Experimental Study Of Oncolytic Herpes Simplex Virus G47δ Combined With Angiogenesis Inhibitor Or MMC On Urothelial Carcinoma Of Bladder

The third-generation oncolytic herpes simplex virus G47δ(G47δ) can kill cancer cells more stronger and faster than the second-generation oncolytic HSV G207,it has already been proved by results of experimental research.The growth of primary and metastatic cancers can be restrained by endogenous angiogenesis inhibitors with merits of little immunogenicity or adverse reactions.Mitomycin C(MMC) as an intravesical chemotherapy drug is widely used to treat urothelial carcinoma of bladder after transurethral resection of bladder tumor(TURBt).It is possible to exert drug synergism to combine oncolytic herpes simplex virus G47δwith angiogenesis inhibitor or MMC on urothelial carcinoma of bladder.Objective1.To investigate whether the Kringle 5(K5) gene could be cloned from the human bladder carcinoma tissue,and to identify biological activity of bacterial expressed recombinant K5 protein.2.To investigate the killing effect of G47δor pcDNA3.1/K5 on bladder cancer cells,and to evaluate the drug synergism of the combination of the two drugs.3.To investigate the drug synergism of G47δand MMC on killing bladder cancer cells.4.To investigate whether the Endostatin(ES) gene could be cloned from the human bladder carcinoma tissue,and the method of ES fused with K5 gene.5.To investigate the correlation of pathologic grade and clinical stage with expression level of VEGF(vascular endothelial growth factor),Angiostatin(AS), ES,K5 in serum,urine,or cancer tissue.Materials and Methods1.Cloning,optimal expression,purification and activity identification of K 5: Total RNA was extracted from human bladder carcinoma tissue,then the K5 cDNA was amplified by RT-PCR.A recombinant prokaryotic expression vector pGEX-5X-1/K5 was constructed and the K5 protein was expressed in engineering bacteria.K5 was purified by tagged GST agarose 4B affinity column chromatography,the anti-proliferative effects of K5 on ECV304 were examined by MTS assay. 2.Combining pcDNA3.1/K5 with G47δon killing bladder cancer cells:DNA were extracted from G47δand wild-type HSV-1,then the target fragments amplified by PCR were identified by agarose gel electrophoresis and gene sequencing technologies; Virus titer was determined by virus plaque test;A eukaryotic expression vector pcDNA3.1/K5 was constructed and pcDNA3.1/K5 was transferred into human urinary bladder carcinoma cell line BIU-87 or EJ by polyfect liposome,subsequently,the cells were infected with G47δ,then the anti-proliferative effects of pcDNA3.1/K5 and G47δon BIU-87 or EJ cell were examined by MTS assay;Recombinant plasmid of pEGFP-n2/K5 was constructed and the plasmid was transferred into cancer cells,and the cells were infected with G47δsubsequently,the location of K5 in cells was observed through fluorescence microscope;The effects of pcDNA3.1/K5 on replication of G47δwas assessed using virus plaque test;K5 mRNA was detected by RT-PCR after pcDNA3.1/K5 was transferred into cancer cells by PolyFect liposome.3.Combining MMC with G47δon killing bladder cancer cells:The cancer cells were infected with G47δ,1h later,MMC was added,the anti-proliferative effects of MMC and G47δon BIU-87 or EJ cells were examined by MTS assay,the combination index (CI)-isobologram of Chou-Talalay analysis was used to analyze virus and drug combinations;The anti-proliferative effects of wild-type HSV-1 and G47δon cancer cell were assessed by MTS assay;The effects of MMC on replication of G47δwas assessed using virus plaque test.4.Cloning of ES,ES fused with K5 gene and eukaryotic expression vector with ES-K5 was constructed:Total RNA was extracted from human bladder carcinoma tissue,then the ES cDNA was amplified by RT-PCR;The fusion gene of ES-K5 was artificially synthesized by splicing by overlap extension(SOE) method and inserted into eukaryotic expression vectors such as pcDNA3.1,pEGFP-n2,then the recombinant plasmids were identified by gene sequencing technologies.5.Detection of the expression level of VEGF,AS,ES,K5 in serum,urine,or cancer tissue:Expression level of VEGF,AS,ES,K5 in serum and urine were detected with ELISA,and expression level of them in cancer tissue was detected with IHC,then correlation of pathologic grade and clinical stage with the expression level of VEGF,AS, ES,K5 was analyzed.Results1.Cloning,optimal expression,purification and activity identification of K 5: The acquired gene was 282bp,and it was inserted into pGEX-5X-1 plasmid successfully; The optimal parameters of expressing K5 in prokaryotie expression system pGEX-5X-1 are: engineering bacteria for E.coli