Anti Tumor Effect Of CD/TK Suicide Gene Driven By VEGFP On Colorectal Neoplasm In Vitro And In Vivo

Objectives: Gene therapy is one of the most effective methods for malignant neoplasms. However many problems such as low transduction efficiency, unsatisfactory antitumor, poor target work and unsafety still remain. In order to enhance antitumor effect and target, the recombinant adenovirus containing cytosine deaminase and type I herpes simplex virus thymidine kinase gene driven by vascular endothelial growth factor promoter (Ad-VEGFp-CDglyTK) was used as vector to infection colorectal cancer LOVO cell line in vitro and in vivo respectively. Then the prodrugs were gave to therapy on colorectal cancer cell line. A prior study was also carried out to evaluate the safety of the double suicide gene therapy system.Methods: (1) In vitro killing effect of the gene therapy system Ad-VEGFp-CDglyTK to colorectal cancer cell line: Recombinant adenovirus Ad-VEGFp-CDglyTK, Ad-CMVp-CDglyTK, Ad-CMVp-CD or Ad-CMVp-TK, which contain Green fluorescent protein (GFP) report gene, infected LOVO cells respectively. Then the LOVO cells were cultured with the prodrug 5-Fluorocytosine and Ganciclovir. The colony forming and survival rate of LOVO cells as well as bystander effect of the therapy system were investigated. The cellular morphology was also observed. (2) In vivo anticancer effect of Ad-VEGFp-CDglyTK oncolorectal cancer cells: The transplantation colorectal neoplasm of nude mice model was constructed. The recombinant adenovirus was multiply injected by microsyringe to animal model which transfect LOVO colorectal cancer cells. Thereafter, 5-Fluorocytosine and Ganciclovir were injected to peritoneal cavity of the animal model. The gravity and growth suppression rate of the solid tumor were calculated. The cellular morphology was also observed.Results: (1) Cellular colony formation rate: The cellular colony formation rate of Ad-VEGFp-CDglyTK, Ad-CMVp-CDglyTK, Ad-CMVp-TK or Ad-CMVp-CD in the treatment of LOVO cells were 15.58%, 17.67%, 22.67% and 24.33% respectively. The cellular colony formation rate of Ad-VEGFp-CDglyTK or Ad-CMVp-CDglyTK,was statistically much lower than that of Ad-CMVp-TK or Ad-CMVp-CD. (2)Cellular survival rate: The cellular survival rates of Ad-VEGFp-CDglyTK, Ad-CMVp-CDglyTK, Ad-CMVp-TK and Ad-CMVp-CD in the treatment of colorectal cancer cell line were 18.22%,18.22%,46.22%and 49.78% respectively. The cellular survival rates of Ad-VEGFp-CDglyTK or Ad-CMVp-CDglyTK, was statistically much lower than that of the others. (3) Bystander effect: Only 10% cell survived when the transfer gene colorectal cells of both single suicide gene groups account for 50%, however about 10% cell survived when the transfer gene colorectal cells of both double suicide gene groups account for 30%.The bystander effects from double suicide gene groups carrying CMV promoter was similar to that of double suicide gene groups carrying VEGF promoter. (4) Results from animal model: The heavy and suppression rate of tumor: The heavy and suppression rate of tumor from different groups varied, of which a 54.21±7.35mg of the lightest tumor heavy and a 89.18%the of highest tumor suppression rate were detected in Ad-VEGFp-CDglyTK group, a 94.50±7.05mg of tumor heavy and a 81.14% of tumor suppression rate were observed in Ad-CMVp-CDglyTK group, a206.7l±9.82mg of tumor heavy and a 58.74% of tumour suppression rate were observed in Ad-CMVp-TK group, a 185.61±9.30mg of tumor heavy and a 62.95% of tumor suppression rate were observed in Ad-CMVp-TK group respectively. (5) Morphological change: In vitro LOVO cells from Ad-VEGFp-CDglyTK group were damaged and drastic apoptosis was seen. By general pathological technique, it could be seen that the tumor cell growth was suppressed in vivo, especially strongest suppression rate in Ad-VEGFp-CDglyTK group. Under electroscope, tumor cells apoptosis from all groups transfect by recombinant adenovirus occurred while tumor cell apoptosis from all groups not transfect by recombinant adenovirus didn't occur. No pathological change of liver, kidney, lungs and heart of nude mice in therapy animals was presented.Conclusions: (1) Double suicide gene driven by VEGF promoter has an exactly evident killing effect to LOVO cell in vitro than that of single suicide gene driven by CMV promoter. (2) Double suicide gene driven by VEGF promoter has a evident antitumor effect on LOVO cell in vivo, which is more evident than those of single suicide gene therapy system and double suicide gene therapy system driven by CMV promoter. (3) The recombinant adenovirus of double suicide gene therapy system driven by VEGF promoter is avirulent in vivo in investigation.