Experimental Study On Double Targeted Therapy Of PAMAM-D Mediated Suicide Gene System In Prostate Cancer Cell Line PC-3

Objective:To investigate the role of folate-modified nonviral vector and PSMA enhancer and promoter targeting for prostate cancer cell line PC-3. To explore the effect of Cx43 gene and gemcitabine on bystander effect in HSV-TK/GCV suicide gene system. To establish a safe and effective system that can deliver and express nonviral vector and suicide gene.Methods:Recombinant plasmid was transfected into prostate cancer cell line PC-3 and HepG2,NIH3T3 with G5-PAMAM-D-fol and G5-PAMAM-D respectively. After extracting RNA of transfected cells, the mRNA expression of TK and Cx43 gene were detected by RT-PCR; after extracting protein of transfected cells, the protein expression of TK and Cx43 gene were detected by western blot. 24h after thansfection, the precursor ganciclovir was added to the transfected PC-3 cells in each group at final concentration 0.1,1,5,10,20,50,100,200,500μg/ml, gemcitabine was only added to group A and group C at concentration 500μmol/L. MTT assay was used to measure OD value and calculate cell inhibition rate in order to evaluate cell proliferation ability after 24 hours incubation. Cell apoptosis was detected by flow cytometry. PC-3 cells were subcutaneously inoculated into nude mice to establish tumor model. Tumors were injected intratumorally with different complexes.24 hours later, GCV (100mg/kg) was injected intraperitoneally once a day in five days. When GCV was injected in the first day, gemcitabine (20mg/kg) was injected in the first group and the third group. The above process was repeated three times in 18 days. The volume of tumors during therapy and the final weight of tumors were investigated.Results:RT-PCR and western blot showed that PC-3 cells express TK and Cx43 mRNA and protein, neither HepG2 cells nor NIH3T3 cells express TK and Cx43 gene. The result of MTT assay showed:OD value had no difference between group I and group J (P>0.05); OD value of group A-H was all significantly smaller than that of group I and J (P<0.05); in group G and H, OD value had no difference (P>0.05). At lower GCV concentration (0.1μg/ml and 1μg/ml), OD value had no difference between group A-F and group G and H (P>0.05). At higher GCV concentration, OD value of group A-F was significantly smaller than that of group G and H (P<0.05); Among group A-F, in comparison with OD value of every two groups, there were significant difference (P<0.05). The cell growth inhibition rate curve reflected:with the increasing of GCV concentration, cell growth inhibition rates in group A-F increased in different extent, the group A increased most obviously. However, the cell inhibition rates in group G and H were both at lower level, and that in group I was nearly zero. Cell apoptosis detection showed:the maximal apoptosis rate of group A was 12.51%(12.37±0.17), which was significantly higher than the other groups (P<0.05); the cell apoptosis rates in group B-F were significantly higher than those in last four groups (P<0.05). Among group B-F, compared with cell apoptosis rate of every two groups, there were significant difference (P<0.05); cell apoptosis rates had no difference between group G,H and group I,J (P>0.05), but the cell apoptosis rates of group G and H were higher than those of group I and J (P<0.05). Through measuring the volume of tumors and drawing tumor growth curve, we can find:in the first four groups, the volume of tumors during therapy was significantly smaller than other four groups (P<0.05). Especially in the first two treatment cycles, the tumor grew slowly and treatment effect decreased with the increasing of the volume of tumors. Among the first four groups, there were significant difference (P<0.05), the volume of tumors of the first group was the smallest. In the first four groups, the final weight of tumors was significantly decreased (P<0.05); and there was significant difference between every two groups (P<0.05), except for the twice group and the fourth group; in the last four groups, the final weight of tumors had no difference (P>0.05).Conclusion:Folate-modified nonviral vector and PSMA enhancer and promoter can target for prostate cancer cell line PC-3 at transfection and expression level, and suicide gene system can effectively kill PC-3 cells. Cx43 gene and gemcitabine can enhance bystander effect. It provides important experimental basis for prostate cancer gene therapy.