BL21,temperature for 37℃,concentration of IPTG for 1 mmol/L,time for 6h;K5 protein,molecular weight of 12 kD,was obtained after digested by thrombin;K5 protein showed effective in specifically inhibiting proliferation of ECV304 and its optimum concentration was 5μg/ml.2.Combining pcDNA3.1/K5 with G47δon killing bladder cancer cells:G47δvirus can incubate very rapidly in Vero cell;Agarose electrophoresis shows:the target fragment of G47δamplified by PCR was between 250bp and 500bp,but the target fragment of wild-type HSV-1 was between 500bp and 750bp,DNA sequences of two viruses were right by gene sequencing analysis,these indicated that the recombinant virus of G47δwas constructed successfully;The virus titer was 2.5×10~7 pfu/ml by virus plaque test;Both pcDNA3.1/K5 and G47δcould restrain the proliferation of BIU-87 and EJ cells, the inhibition effects were positively correlated with their action time and dosage,and combining pcDNA3.1/K5 with G47δcould cause the greater inhibition than one of them; G47δdid not disturb the location of K5 in cytoplasm and karyon;pcDNA3.1/K5 hardly disturbed on replication of G47δ,the number of plaque forming unit was same;K5 mRNA was between 250bp and 500bp,which was accorded with the molecular weight of K5.3.Combining MMC with G47δon killing bladder cancer cells:Both MMC and G47δcould restrain the proliferation of BIU-87 and EJ cells,the inhibition effects were positively correlated with their action time and dosage,and combination index<1 which indicated the drug synergism;G47δcaused the greater inhibition than wild-type HSV-1; MMC hardly disturbed on replication of G47δ,the number of plaque forming unit was same.4.Cloning of ES,ES fused with K5 gene and eukaryotie expression vector with ES-K5 was constructed:The acquired gene was 552bp,and it was inserted into pcDNA3.1 plasmid successfully;The fusion gene of ES-K5 was constructed successfully by the method of SOE,the linker was(Gly4Ser)3,and ES-K5 was inserted into pcDNA3.1 and pEGFP-n2 plasmids successfully.5.Detection of the expression level of VEGF,AS,ES,K5 in serum,urine,or cancer tissue:Expression level of VEGF,AS,ES,K5 in serum and urine were positively correlated with pathologic grade and clinical stage;expression level of them in cancer tissue was positively correlated with pathologic grade too.Conclusions1.Cloning,optimal expression,purification and activity identification of K5:1) K5 gene was cloned successfully from human bladder carcinoma tissue.2) The optimal parameters of expressing K5 in prokaryotic expression system pGEX-5X-1 are:engineering bacteria for E.coli BL21,temperature for 37℃, concentration of IPTG for 1 mmol/L,time for 6h. 3) K5 protein has a large expression level and easy to purify by way of E.coli containing recombinant plasmid pGEX-5X-1/K5.2.Combining pcDNA3.1/K5 with G47δon killing bladder cancer cells:1) pcDNA3.1/K5 and G47δcould restrain the proliferation of bladder cancer cell,the inhibition effects were positively correlated with their action time and dosage,and combining pcDNA3.1/K5 with G47δcaused the greater inhibition than one of them.2) pcDNA3.1/K5 hardly disturbed on replication of G47δ.3) pcDNA3.1/K5 can stably express in cells3.Combining MMC with G47δon killing bladder cancer cells:1) MMC and G47δcould restrain the proliferation of bladder cancer cells,the inhibition effects were positively correlated with their action time and dosage,and combination can produce synergistc effect.2) G47δcaused greater inhibition than HSV-1.3) MMC hardly disturbed on replication of G47δ.4.Cloning of ES,ES fused with K5 gene and eukaryotic expression vector with ES-K5 was constructed:1) ES gene was cloned successfully from human bladder carcinoma tissue.2) Fusion gene of ES-K5 could be inserted into eukaryotic expression vectors.5.Detection of the expression level of VEGF,AS,ES,K5 in serum,urine,or cancer tissue:1) Expression level of VEGF,AS,ES,K5 in bladder cancer was positively correlated with pathologic grade and clinical stage.2) Positive and negative regulation control angiogenesis,and the balance is broken in tumor angiogenesis,the possible reason was that it increased the expression and secretion of both angiogenesis factors and angiogenesis inhibitors,but biological activity of angiogenesis factors is stronger.3) The reason of a rapid progression of metastasis after surgical resection of the primary tumor is that the reduction of production and secretion of antimitotic factors by the cells of the primary tumor that inhibit the proliferation of metastatic cells,but it can not reasonably explain that expression level of angiogenesis inhibitor were positively correlated with pathologic grade and clinical stage.