Experimental Study On Adenovirus-mediated HSV-TK Gene In Combination With Chemotherapeutics For Human Bladder Cancer In Vitro

Background Bladder carcinoma is not only one of the commonest cancers ofurothelial system, but also an important problem of public health, the femalemorbidity of is equal to one quarter of the male morbidity. Bladder carcinoma thefourth common malignant tumors in America. There are more than 200000 patientsdiagnosed with bladder carcinoma and about 12000 patients died of bladdercarcinoma. Transitional epithelial cell cancer accounts for 90% of bladder carcinoma.Metastatic lesions have occurred in about 15% of patients with bladder carcinomawhen diagnosed first; About from 30% to 40% of patients with invasive bladdercarcinoma suffers from distant metastasis after radical cystectomy and about from20% to 30% of superficial bladder carcinoma deteriorates into invasive or metastaticcarcinoma after operation, so the prostecdtive efficacy of therapy for bladdercarcinoma is not satisfactory. Because of the unique bionomics of bladdercarcinoma, the effect of surgical therapy, radiotherapy and chemotherapy forbladder carcinoma is quite limited.The high recurrence rate of superficial bladdercarcinoma after transurethral resection is one of therapeutic problems urgent to besolved. With the development of studies on the suicide gene therapy for bladdercarcinoma, we realized that suicide gene therapy is a promising management forbladder carcinoma. However, there are also shortcomings in gene therapy such as low gene transfection efficiency, poor targeting of gene transduction, toxicity and harm ofgenes and genetic carriers on humanbody, the autoimmune reponse resulted from thecarrier and the killing of tumor cells, additionally, the metabolic product of prodrugonly has lethal effect on tne cells in dividing phase. In order to make up thedisadvantages of suicide gene therapy, more and more scholars dedicate themselves tothe study on the combination of present therapy and suicide gene therapy to search fora new treatment for the recurrence of bladder carcinoma. HCPT and THP are thechemotherapeutics used in the irrigation of bladder to prevent the recurrence ofbladder carcinoma. HCPT, a microamount alkaloid derived from camptothecaacuminata, can interfere the DNA duplication by inhibiting isomerase and hasfavourable synergistic effect with other anticancer drugs without intersecting drugfast, which can used in chemotherapy for many kinds of tumors.There have not beenany relevant reports about the combination of HCPT and TK gene therapy to treatbladder carcinoma with this unique property of HCPT.THPL, a newtypesemisynthesis anticancer drug of the kind of anthracene nucleus, adds a pyran ring onthe aminosaccharide of ADR to increase the activity of anticancer and the drugconcentration in target cell obviously, has less demiperiod and side effects.In thisresearch, we study the lethal effects of TK gene| in combination with low doses ofchemotherapeutics on hunman bladder carcinoma cell line T-24 cells and compare thedifferences in effects between each therapy in order to provide certain experimentalbases for the suicide gene therapy in combination with chemotherapeutics for bladdercarcinoma.Objective 1, To study the lethal effects of adenovirus mediated HSV-TK incombination with low doses of HCPT on the Human bladder carcinoma cells in vitro.2,To study the lethal effects of adenovirus mediated HSV-TK in combination withlow doses of THP on the Human bladder carcinoma cells in vitro. Methods1, The T-24 cells were cultured in vitro.2, The 293 cells were transfected with replication defective adenoviruscontaining TK gene and green fluorescent protein (GFP) gene, then the adenoviruswas amplificated and adenovirus titer was determined by quantifying the GFPpositive cells.3, The T24 cells of human bladder carcinoma were trasfected with TK gene toobtain the T-24 cells transfected with TK gene. After the transfection of Hunmanbladder carcinoma cell line T-24 cells with the CD-TK adenovirus, extract total Rnaof T-24 cells and the infected T-24 cells to carry out RT-PCR and was indentified byelectrophoresis.4, The T24 cells and T 24 cells infected with TK