Effect And Underlying Mechanism Of Caffeine On DNA Damages Of P53~+-wild-type RT4Bladder Cancer Cell Line

Background:Bladder cancer is among the most common disease in urological field, and the global incidence rate of bladder cancer is increasing yearly. Epidemiological data indicate that multiple environmental factors may joint forces in the development of bladder cancer, including the intake of overdose caffeine. It has been suggested that overdose caffeine may enhance the risk of bladder cancer. However, caffeine is one of the most-widely consumed neurostimulators in the world, and the effect of caffeine on the occurrence and development of bladder cancer will have great influence on public health. Thus, the effects and underlying mechanism of caffeine on the development of bladder cancer deserves further investigation. Part I Effect of caffeine on cellular apoptosis in p53+-wild-typeRT4bladder cancer cell lineObjectiveBy using MTT method and Flow cytometry method, to observe the cellularresponses of p53+-wild-type RT4bladder cancer cell line to caffeine, ionic rays (IR) orcombination treatment of caffeine and IR, and further investigate the effect of caffeineon the apoptosis of bladder cancer cells.Material and Methods1.Culture of RT4bladder cancer cells.2.Recovery and cryopreservation of RT4bladder cancer cells.3.Group and different treatment of cells.4.Effect of IR on proliferation of RT4bladder cancer cells (MTT method).5.Effect of combination of IR and caffeine on proliferation of RT4bladder cancer,cells (MTT method).6.Apoptosis of RT4bladder cancer cells (Flow cytometry method).7.Statistical Analysis.Results1.IR treatment enhanced the apoptosis of RT4bladder cancer cells in vitro.2.Caffeine treatment inhibited the damage effect of IR on RT4bladder cancercells.Conclusion:1.IR treatment caused cellular apoptosis in RT4bladder cancer cells in atime-dependent and dose-dependent manner.2.Combinative treatment of IR and caffeine also caused cellular apoptosis in RT4bladder cancer cells in a time-dependent and dose-dependent manner. 3. Treatment with caffeine of1mM led to decrease of apoptosis in ion-irradiated RT4bladder cancer cells, indicating a protective effect of caffeine of1mM on ion-irradiated RT4bladder cancer cells. Part Ⅱ Effect of caffeine on cell cycle in p53+-wild-type RT4bladder cancer cell lineObjectiveTo observe the cell cycle of p53+-wild-type RT4bladder cancer cell line to caffeine, ionic rays (IR) or combination treatment of caffeine and IR, and further investigate the effect of caffeine on cell cycle of p53+-wild-type RT4bladder cancer cells.Material and MethodsTo detect the cell cycle in p53+-wild-type RT4bladder cancer cells by flow cytometry.Results1. IR treatment caused cell cycle arrest at G0/G1phase and G2/M phase, especially at G2/M phase.2.1mM of caffeine treatment also caused cell cycle arrest at G0/G1phase and G2/M phase, and the effect on G2/M phase arrest was dramatically.3. Caffeine decreased the effect of cell cycle arrest in iron-irradiated RT4bladder cancer cells. After treatment of caffeine, cells passed G0/G1phase quickly while arrested at G2/M phase.Conclusion1. G0/G1and G2/M-phase arrest may be associated with the ray sensitivity in RT4bladder cancer cells. Caffeine treatment enhanced G2/M-phase arrest of the cell cycle, and protected the cells against irradiation by inhibiting the apoptosis of RT4bladder cancer cells.2. Caffeine of1mM may promote the occurrence and development of bladder cancer. Part Ⅲ Mechanism of caffeine's effect on DNA damage of p53+-wild-type RT4bladder cancer cellsObjectiveTo observe a series of DNA damage substances such as γH2AX, ATM, ATR, pATM, Chk1, Chk2, p-Chk2, p53, p-p53, p21, PUMA and FAS in p53+-wild-type RT4bladder cancer cell subjected to caffeine, ionic rays (IR) or combination treatment of caffeine and IR, and further investigate the possible mechanism of caffeine on RT4bladder cancer cells.Material and Methods1. To detect the quantity and density of γH2AX foci by immunofluorescence staining.2. To detect the expressions of p53as well as its targets by real time PCR.3. To detect the expressions of p53, p-p53, ATM, p-ATM, ATR, Chk2, p-Chk2, γH2AX, PUMA, p21and caspase-3by Western blotting.Results1. Caffeine inhibited γH2AX by inhibiting upstream regulators.2. Caffeine inhibited the expression of target gene of p53.3. Caffeine inhibited the apoptosis of iron-irradiated RT4bladder cancer cells.Conclusion1. IR treatment might injury RT4bladder cancer cells via ATM-Chk2-p53-Puma signal axis.2. Caffeine inhibited the expression of γH2AX by inhibiting upstream regulators of DNA damage response cascade, such as ATM and ATR.3. Caffeine inhibited the expression of downstream targets of p53, such as TP53, p21, PUMA and FAS. 4. Caffeine might reduce the injury of IR on RT4bladder cancer cells by inhibiting the signal pathways of DNA damage response; therefore have protective effect on RT4bladder cancer cells. Part Ⅳ Effect of caffeine on nude mice with bladder cancerObjectiveRT4bladder cancer cell was cultured in vitro and inoculated to nude mice so that animal model of nude mice with bladder cancer was established. Caffeine was pre-treated in the nude mice and IR was applied to the solid tumors, so that effect and associated mechanism of caffeine on ray-sensitivity of RT4bladder cells was observed.Material and Methods1. Culture of RT4bladder cancer cells.2. Recovery and cryopreservation of RT4bladder cancer cells.3. Establishment of nude mice model with RT4bladder cancer cells.4. Group and different treatment of nude mice.5. Detection of γH2AX and p-ATM by immunohistochemical method.Results1. Successful establishment of nude mice model with RT4bladder cancer cells.2. Caffeine inhibited cell apoptosis of bladder mucosal cell in iron-irradiated normal nude mice.3. Caffeine reduced the ray-sensibility to IR in RT4bladder cancer cells, and inhibited the production of yH2AX upon IR.4. Caffeine reduced the influence of IR on RT4bladder cancer cells, and inhibited the expression of p-ATM.Conclusion1. Animal model of nude mice with bladder cancer can be successfully established using RT4bladder cancer cells.2. Caffeine of1mM inhibited IR-induced cell apoptosis of solid tumors derived from bladder cancer cell line. 3. Caffeine of1mM may inhibit ATM-Chk2-P53-Puma pathway and activate other DNA damage response pathways, therefore have protective effect on p53+-RT4bladder cancer cells.