Study On Rectal Cancer Targeting Gene Therapy In Xenograft Model By Bifidobacterium Infantis Mediated Recombinant HSV TK

Objective: We aimed to study the mechanism of rectal cancer targetinggene therapy by Bifidobacterium infantis (BI) expressing herpes simplexvirus-thymidine kinase (HSV-TK) in combination with Ganciclovir (GCV).Methods: HSV-TK gene in plasmid pBEX-TK was identified by PCR andSDS-PGAE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).Then the plasmid was introduced into BI by electroporation to getgenetically engineered bacteria pBEX-TK/B. infantis (BI-TK). Colorectalcancer cell Colo320was inoculated in subcutaneous tissue of nude mice toestablish xenograft colorectal cancer model. The tumor tissue was injectedwith PBS, BI+GCV and BI-TK+GCV respectively. Nude mice wereeuthanized at48hours posttreatment. The tumors were striped from nudemice, fixed in10%formaldehyde solution and dealt with Dehydration,paraffin embedding, sectioning, routine HE staining. Immunohistochemicalstaining was applied by rabbit anti-human XIAP antibody and rabbitanti-human caspase-3antibody. Professional software (Image-Pro Plus5.1) was used to analyse IOD (Integrated Optical Density) of X-linked inhibitorof apoptosis protein XIAP and apoptosis protein marker caspase-3in tumortissue. The gray value of the expression of XIAP and caspase-3wasanalyzed in different groups.Results: HSV-TK gene was1100bp in shuttle plasmid pBEX-TK identifiedby PCR. The HSV-TK was successfully expressed in prokaryoticexpression system pET32a(+)/BL21(DE3) with60kD protein, pBEX-TKwas transformed into BI by electroporation and the recombinant wassignatured BI-TK. Nude mice subcutaneous colorectal cancer model wasconstructed successfully and growth curve of cancer volume was draw afterColo320was inoculated in subcutaneous tissue of nude mice, and thecancers was transplanted between nude mice successfully. HE stainingresults showed that cancer cells in BI+GCV treatment group andBI-TK+GCV treatment group were significantly less than in PBS treatmentgroup, and cancer cells in BI-TK+GCV treatment group were the least.IOD of XIAP and caspase-3in cancer cell area of immunohistochemicalpictures analysed by Image-Pro Plus5.1showed that the expression ofXIAP levels in BI+GCV treatment group and BI-TK+GCV treatment groupwere significantly lower than in PBS treatment group, and XIAP wasalmost undetected in BI-TK+GCV treatment group. The expression ofactive caspase-3levels in BI-TK+GCV treatment group were significantlyhigher than in other two groups, and the expression of active caspase-3 levels in BI+GCV treatment group was significantly higher than in PBStreatment group.Conclusion: B. infantis in combination with HSV-TK/GCV suicide genesystem could promote apoptosis of cancer cells, the mechanism of which isdown-regulate the expression of XIAP and up-regulate the expression ofactive caspase-3.