWPE-1were transfected by pDsRed2-CXCR4, pGL4.17-CXCR4respectively. The expression of DsRed2and luciferase were detected to evaluate the transcriptional activities of CXCR4promoter.Results:In the prostate cancer cell LNCaP, PC-3the CXCR4promoter has obvious promoting activity. The transcriptional activity of the LNCaP was higher than PC-3(P<0.05). The promoting activity of CXCR4promoter cannot be found in the prostate epithelial cell line RWPE-1almost. CXCR4promoter showed stronger activity in prostate cancer cell than prostate epithelial cell (P<0.001).Conclusion:The high transcriptional activities of CXCR4gene promoter in prostate cancer cell indicate its potential utility as novel candidates for transcriptional targeting of prostate cancer.Part3Specific Anti-tumor Effect of Ad-CXCR4-E1to Human Prostate Cancer CellObjective:Use conditionally replicative adenovirus Ad-CXCR4-El to infect human prostate cancer cell LNCaP, PC-3and normal human prostate epithelial cell line RWPE-1, evaluating the specific anti-tumor effect of Ad-CXCR4-E1to human prostate cancer cell.Methods:The El gene was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) from human embryonic kidney cells (HEK293). The conditionally replicative adenovirus Ad-CXCR4-E1containing CXCR4promotor was constructed and infected into LNCaP, PC-3and RWPE-1cells. The expression of El in LNCaP, PC-3and RWPE-1cells was detected by Western blot. Growth inhibition and oncolytic effect of cells were examined by cytopathic effect, trypan blue staining, MTT, crystal violet staining and Annexin V/PI flow cytometry.Results:The plasmid enzyme digestion and PCR results confirmed that the CXCR4promoter and El region gene was cloned successfully. The recombinant conditionally replicative adenovirus Ad-CXCR4-El was constructed successfully, which was confirmed by PCR. Western-blot showed that prostate cancer cells which infected with Ad-CXCR4-E1expressed high level of E1protein, but E1protein cannot be found in the prostate epithelial cells RWPE-1. cytopathic effect, trypan blue staining, MTT, crystal violet staining and flow cytometry revealed a stronger oncolytic effect on LNCaP and PC-3cells (P<0.05), but normal prostate epithelial cells RWPE-1without significant damage (P>0.05).Conclusion:A novel recombinant CXCR4-conditionally replicative oncolytic adenovirus is constructed successfully, Ad-CXCR4-E1make a specific oncolytic effect on prostate cancer cells LNCaP, but does not work in normal prostate epithelial cells RWPE-1. CXCR4-conditionally replicative oncolytic adenovirus provides us a new strategy for prostate target therapy.