| Polyamines are known to be critically involved in cell growth and have been implicated in the process of cell transformation. On the other hand, the level of polyamine is high in cancer cell and tissues, and rapid tumor growth has been associated with remarkable elevation of polyamine biosynthesis and accumulation. Ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis, was found to increase in cancer cells especially prostate cancers. Some chemotherapeutic agents, such as DFMO, which aimed to inhibit the activity of ODC have appeared and taken on inhibitory effects on tumor growth in vitro and in vivo, though showing dose-limiting toxicity. Taken together, these findings suggest that ODC may provide an important target for the development agents that inhibit carcinogenesis and tumor growth.Prostate cancer is the most frequently diagnosed cancer in men. Metastatic prostate cancer is essentially resistant to systemic cytotoxic chemotherapy, while external beam and radioisotope radiotherapy offers only symptom palliation. Clearly the development of novel therapies, such as gene therapy, is a high priority. In addition, the human prostate is an accessory organ and is not required for potency or urinary continence. Some studies had proved that the prostate gland had almost the highest level of polyamines in the body and prostate carcinomas had even greater elevated polyamine levels. In our earlier studies on human prostate disease, we also proved that expression of ODC was greater in the benign prostatic hyperplasia (BPH)than in normal prostate tissues.Because Adenoviral vectors are among the most promising gene transfer vehicles for direct, in vivo gene therapy for the treatment of a diverse array of human disease. In this study, we constructed a replication-deficient recombinant adenovirus to efficiently deliver a 120bp antisense ODC which is complementary to initiation codon and tested the effect of antisense ODC on prostate cancer. The adenovirus carrying antisense ODC (rAd-ODC/Ex3as) was infected to prostate cancer cells PC-3 and LNCap. Expression of ODC and concentration of polyamines in cells were determined by Western blotting and HPLC. MTT assay was used to analysis the effect on cell growth. Cell cycle was evaluated by FCM and cellular invasion by Matrigeal invasion assay. The relationship of polyamine and CDKI-P21 was also assessed by Western blotting. A nude mouse xenograft model was used to examine tumorigenicity and the effect on the formed prostate tumors.Part 1 Construction and package of an antisense RNA recombinant adenovirus of the third extron in ODC geneMethods:Domain of human ODC cDNA was amplified by RT-PCR from prostate cancer tissues and it was cloned into shuttle vector pAdTrack-CMV by TA clone. Then it was linealized with Pme I and transformed into pAdeasy-1 cell. The identified recombinant adenovirus plasmid pAd-ODC/Ex3as was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. The recombinant adenovirus production was observed by fluorescent microscope and PCR technique was used to detect target gene.Results:A 120bp cDNA was amplified by RT-PCR from prostate cancer tissue and the sequencing result showed it was the domain of the third extron in ODC gene. There were some positive recombinant bacterial clones after transformation ofpAdeasy-1 cell with pAdTrack-CMV-0DC/Ex3as. Fluorescent microscope observation and PCR conformed that pAd-ODC/Ex3as could infect 293 cell and replicate in the cell.Conclusion:The antisense RNA recombinant adenovirus of ODC gene (rAd-ODC/Ex3as) has been constructed and packaged. It provides a sound foundation for further study.Part 2 Inhibition of Prostate Cancer Cells with Antisense RNA of Ornithine Decarboxylase GeneMethods:To study the effect of antisense RNA of the third extron of Ornithine decarboxylase on prostate cancer cells PC-3 and LNCap, both of the cells were infected with rAd-ODC/Ex3as. MTT assay was used to measure the infection rate and observe the growth inhibition, Western Blot technique and HPLC were used to observe the inhibition of ODC and polyamine levdl in infected tumor cells. Flow cytometry was used to analyze cell cycle progress and apoptosis and the relationship of polyamine and CDKi-P21 was measured by Western Blot. The malignant phenotype of PC-3 cells was assessed by Matrigeal invasion assay respectively.Results:Results showed that rAd-ODC/Ex3as could significantly inhibit the growth of PC-3 and LNCap cells and invasive ability of PC-3 by 50MOI and 25MOI without cell toxicity. Through Western Blot, it was found that 45% and 59% of ODC protein expression were inhibited in PC-3 and LNCap cells, and the decreased ODC expression could decrease the polyamine level and induce the overexpression of CDKi—p21. Cell flow cytometry analysis showed that rAd-ODC/Ex3as could lead the infected cell to arrest at Gl phase, but no effect to apoptosis. Matrigel invasionassay showed relatively low invasion.Conclusion:rAd-ODC/Ex3as could interfere with ODC expression and polyamine level, induce Gl arrest by p21 overexpression and suppress the proliferation and invasive ability of prostate cancer cells.Part 3 Inhibition of Prostate Cancer Cells with Antisense RNA of Ornithine Decarboxylase Gene In VivoMethods:Firstly, to detect the effect of the antisense ODC on prostate tumorigenicity: the PC-3 cells were infected with rAd-ODC/Ex3as at a MOI of 50 for 48 hours, harvested, washed three times with PBS, and resuspended in 1640 medium. The cell suspensions (2><106) in a total volume of lOOul were then injected subcutaneously into 6-week-old BALB/C nude male mice. Tumor volume was measured every week and calculated with the following formula: volume=MlxM2xM2><0.5236 (Ml, long axis; M2, short axis). Secondly, to examine the effect of the antisense ODC on the formed prostate tumor: the mouse PC-3 tumors were established by injecting 5><106 cells (in medium) s.c. into nude male mice. When tumors had reached 5~7mm in diameter, the mice were treated with the recombinant adenovirus every third day. Tumor size was measured twice a week.Results:Expression of antisense ODC inhibited the growth of PC-3 cells in vivo, as evidenced by the reduction in tumor incidence and tumor sizes, when compared with those of rAd-GFP treated or no virus-treated tumors. Mice that received rAd-ODC/Ex3 as-treated cells did not develop tumors during a 6-week observation period. The tumor growth rates, calculated by using an exponential curve, were 7.5 and 33.73mm3/day for the rAd-GFP-treated and no virus-treated tumors. AntisenseODC can also inhibit the growth of the formed prostate tumors.Conclusion:Antisense ODC plays a role in the prostate tumorigenicity, on the other hand it has inhibitory effect on the formed prostate tumors.SUMMARYExpression of ODC in PC-3 and LNCap cells were reduced to 45 and 59%, and three polyamines were also decreased by the rAd-ODC/Ex3as treatment. Consequently, cell growth was substantially inhibited and cell cycle arrested at Glphase. Matrigel invasion assay showed relatively low invasion. Marked suppression of tumor formation and tumor growth was observed in the xenograft model. This study suggests that rAd-ODC/Ex3as has the antitumor effect on the human prostate cancer cells.
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