Experimental Study On CD-TK Double Suicide Gene Systems And Genistein For Prostate Carcinoma

Background and Objectives Prostate carcinoma is the most frequentlydiagnosed malignancy, which prevails in Europe and USA, and the second leadingcause of cancer deaths.The incidence of this disease is just next to the lung cancer. Itwas reported that the new case reached to 232090 in 2005, mortality rate got to 30350at the same time. The incidence of prostate carcinoma was lower in our country thanin Europe or USA. But thanks to regulation of dietary components, the agingpopulation and the development of diagnosis in our country and the other Asiancountry over the recent years, the number of prostate cancer is ascending year afteryear. In earlier stages the prostate cancer patients can be cured or maintainedsuccessfully by standard medical treatments including surgical, incretion approach etal,but there are still a series of side-effect such as endocrine,erectile dyefunction,recurrence et al. Even more there are still no effective therapy for those who haveadvanced disease when being diagnosed or who have failed the primary curativeattempts. Therefore, there is an urgent need to develop novel systemic strategies forthis major health problem. One novel approach against prostate cancer is genetherapy.This strategy is promising.CD gene codes Cytosine deaminase, a n enzyme found in a variety of bacteria and fungi, is also able to deaminat the nontoxic prodrug 5- fluorocytosine (5FC) tothe toxic metabolite 5- fluo rouracil (5FU). FU), which inhibits RNA and DNAsynthesis during the S phase of the cell cycle.HSV-TK gene codes thymidinekinase, which is able to initially phosphorylate GCV,then subsequent incorporationinto DNA and cause turnout cell death.All over the world many scholars utilizevectors such as adenoviral or retrovirus to transfer CD and TK genes into liver canceror cholecyst cancer's cells, and then kill them. Although many studies havedemonstrated particular advantage, the therepeutic effect had not so much. It waspartially due to low gene delivery or low transgene expression. At the same time therewere some difference between in vivo and in vitro.Genistein,one of the predominant soy isoflavones, its chemic name is 4,5, 7-trihydroxyisoflavone. It is present in fruits, vegetables,especially in soy.Genisteininhibits protein tyrosine kinase (PTK), which is involved in phosphorylation oftyrosyl residues of membrane-bound receptors leading to signal transduction, and itinhibits topoisomeraseâ…¡, which participates in DNA replication, transcription andrepair.Genistein can kill cell by inducing the cells apoptosis.So far there is not any report about therapy with CD-TK double suicide gene andgenistein.To explore the therapeutic effect, we used CD-TK double suicide geneand/or genistein to inhibit the RM-1 cells.It will helpful to provide some experimentalproof for prostate cancer' therapy.But GEN was generally intaken by food. In thisway GEN would be depleted partly by metabolize.So finally it was hard to control thedrug quantity, which reached to the tumor.Materials and Methods:1,Amplification, identification and the titer determination of the replicationdefective adenovirus vector expressing CD-TK double suicide fusion gene;Infect theHEK293 cells with the adenovirus vector expressing CD-TK double suicide fusion gene and green fluorescent protein(GFP) gene to amplify the recombinantadenoviruses;2,use the PCR assay to confirm that the adenovirus contains CD-TK doublesuicide fusion gene,and quantify the GFP positive cells to determine the titer of therecombinant adenovirus.Afer the transfection of RM-1 cells with the CD-TKadenovirus,the total mRNA of the infected RM-1 cells were extracted. RT-PCRassay to detect the expression of CD-TK fusion gene in the RM-1 cells transfected bythe CD-TK adenovirus;3,Incubate CD-TK adenovirus-transfected RM-1 cells with the differentconcentration of GCV,5-FC,GEN for 72 hours,use the MTT assay to determine thesurvival rates of different groups;4,The animal models were established by subcutaneously injecting about 1×10⁶RM-1 cells on the C57BL/6 female mice.24 mice with about 80 mm³ of the tumourvolume were selected,and divided into the 4 groups randomly:â‘ The control;â‘¡genistein injected intraperitioneslly;â‘¢GCV+5-FC injected intraperitioneally;â‘£GCV+5-FC +genistein injected intraperitioneally.The adenovirus was injecedeintratumorally with the dose of 100μl/d/mouse/time×3 days.During the course ofthe treatment,the volumes of the tumors were measured(volume=width²Ã—length×0.5236).The tumor tissue were analyzed by histopathology andimmunohistochemistry.5,The datas were dealed with SPSS10.0,analysed by Univaiate.Result The recombinant adenovirus were successfully amplified in HEK293cells and confirmed by the PCR assay to contain the CD-TK fusion gene,and its titerwas about 1.58×10¹⁰PFU/ml. The RT-PCR assay showed that the CD-TKadenovirus-transfected RM-1 cells expressed both CD and TK mRNAs.With theincreased concentration of the GCV and 5-FC,the survival rates of CD-TK adenovirus transfected RM-1 decreased.When the concentration of GCV and 5-FC was 125μg/ml and 160μg/ml,the survival rates were 47.27±±9.41%,71.56±1.70%,18.45±4.24 % for GCV,5-Fc and GCV+5-Fc group respectively.In comparison with theGCV,5-Fc group, the survival rates of GCV+5-Fc group were significantly low(P＜0.05).The results showed that the CD-TK double suicide gene system was betterthan the single suicide gene system. With the increased concentration of GEN,andmake the GCV,5-FC constanteously, the survival rates of CD-TK adenovirustransfected RM-1 decreased. In comparison with the GEN group, the survival rates ofGCV+5-Fc+GEN group were significantly low(P＜0.05).At the end of experiments,the average tumour volume of GEN-treated group was 359.06±53.17mm³,smallerthan that of the control proup significantly(1008.73±126.73 mm³)(P=0.00). Theaverage tumour volume of GCV+5-Fc+GEN-treated group was 25.31±9.24 mm³,smaller than any others(P=0.00).The same result can be found between GCV+5-Fc+GEN-treated group and GCV+5-Fc-treated group.Conclusion:1,The adenovirus expression vector can effectivelly introduce the CD-TKdouble suicide fusion gene into the RM-1 cells.2,The CD-TK double suicide gene systems had the synergistic effect on RM-1cells. The double suicide gene systems had stronger effect than the single suicidegene system.3,GEN could inhibit RM-1 cells in vitro.Moreover it could augment the killingeffect of CD-TK double suicide fusion gene system on RM-1 cells in vitro.4,GEN could enhance the inhibitory effect of CD-TK double suicide fusiongene system on the murine subcutaneous prostate cancer.5,The CD-TK double suicide gene systems and GEN induced the RM-1 cellsapoptotic by down-mediate the bcl-2.