The Construction Of A Bladder Cancer-specific Chimeric CAR Independent Oncolytic Adenovirus And Its Effect Against Cancer Cells

Objective:Bladder cancer is one of the most common malignancies in urinary system diseases,and along with the increase of air pollution and the number of smokers,the incidence of bladder cancer raise year by year.The general treatment of bladder cancer are surgery,radiotherapy,chemotherapy and perfusion therapy,but these methods still have shortcomings of the low long-term cure rate and high recurrence rate.In recent years,gene therapy has become an emerging treatment for bladder cancer,which construction the oncolytic adenovirus by carrying tumor suppressor gene to targeting bladder cancer cells is a new research direction.In most of the studies on the biological treatment of oncolytic adenovirus for bladder cancer,the often used biological carrier is adenovirus serotype 5(Ad5),which play the oncolytic action mainly through the identification of coxsackie adenovirus receptor(CAR)in the bladder cancer cell surface.However,for some poorly differentiated tumor cells,the expression of CAR in the cells surface is low,thus hindering the role of Ad5 against bladder cancer.Therefore,this study constructs a CAR-independent chimeric bladder cancer-specific oncolytic adenovirus Ad5/F11p-PSCAE-UPII-E1 A,which can enter the tumor cells by CD46 molecules broad spectrum expression in the bladder cancer cells surface to play the oncolytic action.Cisplatin is a more commonly used chemotherapeutic agent in the treatment of bladder cancer.So,we investigated whether Ad5/F11p-PSCAE-UPII-E1 A combined with cisplatin had synergistic antitumor effects through a series of in vitro experiments.The construction of chimeric oncolytic adenovirus and its combination with cisplatin provide a new idea for the gene therapy of bladder cancer and expand the field of bladder cancer treatment.Methods: The skeletal plasmid Ad5/F11 p,chimed the fiber tail domain of Ad5 and the fiber shaft and knob domains of Ad11 p,were cotransfected with the shuttle plasmid Rp-PSCAE-UPII-E1 A into Escherichia coli BJ5183 for homologous recombination to form recombinant plasmids pAd5/F11p-PSCAE-UPII-E1 A.And the recombinant plasmids were identified by the experimental method of restriction endonuclease and polymerase chain reaction(PCR).According to the pAdEasy-1 system,the recombinant plasmid pAd5/F11p-PSCAE-UPII-E1 A was packaged in HEK293 cells to obtain chimeric oncolytic adenovirus Ad5/F11p-PSCAE-UPII-E1 A.The chimeric oncolytic adenovirus Ad5/F11p-PSCAE-UPII-E1 A were identified by PCR method,and the correct virus were purified,amplified and measured titer.The newly constructed oncolytic adenovirus Ad5/F11p-PSCAE-UPII-E1 A was applied to 5637,EJ and T24 bladder cancer cells.The CCK-8,Quantitative real-time PCR,Western blot,RNA interference technique and flow cytometry were used to observe the anti-tumor effect of Ad5/F11p-PSCAE-UPII-E1 A on bladder cancer cells with different expression of CAR,and to explore the effect of Ad5/F11p-PSCAE-UPII-E1 A on the cell cycle.Finally,the anti-tumor effect of Ad5/F11p-PSCAE-UPII-E1 A combined with cisplatin on 5637,EJ and T24 bladder cancer cells was studied by in vitro experiments.The expression of Fas,Bax,Bcl-2 and cleaved Caspase 3 were measured to explore preliminarily the mechanism of Ad5/F11p-PSCAE-UPII-E1 A combined with cisplatin against bladder cancer.Results:(1)We constructed the correct recombinant plasmid pAd5/F11p-PSCAE-UPII-E1 A according to the homologous recombination between skeletal plasmid Ad5/F11 p and shuttle plasmid Rp-PSCAE-UPII-E1 A.And the CAR-independent chimeric oncolytic adenovirus Ad5/F11p-PSCAE-UPII-E1 A was packaged according to the pAdEasy-1 packaging system.After purification,amplification and determination,the harvested virus titer is 1 × 1010 PFU / ml.(2)The qRT-PCR showed that the expression of CAR in T24 cells was low,while 5637,EJ and T24 cells showed high expression of CD46.(3)Ad5/F11p-PSCAE-UPII-E1 A can enter 5637,EJ cells with high expression of CAR and T24 cells with low expression of CAR to replicate and play anti-tumor effect,and the killing effect is better than Ad5-PSCAE-UPII-E1 A.No killing effects were observed for the people normal urinary tract epithelial cell line SV-HUC-1.(4)The chimeric oncolytic adenovirus Ad5/F11p-PSCAE-UPII-E1 A enters into bladder cancer cells to replicate and play anti-tumor effect had no relevance with CAR expression.After infected with Ad5/F11p-PSCAE-UPII-E1 A,the expression of E1 A protein and the anti-proliferative effect were no difference between untreated with CAR-SiRNA cells(EJ,5637,T24)and treated with CAR-SiRNA cells(EJ-CAR-Si RNA,5637-CAR-Si RNA,T24-CAR-SiRNA).(5)For 5637,EJ and T24 cells,Ad5/F11p-PSCAE-UPII-E1 A can block most of the bladder cancer cell cycle in G1 phase.(6)There were synergistic anti-tumor effect between Ad5/F11p-PSCAE-UPII-E1 A and cisplatin on 5637,EJ and T24 bladder cancer cells.(7)Ad5/F11p-PSCAE-UPII-E1 A combined with cisplatin could induce the early apoptosis of bladder cancer cells,and the number of early apoptosis cells was higher than that of cisplatin alone group and Ad5/F11p-PSCAE-UPII-E1 A single group.(8)The combined effect between Ad5/F11p-PSCAE-UPII-E1 A and cisplatin can up-regulate the expression of Fas,Bax and cleaved Caspase 3 genes and down-regulate the expression of Bcl-2 gene for 5637,EJ and T24 bladder cancer cells.Conclusion: We constructed a CAR-independent chimeric bladder cancer-specific oncolytic adenovirus Ad5/F11p-PSCAE-UPII-E1 A,which can be used for the following research.The chimeric virus Ad5/F11p-PSCAE-UPII-E1 A can enter the 5637,EJ and T24 bladder cancer cells to play the anti-tumor effect,and the killing effect is better than Ad5-PSCAE-UPII-E1 A.Ad5/F11p-PSCAE-UPII-E1 A can block bladder cancer cell cycle in G1 phase.Ad5/F11p-PSCAE-UPII-E1 A combined with cisplatin had synergistic anti-tumor effect,and can induce apoptosis of bladder cancer cells by extrinsic and intrinsic apoptotic pathways.The construction of chimeric oncolytic adenovirus and its combination with cisplatin provide a new idea for the gene therapy of bladder cancer,and extend the field of bladder cancer treatment,which provides a theoretical basis for the next research.