| Bladder cancer is the most common tumor in urinary tract tumor,the recurrence rate is higher after operation.It is important to prevent the recurrence in the research.we have obtained the comfirm effect to combint different mechanism drugs,the study that Interaction of Mitomycin C with Nimesulide in bladder cancer is little.Objective:This study was designed to valuate the potential role of Nimesulide(NIM), a selective Cyclooxygenase-2(Cox-2) inhibitor,Combined with Mitomycin-c(MMC) on proliferation,apoptosis and cell cycle of Bladder cancer cell lines EJ. To investigate expressions of related genes Bax and Bcl-2in Bladder cancer EJ cell lines.Methods:1. Human Bladder cancer cells line EJ was chosen as the trail material and the treated time was24h,48h,72h.2. Experimental sub-groups:By the randomized principle, the control group that is untreated group (normal cultured EJ cells) and experimental group that was divided into3groups MMC group was treated by2mg/L MMC,NIM group was treated by300umol/L NIM, and combinatied group was treated by MMC (2mg/L)+NIM (300umol/L)3. After cells treated, control group and experimental groups continued to cultivate for24h,48h,72h, observing the proliferative inhibition of EJ Bladder cancer cells line by A values from MTT test.4. The control group and the experimental groups were treated by24h,48h,72h,Detect the percentage of apoptosis and cell cycle of EJ Bladder cancer cells line by flow cytometry(FCM). 5. The control group and experimental groups were treated with48h, to observe the protein expression of cell apoptosis associated genes Bax and Bcl-2was examined by S-P immunohistochemistry technique.6. Statistical analysis:statistical process was used by SPSS11.5of statistical software,all the datas were indicated by mean±tandard deviation (x±s). Analysis of variance was used by factorial design. P<0.05was considered statistically different between of them.Results:1.Detect the proliferative inhibition of EJ cells by MTT:After treated24h,48h,72h, at different culture time, the average number of EJ cell decreased, The experimental groups including MMC group, NIM group and the combinatied group, compared with control group differences was significant (P<0.05) with the same factor, At the time of72h, the differences of the A values were significant statistically compared with the time of24h and48h (P<0.05).At the same treated time, the combinatied group witch was treated by NIM and MMC compared with NIM group and MMC group,the differences was significant (P<0.05). It showed that experimental treatment could restrain EJ cell to grow. Inhibiting effect of the combinatied group is obviously(P<0.05), comparing with NIM group and MMC group. With the extension of treated time, the number of survival cells decreased obviously in the combinatied group after treated72h.2.Detect the effect of EJ cell lines by FCM:2.1Detect the percentage of apoptotic on EJ cellsAfter treated24h,48h,72h, at different culture time, the differences between the experimental groups including MMC group and the combinatied group and the control group were significant (P<0.05). NIM group compared with the control group, the differences had no statistical significance (P>0.05). In the same factor, with the culture time extended, the percentage of apoptotic increased in the MMC group and the combinatied group, yet the differences had no statistical significance (P>0.05) among these groups.In the NIM group,there is no changes in the percentage of apoptotic (P>0.05) 2.2the percentage of mortality of EJ cells:After treated24h,48h,72h,at the same culture time, the differences of mortality of EJ cells between the experimental groups and the control group were significant (P<0.05). At the same factor, with the culture time extended, the percentage of mortality increased in the experimental group, the mortality of EJ cells increased obviously in the combinatied group after treated72h, the differences had statistical significance (P<0.05).3.Detect the cell cycle of EJ cell lines by FCM:After treated24h,48h,72h, at the same culture time, the differences of the percentage of G1cells between the experimental groups---including NIM group and the combinatied group---and the control group were significant (P<0.05). MMC group compared with the control group, the differences had no statistical significance (P>0.05). At the same factor, with the culture time extended, the percentage of G1cells increased in the NIM group and the combinatied group,yet the differences had no statistical significance (P>0.05) among these groups. It showed that NIM influence cell cycle and blocked in G1delay.4.Expression of Bax and Bcl-2after48h by immunohistochemistry:The immunohistochemistry results indicated that the levels of Bax proteins increased in MMC group,NIM group and the combinatied group compared with the control group, the differences had the statistical significance (P<0.05), the expression rates of the protein in the combinatied group compared with NIM group, MMC group, the differences were significant (P<0.05). but the expression rates of Bax proteins had no significant difference (P>0.05) between NIM group and MMC group.While the levels of Bcl-2proteins decreased in MMC group, NIM group and the combinatied group compared with the control group, the differences had the statistical significance (P<0.05), the expression rates of the protein in the combinatied group compared with NIM group, MMC group, the differences was significant (P<0.05).but the expression rates of Bcl-2proteins had no significant difference (P>0.05) between NIM group and MMC group. The expressions of Bcl-2proteins in the combinatied group was negative.Conclusions:NIM can enhance drug toxicity of MMC to inhibited proliferation of Bladder cancer cell lines EJ.The sensitivity of bladder cancer cell EJ to mitomycin C can be enhanced by selective Cox-2inhibitors NIM, It might be one of the mechanisms that NIM influence cell cycle and resulted in G1delay.MMC combined with NIM could induce apoptosis of bladder cancer EJ cells by up-regulation of espression of Bax and down-regulation of Bcl-2. |
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