The Study On Killing Effect Of KDR Promoter Driving Double Suicide Gene On Human Colorectal Cancer SW480 Cells

Background: Human colorectal carcinoma (HCC) is one of the most normal cancers in elementary organs in humans. Morbility of colorectal carcinoma has rise in recent years, it hurt human health seriously. The main intervention is surgical operation, chemotherapy and radiotherapy. The biotherapy has become new hot point recently, the suicide gene is the most noticeable research field, and it is an effective and more promising genetic therapy in tumor. Suicide gene also call drug sensitive gene, and it can transfer the prodrug to toxic drug to kill the tumor cells. The methods include direct killings and bystander effects, at same time have little hurt to normal cells and relive the side effect of whole body. TK gene-herpes simplex virus thymidine kinase can transfer the ganciclovir (GCV) to cytotoxicity to stop the sythesis of DNA in target cells and kill it. CD gene–colicytocine deaminase can transfer 5-FC to toxic 5-FU to kill the target cells.No matter the single suicide gene TK or CD when they are used as anti-tumor therapy will partly induce drug resistance. There are different sensitive to drug for different kind of tumor, however double suicide gene union therapy will increase the death rate of tumor and improve the treatment effect, enlarge the range of treatment and decrease the rate of drug resistance.Kinase domain insert containing receptor-KDR has high level expression in the vascular endothelial cells of active proliferation tumor , but has low level or no expression in the normal tissues. The suicide gene with KDR promoter will make the suicide gene specific expression in the tumor tissues, increase the target treatment, decrease the immuno-reaction and avoid the side effect to the other organs.So we built the co-expression adenovirus vector containing TK gene, CD gene and KDR promoter, co-expression adenovirus vector containing TK gene, KDR gene, and co-expression adenovirus vector containing CD gene, KDR gene. In order to investigate the killing effect to the colorectal cancer SW480 cells.Objective: To investigate the killing effect to colorectal SW480 cells of the co-expression adenovirus vector containing double suicide genes. Using the re-compound adenovirus pAdKDR-TK/CD united with GCV and 5-FC to observe the killing effect to colorectal cancer cells, ECV304 cells and LS174T cells, also to know the target killing effect of KDR promoter. Compared with the single suicide gene and double suicide genes to get further information about synergistic action between double suicide genes and combined prescription. Utilize the double suicide genes radiated byγ-ray to observe it's co-inhibition role and double suicide genes and prodrug's radiosensitization.. These can lay foundation for the further clinical experimental study.Metheds: The thymidine kinase-TK gene, colicytocine deaminase-CD gene and Kinase domain insert containing receptor-KDR gene amplified by using PCR means. They were cloned into eukaryotic expression vector pcDNA3.1 (+) to construct the pKDR-TK plasmid, pKDR-TK plasmid and pKDR-TK/CD plasmid, using the gene recombinant technology to transfect the fusion gene to adenovirus and constructed the co-expression vector with TK, CD and KDR genes, and named pAdKDR-TK, pAdKDR-CD and pAdKDR-TK/CD respectively.Recombinant plasmid pAdKDR-TK, pAdKDR-CD and pAdKDR-TK/CD were packed by 293 cells, and selected by G418 mediated by lipoplast, till obtain the positive clone. And then enlarged cultivation and tested the titer of virus by NIH3T3, transfected the recombinant adnovirus to colorectal SW480 cells, selected the positive clone, and the infected SW480 cells were assessed by PCR, RT-PCR.The killing effect of GCV, 5-FC on gene–modified cells were assessed by observing growth state of to ECV304 cells, SW480 cells and LS174 cells, MTT and FCM.; And comparison of killing effect of GCV, 5-FC, GCV+5-FC on colorectal SW480 cells were assessed.The increasing function ofγ-ray on gene-modified rate of colorectal SW480 cells, the radiosensitizing effect ofγ-ray to prodrug GCV, 5-FC and GCV+5-FC on colorectal SW480 cells, and the cooperated synergistic action of prodrug GCV, 5-FC and GCV+5-FC on colorectal SW480 cells withγ-ray radiation were observed.Results: By sequencing,restriction digestion and PCR we confirmed that the sequence, length, position and orientation of inserted genes were all correct. The target vector pAdKDR-TK, pAdKDR-CD and pAdKDR-TK/CD were constructed successfully. PCR and RT-PCR demonstated that TK gene, CD gene and KDR gene were integrated into the SW480 cell genome and expressed at the mRNA