Study On Gene Therapy For Human Colorectal Cancer By Herpes Simplex Virus Thymidine Kinase/gancyclovir System

Background: Human colorectal carcinoma (HCC) is one of the most lethal cancers in humans. The majority of patients died of local recurrence despite aggressive medical intervention including surgery, radiotherapy, chemotherapy. Recent attention has been focused on gene therapy of tumors. One particularly appealing approach called suicide gene therapy is to incorporate the viral gene which codes for the enzymethymidine kinase into the replicating cancer cells so that the viral-derived thymidine kinase can converts the pro-drug ganciclovir (GCV) to form a toxic anabolite, then the toxic anabolite will be selectively produced in the tumor cells to generate a selective antitumor effect. Objective: We studied whether GCV could induce apoptosis of HSVtk+/LoVo cells in vitro. In order to assess whether 5-FU may improve the efficiency of the HSVtkIGCV system, we tested these in association with GCV in the HSVtk-expressing human colon cancer cell line (HSVtk+/LoVo cells) in vitro and in vivo. Methods and Results 1. Revival and cultivation of HSVtk+/LoVo cells In our previous experiment we had gained the HSVtk+/LoVo cell culture which were selected in RPMI164O culture medium containing lOOngIml G41 8. In this study HSVtk+/LoVo cells grew well in RPMII64O culture medium after they were revived. 2. Study on the apoptosis in human colorectal cancer LoVo cells induced by HSVtkIGCV system in vitro After administration of GCV apoptotic HSVtk+/LoVo cells were observed by light microscope, fluoroscope, electron microscope. Cell cycle was analyzed using flow cytometry. The percentage of apoptosis was enhanced following time. GCV showed a remarkable cell cycle specificity. The percentage of LoVo cells in G0/G1 phase decreased from 42.3% to 31.3%, and the percentage of LoVo cells in S phase enhanced from 3 8.7% to 62.0%. 3. Synergistic use of Herpes Simplex Virus-Thymidine Kinase I Ganciclovir system and 5-FU for the treatment of human colorectal cancer (HCC) in vitro HSVtk+/LoVo cells were seeded in the presence of GCV or 5-FU or GCV+5-FU at a density of 3 X 103/ml in 96-well tissue culture plates. Cells were quantitative by using MTT assay. After 24 hours?incubation, MTT(20u1) was added to each well. After 4 hours of incubation at 370C with MTT, the unreacted MTT and medium were removed and 1 SOul/well dimethy sulfloxide (DMSO) was added to solubilize the MTT formazan. After gentle agitation for 10 minutes, the optical density of each well was measured with microplate reader (Model 960,Biorad Inc.) equipped with a 570-nm filter. The spectropnotometer was calibrated at 0 absorbance using wells that had only contained medium and MTT. 0D570 of each well was measured. The survival rate was determined by comparing 0D570 of treatment groups with that of control groups. The effect of synergistic use of HSVtkIGCV and 5-FU for the treatment of HSVtk+/LoVo cells in vitro was better than other groups (P<0.05). 4. Experiments in vivo HSVtk+/LoVo cells were suspended in phosphated-buffed saline at a concentration of 1 X 108 cells/ml and injected subcutaneously (1 OOul inoculum volume) into the right back of syngeneic female BALB/C nude mice (4-5 weeks old , living in SPF condition...