Construction Of Recombinant Adeno-associated Virus Expressing Apoptin Providing Anti-tumor Effect To Human Bladder Cancer In Vitro And In Vivo

Objective1. To construct and prepare the recombinant adeno-associated virus (AAV) expressing apoptin and evaluate the biological activities in vivo and in vitro.2. To investigate a method of the preparation and purification of the apoptotic protein polyclonal antibody and to make an immunological evaluation of the effectiveness and specificity of the purified antibody.3. To analyze the characterization of rAAV-VP3to human bladder cancer cell lines and the potential mechanism of its anti-tumor effect.4. To assess and explore the mechanism and outcomes of gene therapy to Balb/c nude mice of human bladder cancer model by recombinant adeno-associated virus delivering VP3(rAAV-VP3). Methods1. In this study the AAV Helper-Free System was used to generate the recombinant adeno-associated virus expressing VP3gene. The VP3gene was cloned into the expression vector pAAV-MCS and the recombinant plasmid pAAV-VP3is confirmed by double enzymatic digestion using EcoR I and Sal I. The recombinant expression plasmid is co-transfected into the AAV-293cells with pHelper and pAAV-RC to produce recombinant AAV viral particles. The morphology of the virus particles were examined by electron microscopy. The physical titer of recombinant AAV was measured through Digoxigenin labeled CMV probe dot blot method, and the recombinant virus was verified by PCR of the exogenous interest genes of apoptin. RT-PCR and Western blot were performed to detect the transcription and expression of apoptin.2. VP3gene was cloned into the prokaryotic expression vector pET8a. The prokaryotic expression vector pET8a-VP3was employed to express VP3protein in BL21which was induced by IPTG, and the protein was extracted from the cell and purified. The purified VP3protein was applied to immunize the New Zealand rabbits combined with the Freund's complete adjuvant or incomplete adjuvant by multi-points subcutaneous injection. ELISA test was performed to measure the titer of the antibody after immunization. Two days after the measurement the whole blood was harvested by cardiac puncture and the serum was separated from the whole blood. The immunglobin IgG was purified by Protein a method from the antiserum.3. Concerning on the anti-tumor activity of rAAV-VP3in vitro, a variety of methods have been applied to the assays. Firstly, we observed the different MOI influence of rAAV-VP3to the growth of the bladder cell lines T-24and EJ, and determined the optimal infection MOI. Then, RT-PCR and Western Blot were performed to detect the expression of the Apoptin on mRNA level and protein level respectively. The anti-tumor effect of the virus was confirmed by employing transmission electron microscope and DNA ladder based method. To further investigate the apoptosis of the T-24and EJ cells after infection, we analyzed the cell cycle by using flow cytometry and apoptosis by Annexin V-FITC/PI double staining. Some bonus assays including the evaluation of the effect on expression of surviving and activation of caspase-3pathway have also been carried out by Western blot and Elisa.4. Gene therapy evaluation of the recombinant adeno-associated virus expressing VP3(rAAV-VP3) of the Balb/c nude mice model of human bladder cancer:We have established the Balb/c nude mice model of human bladder cancer. The animal models were divided into4groups, including negative control group, rAAV-eGFP treated group as control, MMC treated group and VP3infusion group. The dosage of the injection was1×1010v.g./50μl/mouse, and positive control was lmg/kg. The virus and drugs were delivered by multiple injection of the tumor cells. The anti-tumor effect of rAAV-VP3to Balb/c nude mice model of human bladder cancer was analyzed by growth curve of the tumor, calculation of the average tumor weight and histochemistry of the tumor section. The comparisons of the outcomes were performed by observation of the general condition and the survival ratio in each group. The staining of the histochemistry were used to detect the expression efficiency of the VP3in tumor cells after rAAV-VP3infection. The counting of the immuno-positive cells was also applied to compare the differential expression pattern of Ki-67,C-erbB-2,Rb,nm23between rAAV-VP3delivered group and control group.Results1. The expression vector pAAV-VP3was successfully constructed and the sequence of the VP3was confirmed by DNA sequencing. The rAAV-VP3were obtained by three plasmids co-transfection into AAV-293cells after72hours. The recombinant adeno-associated virus has a high titer of5.1×1011v.g./ml, and virus particles can be observed by electron microscope. By