Study On The Influence Of Hydroxycamptothecin And Smac Gene Regulated By The Prostate Specific Antigen Promoter/Enhancer On Prostate Cancer Apoptosis

PART 1 Construction and Identification of Recombinant Plasmids with Prostate-Specific Antigen Enhancer and Promoter in Gene Expression RegulationOBJECTIVE: To study the expression efficiency and specificity of prostate specific antigen (PSA) enhancer/promoter in a possible targeted gene therapy scheme for prostate cancer.METHODS: Genomic DNA was obtained from prostate cancer tissues. PSA enhancer and promoter sequences were amplified using PCR method, then the two fragments were cloned into plasmid pEGFP-1 to construct the expression vectors. After transfecting into different cell lines, the expression status was observed in fluorecent microscopic examination method.RESULTS: The recombinant plasmids (pPSAE-EGFP, pPSAP-EGFP and pPSAE-PSAP-EGFP) were successfully constructed. In prostate cancer cell PC-3, pPSAE-PSAP-EGFP expressed more GFP than pPSAP-EGFP and the green fluorescence was also observed in pPSAE-EGFP, which showed that PSA enhancer not only could notably increase the transcription efficiency of PSA promoter but also has modulating ability singly used.CONCLUSION: The expression efficiency and specificity of gene could be inreased by PSA enhancer/promoter combination which should provide trial evidences for clinical gene therapy of prostate cancer. PART 2 Study of Hydroxycamptothecin Promoting Apoptosis of Prostate Cancer Cell Line PC-3OBJECTIVE: To observe the effects of hydroxycamptothecin (HCPT) on the apoptosis of prostate cancer cell line PC-3 and to explore the possible mechanisms.METHODS: The influence of different concentrations (1×10-1, 1×10-2, 1×10-3, 1×10-4 mg/ml) of HCPT on PC-3 cells proliferation at different time (12, 24, 48 h) was assayed respectively by tetrazolium (MTT) assay. The morphologic changes of apoptosis cells were observed by acridine orange/ethidium bromide dyeing. The DNA of apoptotic cells were analyzed with agarose gel electrophoresis. The apoptosis ratios of HCPT on prostate cacer cells were analyzed with flow cytometry (FCM).RESULTS: The growth of PC-3 was inhibited by HCPT in time and dose dependence. The values of IC50 were 6.50×10-2 mg/ml (12 h), 2.35×10-2 mg/ml (24h) and 5.31×10-3 mg/ml (48h) respectively. The typical apoptosis cells in fluorescence microscope showed budding phenomena and apoptotic body. And the DNA ladder was observed in ultraviolet light. FCM analysis exhibited that the apotosis ratios of PC-3 cells were distributed in peak shape which reached the maximal point (35.76 %) at 1×10-3 mg/ml.CONCLUTION: HCPT could suppress PC-3 cells proliferation significantly in different ways. However, the mechanism is not clear and need further studies. PART 3 Study on the Influence of Hydroxycamptothecin and Smac Gene Regulated by the Prostate Specific Antigen Promoter/Enhancer on PC-3OBJECTIVE: To construct an eukaryotic expression vector containing Smac gene controlled by human prostate-specific antigen enhancer/promoter and observe the effect of apoptosis induced by recombinant plasmid and low dose hydroxycamptothecin.METHODS: Construct the recombinant vector with molecular biology technique. After transfection into prostate cancer cell line PC-3 and adding low dose HCPT, the status of cells was observed. And then, the in vitro cellular growth activities were assayed by MTT colorimetry and the apoptosis was measured by flow cytometry. Cellular Smac gene, XIAP gene, and caspase-3 gene expression were determined by reverse transcription-polymerase chain reaction.RESULTS: The recombinant plasmid of pPSAE-PSAP-Smac was successfully constructed. And only the prostate cancer cell line PC-3 was suppressed after transfection with pPSAE-PSAP-Smac (P<0.05). RT-PCR results showed that Smac mRNA levels were significantly increased (P<0.05) and the expression of Smac regulated by PSA promoter/enhancer was higher comparing to the CMV promoter-driven control vectors (P<0.05). Moreover, we also found that the expression of XIAP in pPSAE-PSAP-Smac group and (HCPT+ pPSAE-PSAP-Smac) group were decreased 67.53% and 76.95% respectively comparing to normal group. However, caspase-3 mRNA levels were as much as 3.94 folds and 4.31 folds comparing to normal control. The growth inhibition rates of PC-3 cells treated with HCPT, pPSAE-PSAP-Smac and (HCPT+ pPSAE-PSAP-Smac) were 25.39%, 30.76% and 70.91% respectively. The cellular apoptosis rates in (HCPT+ pPSAE-PSAP-Smac) group were increased by 22.84 % and 20.02 % (P < 0.01) respectively compared with HCPT group and pPSAE-PSAP-Smac group.CONCLUTION: An expression vector containing Smac gene based on elements of the PSA gene regulatory sequences has been developed and shown to function in prostate cancer cell lines. The Smac can inhibit IAPs and enhance caspase activity. Even more, it also increases the apoptosis rate companied with low dose HCPT which provides a solid platform for prostate cancer therapy.