Experimental Study Of Dual-regulated Oncolytic Adenovirus Carrying SPAG9-shRNA Combined With Docetaxel For Prostate Cancer

Part Ⅰ:To Construct the Oncolytic Adenovirus with Double Regulation and SPAG9 shRNAObjective: To construct oncolytic adenovirus DD3-ZD55-SPAG9 which has double regulate and load the sperm associated antigen 9(SPAG9)Short hairpin RNA(shRNA),and to verify the effect of regulating the expression of SPAG9 protein.Methods: 1.The vector plasmid p DD3-ZD55-SPAG9 shRNA carrying the DD3-ZD55 promoter and SPAG9 shRNA and the backbone plasmid p BHGE3(containing the adenoviral right arm sequence)were co-transfected into HEK-293 cell lines by Lipofectamine TM2000.When packaged into oncolytic adenovirus DD3-ZD55-SPAG9,it was verified by agarose gel electrophoresis.2.After correct amplification,the virus was amplified in large quantities and purified by Cs Cl density gradient centrifugation.Half of tissue culture infective dose(TCID50)method was used to determine the virus titer.3.Western Blot was used to detect the regulation effect of DD3-ZD55-SPAG9 on SPAG9 protein in PC-3 and DU145 cellsResults: 1.The oncolytic adenovirus DD3-ZD55-SPAG9 was successfully constructed.After amplification and purification,the virus titer of DD3-ZD55-SPAG9 was 9.46×108 pfu/ml.2.Western Blot results showed that DD3-ZD55-SPAG9 could effectively silence the expression of SPAG9 protein in PC-3 and DU145 cells,and the difference was statistically significant compared with the control group(p<0.05).Conclusion: The oncolytic adenovirus DD3-ZD55-SPAG9 was successfully constructed,and the expression of SPAG9 protein in PC-3 and DU145 cells can be effectively regulated.Part Ⅱ:DD3-ZD55-SPAG9 combined with Docetaxel for the treatment of castration-resistant prostate cancer in vitroObjective: To observe the therapeutic effect of DD3-ZD55-SPAG9 combined with Docetaxel(DTX)on castration-resistant prostate cancer in vitro.Methods: 1.Crystal violet staining and CCK-8 were used to observe the toxicity and proliferation inhibition of PC-3 and DU145 cell and normal prostate cell WPMY-1.2.Scratch assay and Transwell invasion assay were used to detect the inhibition of PC-3 and DU145 cell migration and invasion.3.Hoechst-33258 staining assay was used to detect the apoptosis of PC-3 and DU145 cells in each group.4.The expression of SPAG9,invasion and apoptosis related proteins in PC-3 and DU145 cells was detected by Western Blot assay.Results: 1.The results of crystal violet staining showed that the cytotoxicity of PC-3 and DU145 cells in each virus treatment group was time and titer dependent,while DTX was concentration and time dependent.After 48 hours treatment,high concentration of DTX(>4ng/ml),high titer ZD55-SPAG9(MOI>20)DD3-ZD55-SPAG9 had a cytotoxic effect on WPMY-1 cells,while DD3-ZD55-SPAG9 had a cytotoxic effect on MOI>50,suggesting that DD3-ZD55-Sp AG9 had higher safety than the control virus.2.CCK-8 results showed that with the prolongation of viral and drug action time,the inhibitory effect on the proliferation of prostate cancer cells was gradually enhanced,and with the increase of viral titer and drug concentration,the inhibitory effect on the proliferation of cells was also gradually enhanced.After 48 hours of infection,the proliferation inhibition of prostate cancer cells in the combination group was significantly higher than that in the monotherapy groups.After 48 hours,the proliferation of WPMY-1 cells in all treatment groups was not significantly different from that in the control group.3.Scratch experiment results showed that after 24 hours,the percentage of scratch healing area in PC-3 and DU145 cells in each treatment group was lower than that in the control group,and the scratch healing area in the combination group was significantly lower than that in the monotherapy group.4.Transwell experiment results showed that after 48 hours,the number of cells penetrating the compartment membrane in PC-3 and DU145 cells in each treatment group was lower than that in control group,and the number of cells penetrating the compartment membrane in the combination group was significantly lower than that in the single treatment group.5.Hoechst-33258 staining results showed that after 48 hours,the apoptosis rate of each treatment group was higher than that of the control group,and the apoptosis rate of the combined group was significantly higher than that of the single group.6.Western Blot results showed that the expression of SPAG9 protein in all treatment groups was lower than that in the PBS group,the expression of invasion-related protein E-cadherin was higher than that in the PBS group,the expression of Vimentin and MMP-2 was lower than that in the PBS group,and the expression of apoptotic proteins Caspase-3 and Caspase-8 were higher than that in the PBS group.The expression levels of SPAG9,Vimentin and MMP-2 in the combination group were significantly lower than those in the single treatment group,while the expression levels of E-cadherin,Caspase-3 and Caspase-8 were significantly higher than those in the single treatment group.Conclusion:DD3-ZD55-SPAG9 can effectively inhibit the proliferation,migration and invasion of prostate cancer cells in vitro,and can effectively induce the apoptosis of PC-3 and DU145,which is slightly lower than that of ZD55-SPAG9,but has a higher safety against WPMY-1 cells.One of the mechanisms of inhibiting the invasion ability of prostate cancer cells may be related to the up-regulation of E-cadherin and down-regulation of Vimentin and MMP-2 expression through the regulation of Epithelial-mesenchymal transition(EMT)process related proteins after the silencing of Spag9 gene expression.The apoptosis induction mechanism of prostate cancer cells may be realized by promoting endogenous apoptosis mediated by caspase8 and caspase3.Docetaxel can play a synergistic role and enhance the anti-tumor effect of DD3-ZD55-SPAG9.Part Ⅲ : Oncolytic adenovirus DD3-ZD55-SPAG9 combined with Docetaxel in the treatment of xenograftObjective:To observe the therapeutic effect of DD3-ZD55-SPAG9 combined with docetaxel on xenograft in nude mice.Methods: 1.To establish a mouse xenograft model of prostate cancer PC-3 cells.The effects of different treatment groups on the growth of the xenograft were monitored.Hematoxylin-eosin staining(HE)was used to detect tumor growth in each group.2.Td T-mediated d UTP nick end labeling(TUNEL)was used to detect apoptosis of transplanted tumor cells.3.Immunohistochemistry and Western Blot were used to detect the expression of invasion related proteins E-cadherin,Vimentin and MMP-2 in the xenograft cells of each group.Results: 1.The xenograft in nude mice was successfully constructed.Tumor growth rate in each treatment group was lower than that in the control group.HE results showed that there was apoptosis and necrosis of tumor cells in all treatment groups,especially in the combination group.2.TUNEL results showed that there was significant apoptosis of tumor cells in all treatment groups,and the apoptosis was more obvious in the combination treatment group than in the monotherapy group.3.Immunohistochemistry and Western Blot results showed that the protein expressions of SPAG9,Vimentin and MMP-2 were decreased in each treatment group,while the protein expression of E-cadherin was increased,and the expression regulation of the above proteins was more significant in the combination group.Conclusion: DD3-ZD55-Sp AG9 can effectively inhibit the growth of xenograft,promote the apoptosis of xenograft cells,inhibit the expression of Sp AG9,Vimentin and MMP-2 protein,and promote the expression of E-cadherin protein.But the effect was slightly lower than ZD55-SPAG9.The combination of DTX can achieve better therapeutic effect than monotherapy.