Investigation On The Effect Of Querectin On Bladder Cancer Cell

Objective to study growth inhibitory effect of quercetin on bladder cancer cell and quercetin-mediated apoptosis, and to determine the molecular mechanism of quercetin on bladder cancer cell.Methods The three bladder cancer cell lines(EJ,T24 and J82) were treated by different concentrations of quercetin for 24h and 48h, respectively. Cellular proliferation was determined by the MTT assay. Colony formation analysis and growth curve were analyzed to evaluate the inhibitory effect of quercetin on EJ. FCM was used to detect the cell cycle of EJ, T24 and J82 cell lines treated with 100 μ mol/L quercetin for 24h compared with control. Hoechst33258 and TUNEL assay was assessed quercetin-induced apoptosis. Immunocytochemical method was defined the expression of p53,survivin and PTEN protein of EJ, T24 and J82 cells treated with 100 μ mol/L quercetin for 24h. MSP-PCR was used to evaluate the methylation status of several genes in T24 cell line. Results (1) MTT analysis shows that quercetin inhibits the growth of bladder cancer cell in a dose and time-dependent manner. The inhibition occurred in the three bladder cance cell lines. An approximate 50% reduction in cell viability was seen at the dose of 100 μ mol/L after 48h of incubation. (2) Colony formation assay shows that the level of colony formation of EJ cells was significantly inhibited by the increased concentration of quercetin. The counting of colony formation number shows a significantly dose-dependent reduction by quercetin. At the same time the growth curve shows similar result. High concentrations(150-200μ mol/L) of quercetin almost completely blocked the cell growth. (3) FCM shows that the three bladder cancer cell (EJ,T24 and J82) cycles were blocked at G₁/G₁. (4) Hoechst33258 detects an increased level of nuclear fragmentation and apoptotic bodies and the results of the in situ detection of apoptosis (TUNEL) shows a great number of brown-stained nuclei in quercetin-treated group.(5) Immunocytochemical assay shows that the expressin of mtp53 and survivin proteins in treated cells was significantly decreased compared with the control. But both control and treated cells shows a similar level in expression of PTEN protein. (6) MSP-PCR shows both control and treated groups contain the PCR products in the methylation and unmethylated genes. However the signal intensity of methylated genes was strongly reduced in the presence of quercetin. In comparision with the control group (untreated cells), the reduction was about 35%,57% and 73% for Erb,RASSFlA and pie0*4*, re+6spectively. Conclusion The phytochemical, quercetin can inhibit the growth and proliferation of bladder cancer cells and induce apoptosis, which is associated with a decreasing DNA methylation levels of pi6, RASSF1A and Erb genes, and a reduction in the expression of mtp53 and survivin protein. So quercetin is a potential chemopreventive and chemotherapeutic agent for bladder cancer. Further research is needed to investigate the downstream molecular mechanism associated with the demethylated-genes as well as those related to the cell cycle.