level.There were no significance differences on infected rate between ECV304 cells, SW480 cells and LS174T cells, the objective gene was observed among modified-gene cells which infected AdKDR-CD/KT except the LS174T cells. The ECV304 cells and SW480 cells were more sensitive to prodrug and the killing rate was increased accompanying with the increasing of prodrug quantity, and there was no significance difference between them (P>0.05). On other hand the LS174T cells which infected by adenovirs was low sensitive to prodrug, compared with two others there was significance difference (P<0.001). The bystander effect of ECV304 cells and SW480 cells was more obviousness than that of LS174T cells, and when the MOI was 50 the survival rate of SW480 cells and ECV304 cells was (31.3±5.64)%, (33.5±3.5.4)% respectively, the survival rate of LS174T cells was (98.1±0.95)%. There was significance difference between them (p<0.0025).At concentrations of GCV and 5-FC were: 100mg/ml and 1000mg/ml respectively 24h after treatment, cell cycle of transfected cells was monitored. DNA content distribution indicates that the locations of different cells in the cell cycle, control group and prodrug-treated group SW480 cells, the ratios of S phase cells were 34.30±0.9%, 58.19±0.7% (P<0.001), respectively, but that of G1 phase were 41.45±0.3%, 38.26±0.2% (P<0.005), G2 phase 24.25±0.5%, 8.95±0.4% (P<0.0005) respectively.Different concentration of GCV+5-FC had different killing effect to SW480 cells, and there was better killing effect treated with GCV and 5-FC respectively, there was no significant difference between them (p>0.5). But there was more better killing effect treated with GCV+5-FC than that of the GCV or 5-FC (p<0.01). No matter the SW480 cells infected with TK gene or infected with CD gene treated with GCV or 5-FC, the survival rate was decreased accompanying with the increasing of prodrug concentration, and there was no significance difference. They had resemble killing effect when the SW480 cells infected with TK or infected with CD treated with GCV+5-FC, and there was more strong killing effect of SW480 cells which infected with double suicide gene TK/CD than that of single double suicide gene (p<0.001). The killing effect of was more obvious when the concentration of GCV/5-FC at 80/1000mg/ml, and it is can conclude that the double suicide gene treated with recombinant prodrug had synergistic effect. The bystander effect of TK gene and CD gene had no significance, and then the bystander effect of double suicide gene TK/CD were more obvious than that of single suicide gene (TK, CD) (p<0.005).Non gene infected SW480 cells were blended with SW480 cells which infected with TK/CD double suicide gene on different MOI, radiated byγ-ray, 1h per day, 3 days later, it had higher infected rate than that of control group which had noγ-ray radiation (P<0.01). Compared with the killing effect on SW480 cells infected with double suicide gene TK/CD and SW480 cells with non gene-infected radiated byγ-ray, the experimental group was more sensitive than the control group (P<0.001). When the dose ofγ-ray was 0.2Gy, the survival rate of control group was 169.5 times of the experimental group's. It implied that SW480 cells transfected by pAdKDR/CD/TK were more sensitive to prodrug andγ-ray than that of non-transfected. The sensitization detected by cells clone experiment show that relative clone rate of group (G+F+γ) and group(γ) is 16.8% and 59.6% respectively, it indicated that sensitivity of group (G+F+γ) was built up apparently and had significant statistical meaning (P<0.0025). The relative clone efficiency of group (G+F+γ), (G+γ) and (F+γ) was 16.8%, 43.8% and 46.0%, this indicated that the sensitivity toγ-ray of group (G+F+γ) was increased compared with that of group (G+γ) and (F+γ). The sensitivity toγ-ray of double suicide gene is much higher than that of single's (P<0.001).Conclusions: The successful construction of co-expressive vector containing KDR, TK, CD gene had provided the experimental basis for the recombinant therapy of colorectal cancer. The fusion gene built up with KDR promoter had selective killing effect. The prodrug GCV and 5-FC had vitro killing effect on SW480 cells transfected by fusion gene, and the killing effect was augment when the quantity of prodrug was increased. The recombinant prodrug GCV+5-FC had synergic killing effect, and the double suicide gene had stronger bystander effect than that of single suicide gene's. Furthermore the prodrug can induce apoptosis of SW480 cells vitro. Theγ-ray can increase the rate of transfection of SW480 cells obviously, enhance the killing effect to cells for prodrug, and had radio-sensitizing effect.