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目的探讨三氧化二砷(As_2O_3)诱导膀胱癌凋亡过程中蛋白激酶C(PKC)和环磷酸腺苷(cAMP)水平的改变及其在膀胱癌凋亡中的作用。方法应用原位末端转移酶标记技术(TUNEL)、放射免疫、酶联免疫、免疫组化和Western blot等方法分别检测As_2O_3作用后膀胱癌T24细胞凋亡、凋亡过程中PKC和cAMP改变、caspase 3蛋白表达及caspase 3激活。结果药物作用24 h后,凋亡细胞呈现棕黄色浓染和绿色荧光。对照组细胞凋亡率0.86%,PKC总量2.55pmol·min~(-1)·μg~(-1),cAMP含量22.56pmol/ml,caspase 3蛋白表达率8.01%。与对照组相比,5μmol/L和10μmol/L As_2O_3组细胞凋亡率分别升高8.51倍和13.33倍,PKC总量分别降低59.22%和64.71%,cAMP含量分别升高5.34倍和4.23倍,caspase 3蛋白表达率分别上调3.96倍和6.76倍,差异有统计学意义(P＜0.05)。与As_2O_3组相比,5μmol/L和10μmol/L As_2O_3+100nmol/L PKC激动剂佛波酯(PMA)组细胞凋亡率分别降低40.22%和45.58%,PKC总量分别升高1.00倍和0.76倍,cAMP含量分别降低8.37%和31.46%,caspase 3蛋白表达率分别下调51.09%和65.03%。As_2O_3组均可显著激活caspase 3活性,As_2O_3+100nmol/L PMA组对caspase 3激活明显弱于同浓度As_2O_3组。结论As_2O_3可通过降低PKC和升高cAMP水平启动膀胱癌T24细胞凋亡,诱导细胞凋亡。
Objective To investigate the changes of protein kinase C (PKC) and cyclic adenosine monophosphate (cAMP) in the apoptosis of bladder cancer induced by As 2 O 3 and its role in the apoptosis of bladder cancer. Methods TUNEL, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and Western blot were used to detect the apoptosis of T24 cells after treatment with As 2 O 3. The changes of PKC and cAMP, caspase 3 protein and caspase 3 activation. Results After 24 h, the apoptotic cells showed brownish yellow and green fluorescence. The apoptosis rate of control group was 0.86%, the total amount of PKC was 2.55 pmol · min -1 (μg -1), the cAMP content was 22.56 pmol / ml and the expression of caspase 3 protein was 8.01%. Compared with the control group, the apoptosis rates of 5μmol / L and 10μmol / L As_2O_3 groups were increased by 8.51 times and 13.33 times respectively, the total amount of PKC decreased by 59.22% and 64.71% respectively, and the cAMP content increased by 5.34 and 4.23 times, The protein expression of caspase 3 was up-regulated 3.96-fold and 6.76-fold, respectively, with significant difference (P <0.05). Compared with the As 2 O 3 group, the apoptotic rate of PMA group decreased by 40.22% and 45.58%, respectively, and the total PKC increased by 1.00 times and 0.76 times respectively in 5μmol / L and 10μmol / L As 2 O 3 + 100nmol / L PMA group Fold, cAMP content decreased by 8.37% and 31.46%, respectively. The expression of caspase 3 protein decreased by 51.09% and 65.03% respectively. As 2 O 3 group can significantly activate caspase 3 activity, As 2 O 3 + 100nmol / L PMA group of caspase 3 activation was weaker than the same concentration As_2O_3 group. Conclusion As 2 O 3 can activate apoptosis and induce apoptosis of bladder cancer T24 cells by decreasing PKC and increasing cAMP levels.