An Experimental Study Of The Cytotoxic Effects By PAMAM-D Mediated Dual-target Suicide Gene System On The Prostate Cancer Cell Line LNCaP

PartⅠConstruction and identification of prostate-specific recombinant plasmid pIRES-PSMAe/p-TK-Cx43 containing double genesObjective:To construct the prostate-specific recombinant plasmid pIRES-PSMAe/p-TK-Cx43 containning double genes and lay the foundation for experimental research of gene therapy for prostate cancer.Methods:First, Cx43 gene was amplificated and cloned into pMD19-T Simple plasmid;Second, HSV-TK gene was synthesized and cloned into multiple clone site(MCS) A of the eukaryotic plasmid pIRES.The new plamid was named pIRES-TK;Third, PSMAe/p was obtained and cloned into pIRES-TK by replacing CMV promoter. The new plamid was named pIRES-PSMAe/p-TK;Fourth, Cx43 gene was cloned into the MCS B of pIRES-PSMAe/p-TK and the new plamid was named pIRES-PSMAe/p-TK-Cx43.This plasmid was identified by double digestion with SalⅠ/NotⅠand sequenced;Finally, LNCaP cells were transfected by the plasmid pIRES-PSMAe/p-TK-Cx43 and the mRNAs expression of HSV-TK gene and Cx43 gene was observed by RT-PCR assay.Results:All plasmids synthesized in this part were double digested respectively and the specific bands of the inserted genes were observed by Agarose gel electrophoresis. pIRES-PSMAe/p-TK-Cx43 was in line with the expected design by DNA sequencing. The mRNAs of TK gene and Cx43 gene were successfully expressed by RT-PCR assay after the transfection in LNCaP cells by pIRES-PSMAe/p-TK-Cx43.Conclusion:Prostate-specific recombinant plasmid pIRES-PSMAe/p-TK-Cx43 containing HSV-TK gene and Cx43 gene was constructed successfully.PartⅡAn experimental study of the cytotoxic effects by PAMAM-D mediated dual-target suicide gene system on the prostate cancer cell line LNCaP:in vitro and in vivoObjective:To set up a novel delivery and expression system containing non-viral gene vector G5-PAMAM-D-fol and HSV-TK/GCV suicide gene, and to explore the cytotoxic effects on the prostate cancer cell line LNCaP by this system.Methods: (1) In vitro experiment:The recombinant plasmids (pIRES-PSMAe/p-TK-Cx43, pIRES-PSMAe/p-TK, pIRES-TK) constructed in part I were transfected into the prostate cancer cell line LNCaP by two kinds of non-viral gene vectors (G5-PAMAM-D-fol, G5-PAMAM-D).The cells were divided into 10 groups according to different disposals. The mRNAs expression of TK and Cx43 gene were detected by RT-PCR and the proteins expression of TK and Cx43 were detected by western blot.Then MTT assay was used to evaluate cell proliferation ability after 24 hours incubation. Cell apoptosis was detected by flow cytometry after the application of Annexin V-FITC staining.(2) Animal experiment of in situ gene therapy:The prostate cancer cell line LNCaP was subcutaneously inoculated into BABL/C node mice to establish tumor model.The mice were divided into 8 groups according to different disposals.Tumors were injected intratumorally with different complexes.The volume of tumors during the whole therapy and the final weight of tumors were investigated.The proteins expression of TK and Cx43 in tumor tissue were detected by western blot. Pathological examination was performed to the tumors.Results:(1)In vitro experiments:①RT-PCR and Western blot analysis showed that TK genes were expressed in Group A, B,C, D, E, F and Cx43 genes were expressed in Group A, B.But none of the two genes were expressed in group G, H, I, J.②The 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 low GCV concentration (0.1μg/ml and 1μg/ml),OD value had no difference between group A-F and group G or H (P>0.05).At high GCV concentration(>5μg/ml), 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 curve of cell growth inhibition rate reflected: with the increasing of GCV concentration, cell growth inhibition rates in group A-F increased in different extent and 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.③Cell apoptosis detection showed:the maximal apoptosis rate of group A was 15.02±0.39%, which was significantly higher than the other groups (P<0.05);the 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);the cell apoptosis rates in group B-F were significantly higher than those of 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).(2) Animal experiment of in situ gene therapy:In the first four groups(treatment groups), the volume of tumors during the whole therapy was significantly smaller than that of other four groups (control groups) (P<0.05). Especially in the first two treatment cycles, the tumor grew slowly. But the 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 group 1 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 group 2 and group 4; in the last four groups (control groups), the final weight of tumors had no difference (P>0.05).After the treatment, the protein of TK had been detected by Western blot in groupl,2,3,4 and the protein of Cx43 had been detected by Western blot in groupl,2.Pathological examination showed the first four groups had significant anti-tumor effect.Conclusion:G5-PAMAM-D-fol have high targeting to LNCaP cells and low cytotoxicity as a non-viral gene vector. The prostate cancer cell line LNCaP can be successfully transfected by recombinant plasmid pIRES-PSMAe/p-TK-Cx43.Both Cx43 gene and gemcitabine can enhance "bystander effect" of suicide gene. We already set up a novel delivery and expression system preliminarily, which containing non-viral gene vector G5-PAMAM-D-fol and HSV-TK/GCV suicide gene. The system is so safe and effective that it can improve the cytotoxic effects of HSV-TK/GCV suicide gene on the prostate cancer.