were cultured in vitro withdifferent concentrations of HCPT. The concentration of HCPT was 0, 0.001, 0.01,0.1, 1, 10, 50, 100, 200 mg/L respectively. The cell inhibition rate when hunmanbladder carcinoma cells were affected by HCPT alone for 72h were observed byMTT assay.5, The T24 cells and T 24 cells infected with TK were cultured in vitro withdifferent concentrations of THP. the concentration of THP was 0, 0.1, 1, 10, 100mg/L respectively. The cell inhibition rate when hunman bladder carcinoma cellswere affected by THP alone for 72h were observed by MTT assay to determine theeffective concentration of THP.6, According to the effective concentration of HCPT, We selected theconcentration gradients with less cell killing effect, 0.01, 0.05, 0.1, 0.5, 1 mg/Lrespectively. On base of the previous experimental results, the concentration of GCVwith less lethal effect on T-24 cells, 0.25mg/ml, was selected. With the normal T-24 cells as the blank control group, the survival rate after 24h, 48h and 72h of eachgroup were observed by MTT assay.7, According to the effective concentration of THP, We selected theconcentration gradients with less cell killing effect, 1, 5, 10, 15, 20 mg/L respectively.Then the transfected T-24 cells were treated by HSV-TK/GCV gene in combinationwith THP with different concentrations. With the normal T-24 cells as the blankcontrol group, the survival rate after 24h, 48h and 72h of each group were observedby MTT assay.Results1,The recombinant adenoviruses were successfully amplified in HEK 293 cellsand its titer was about 1.58×10¹⁰ PFU/ML after purified.2,The RT-PCR assay showed that the CD-TK adenovirus-transfected T24 cellsexpressed both CD and TK mRNAs.3,The inhibition rate of HCPT on T-24 cells increased gradually as theconcentration increased, the rate was 13.63% when the concentration was 0.01 mg/L.the rate increased to 64.44% when the concentration was 50 mg/L, but there was nosignificant increase in inhibitory action when the concentration was higher than 100mg/L (P=0.117). It showed that can inhibit the growth of hunman bladdercarcinoma cells with concentration dependent.4,The inhibition rate of THP on T-24 cells was less than 30% when theconcentration was less than 1 mg/L. The inhibition curve became steep obviouslywhen the concentration was from 1 mg/L to 100 mg/L. The inhibition rate was 62.94% at 50 mg/L, and 93.22% at 100 mg/L. The inhibition on T-24 cells increasedobviously with increase of the concentration of THP (P=0.00).5,When GCV in combination with HCPT were added, the survival rate of theinfected hunman bladder carcinoma cells in each group decreased significantly(p= 0.00), compared to the group added GCV alone. The killing efficiency in therapeuticalliance group increased obviously when the concentration of HCPT was higher andthe action time was longer. The survival rate of the gourp added 0.5mg/ml GCValone was 34.6% in 72h, The survival rate was only 8.07% of the group added GCV(0.25mg/ml) in combination with HCPT (10mg/L) (P=0.00). It indicated that thelethal effets of HSV-TK/GCV in combination with low doses of HCPT on the humanbladder carcinoma cells were better than those of HSV-TK/GCV alone.6,The GCV in combination with THP also had obvious killing effect on thebladder cancer cells, was better than GCV alone significantly (p=0.00) and increasedobviously when the concentration of HCPT was higher. It also showed that the lethaleffect of HSV-TK/GCV in combination with low doses of THP enhanced the lethaleffect of the therapeutic group and had correlation with the concentration and time asthe GCV in combination with HCPT group.Conclusion1,HSV-TK can be inducted into the human bladder carcinoma cells byadenovirus vector and express.2,HCPT can have lethal effect on the human bladder carcinoma cells(T-24cells) in vitro.3,THP can have lethal effect on the human bladder carcinoma cells(T-24 cells)in vitro.4,HSV-TK/GCV gene in combination with low doses of chemotherapeutics-HCPT can enhance the lethal effect of suicide gene on the human bladdercarcinoma cells.5,HSV-TK/GCV gene in combination with low doses of chemotherapeutics-THP can enhance the lethal effect of suicide gene on the human bladdercarcinoma cells.