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[ ABSTRACT] AIM:To construct a conditionally replicating adenovirus vector activated by CXCR4 promoter and to evaluate its ability of lysing the lung cancer cells specifically.METHODS:Human CXCR4-E1A gene amplified by PCR was cloned into the shuttle plasmid pDC316-GFP to construct the recombinant shuttle plasmid pDC316-CXCR4-GFP.The recombinat shuttle plasmid and adenovirus genomic plasmid pBHG-lox-E1, 3Cre were transfected into 293 cells to construct the recombinant adenovirus CRAd-CXCR4-GFP.PCR was used to detect the target gene fragments, and the viral titer was determined.A549 cells with the highest mRNA expression of CXCR4 were screened out from 5 kinds of lung cancer cell lines by real-time PCR.CXCR4 promoter activity and adenovirus replication numbers were detected in A549 cells after transfection of CRAd-CXCR4-GFP and Ad-NULL.CRAd-CXCR4-GFP and Ad-NULL were transfected into A549 cells and 16HBE cells, the apoptotic rates were detected by flow cytometry and the viability was analyzed by CCK-8 assay.RE-SULTS:The recombinant plasmid pDC316-CXCR4-GFP was constructed successfully.Green fluorescence was observed in 293 cells under fluorescent microscope after co-transfection of pDC316-CXCR4-GFP and pBHG-lox-E1, 3Cre at 11 d. Green fluorescence was observed in 293 cells after infection of amplified 3rd generational adenovirus.PCR showed that the purpose gene was successfully integrated in recombinant adenovirus genome.The virus in the supernatant reached a titer of 1 ×1013 PFU/L.The mRNA expression of E1A and E4 in the A549 cells after transfection of CRAd-CXCR4-GFP was markedly increased compared with Ad-NULL group.Compared with Ad-NULL group and empty control group, the apoptotic rate and the viability of A549 cells in CRAd-CXCR4-GFP group had no significant difference in the first 4 d, the apoptotic rate increased significantly at 5 d, and the cell viability declined significantly at 5 d, but the apoptotic rate and the viability of 16HBE cells in each group had no significant difference within 5 days.CONCLUSION: The conditionally replicating adenovirus vector CRAd-CXCR4-GFP has been successfully constructed, which has the ability of lysing lung cancer cells specifically.
ABSTRACT
Objective To investigate the effect of RNAi-mediated survivin gene with conditionally replicating adenovirus Ad-de-lE1b55KD-shRNA/survivin-EGFP silencing on propagation and apoptosis of colon carcinoma cell lines HT-29 .Methods Ad-de-lE1b55KD-shRNA/survivin-EGFP was transfected to transplantation tumor on athymic mouse ,replication defective adenovirus and liposome vector which was contained the same shRNA as Ad-delE1b55KD-shRNA/survivin-EGFP were as control one .Then the change of transplantation tumor on athymic mouse was tested ,and TUNEL was used to assay the apoptosis rate of transplantation tumor cells .Results After transfection of Ad-delE1b55KD-shRNA/survivin-EGFP ,the quality of transplantation tumor on athymic mouse were reduced and the apoptosis rate of transplantation tumor cells were higher than replication defective adenovirus and lipo -some vector which was contained the same shRNA (P<0 .05) .Conclusion The effect of Ad-delE1b55KD-shRNA/survivin-EGFP to colon carcinoma cell on propagation and apoptosis were higher than replication defective adenovirus and liposome vector .
ABSTRACT
Gene-modified replication-competent adenoviruses (Ads) are emerging as a promising new modality for the treatment of cancer. We have previously shown that E1B 19kDa and E1B 55kDa gene deleted Ad (Ad-deltaE1B19/55) exhibits improved tumor-specific replication and cell lysis, leading to potent anti-tumor effect. As an additional effort to increase cancer cell-selectivity of replicating adenovirus, we have first generated eleven E1A-mutant Ads (Ad-mt#1~#11) with deletion or substitution in retinoblastoma (Rb) binding sites of E1A. Of these viruses, Ad-mt#7 demonstrated significantly improved cytopathic effect (CPE) and viral replication in a cancer cell-specific manner. To further increase the cancer cell-specific killing effect of Ad-mt#7, both E1B 19kDa and E1B 55kDa genes were deleted, resulting in an Ad-deltaE1Bmt7. As assessed using CPE assay, MTT assay, and immunoblot analysis for Ad fiber expression, Ad-deltaE1Bmt7 exerted markedly enhanced cancer cell-specific killing effect as well as viral replication in comparison to either Ad-mt#7 or Ad-deltaE1B19/55. Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-deltaE1Bmt7. In summary, we have developed an oncolytic adenovirus with significantly improved therapeutic profiles for cancer treatment.
Subject(s)
Animals , Humans , Mice , Adenoviridae , Apoptosis , Binding Sites , Homicide , Mice, Nude , RetinoblastomaABSTRACT
Objective To construct the conditionally replicating adenovirus vector Ad-delE1b55kD-shRNA/Survivin-EGFP that can transfects into HT-29 effectually and selectively and contains shRNA targeting to human Survivin gene.Methods EGFP geoe and shRNA gene were inserted into pAd-delE1b55kD,then pAd-delE1b55kD-shRNA/Survivin-EGFP was obtained.The plasmid and pBHGE3 were cotransfected into HEK-293 cell to obtain the conditionally replicating adenovirus vector Ad-delE1b55kD-shRNA/Survivin-EGFP.Then DNA of the adenovirus vector was extracted and identified.The transfection efficiency of the vector to HT-29 and LO2 was detected.The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot.Results EGFP gene and shRNA gene targeting to human Survivin mRNA were inserted into conditionally replicating adenovirus vector successfully,proven by sequencing.The transfection efficiency to HT-29 of Ad-delE1b55kD-shRNA/Survivin-EGFP was significantly higher than replication defective adenovirus and liposome vector(P