infection the Vero cells with rAAV-VP3, the transcription and expression of VP3gene was detected by RT-PCR and Western blotting.2. The immunglobin IgG was purified by Protein A method from the antiserum. The final purified antibody titers were up to1:243,000. The immunological evaluation of the polyclonal antibody specific binding was assayed by using recombinant adeno-associated virus rAAV-VP3infected different cell lines. First, it was used to detect the VP3gene expression by immunofluorescence in human bladder cancer cells T24and EJ, also in Vero cells. Apoptin was observed in T24, EJ cells, mainly located in the nucleus, whereas in Vero cells were localized in the cytoplasm. Secondly, the specific binding of the purified antibodies to VP3protein in different human bladder cancer cells were detected by Western blotting.3. The anti-tumor effect of the rAAV-VP3has been observed by MTT assay. We infected the T-24and EJ cell lines with the MO1:2.0×104v.g./cell,1.0×105v.g./cell,2.0×105v.g./cell,5.0×105v.g./cell and1.0×106v.g./cell respectively for72hours. Interestingly, the evidences indicated that the most significant anti-tumor effect was observed with MOI=5.0×105v.g./cell, and no further better effect could be produced with high MOI. The proliferation of the bladder cells T-24and EJ have been blocked after infection, and obvious results could be obtained as time went on. The values of OD490have increased by small magnitude and gradually decreased after60hours(P<0.01). Some classical morphology of apoptosis including the shrunk of nucleus and appearance of apoptotic body have been observed by transmission electron microscope and100bp DNA ladders have also been visualized in cells infected with rAAV-VP3. The transcription of expression of VP3could be detected by RT-PCR and Western Blot. We have found that the cell ratio of S phase decreased and G2/M was blocked compared with control group after rAAV-VP3infection(P<0.01). The FACS analysis also shown that the percentage of the apoptotic cells have increased after the infection through double staining Annexin V-FITC/PI. Moreover, significant inhibition of Survivin has been observed by VP3, and high expression level of Survivin were in control groups. Active form of caspase-3has been detected in T-24and EJ cells on3days,4days and5days after infection by Elisa based methods, and the significance has been validated by statistical analysis with the comparison of control groups(P<0.01).4. The adeno-associated virus rAAV-VP3could significantly inhibit the growth of the tumor in animal model. The inhibitory efficiency of the rAAV-VP3was coupled with high survival ratio, and the median survival time of55days. An increasing expression level trend of VP3could be detected after infection, and the maximum was attained at21days. The strong expression could be maintained for a long time. Importantly, the expression of VP3in vivo could dramatically downregulate the expression of Ki-67,C-erbB-2, and upregulate the expression of Rb,nm23in target cells.Conclusion1. The rAAV-VP3we have prepared have played good biological activities both in vitro and in vivo.2. The polyclonal antibody we produced has a strong sensitivity and specificity. The results indicated that the location of VP3in normal cell was cytoplasm, and in tumor cell was nucleus.3. The optimal MOI of rAAV-VP3to T24and EJ was5.0×105v.g./cell, and the growth of the bladder cancer cells were significantly inhibited, which mainly target at phase S and phase G2/M. The inhibition of phase G2/M could cause the inhibition of synthesis and degradation of DNA. The expression of VP3mediated by rAAV could downregulate the endogenous expression of Survivin. It has demonstrated that antagonism exists between Apoptin and Survivin, which may be one of the mechanisms that Apoptin could induce the apoptosis in tumor cells. Furthermore, the evidence we obtained indicated that caspase-3activation was one of the critical molecular signal in Apoptin-induced apoptosis pathway. 4. The delivery of VP3by using rAAV could significantly inhibit the growth of tumor, and reduce the invasion and formation of tumor embolus in the normal tissue nearby. The gene therapy by employing VP3could elevate the survival ratio of the nude mice, which was advantageous over anti-tumor drug MMC. The expression of VP3has indicated gradual increase trend in vivo. The mechanism of the anti-tumor effect may be related to the inhibition of the expression of Ki-67and C-erbB-2, and upregulation of the anti-tumor gene Rb, nm23.5. AAV-mediated VP3gene administration may be a new therapeutic technique for the treatment of bladder neoplasms. It will supply a foundation for further studies of anti-tumor effect of bladder cancer.