丝裂霉素C与C2-神经酰胺协同抑制人膀胱癌细胞生长及其机制的研究

目的评价丝裂霉素 C 与 C2- 神经酰胺联合应用对人膀胱癌细胞的作用效果, 并探讨其机制。方法不同浓度丝裂霉素 C 与 C2- 神经酰胺单独及联合作用于人膀胱癌 BIU- 87 细胞后, 应用噻唑蓝( MTT) 法检测药物细胞毒性, 通过计算合用指数( CI) 分析协同作用, 流式细胞术( FCM) 检测 BIU- 87 细胞凋亡率, 吖啶橙( AO) 荧光染色观察凋亡形态学变化, Western blot 检测细胞色素 C 在细胞内分布变化, 并检测半胱天冬氨酸蛋白酶 3 ( Caspase- 3) 活性改变。结果单独应用时丝裂霉素 C 与 C2- 神经酰胺的中效浓度分别是 159μmol/L 和 28 μmol/L, 联合用药时下降为 55 μmol/L 和 11 μmol/L, CI=0.74。丝裂霉素 C 与 C2- 神经酰胺单独及联合应用均可导致 BIU- 87 细胞出现凋亡的形态学变化。两种药物联合应用时的凋亡率高于各自单用( P <0.05) 。线粒体细胞色素 C 含量在丝裂霉素 C 与 C2- 神经酰胺单独及联合应用时均较对照组减少, 联合用药时减少最为明显, 细胞浆内细胞色素 C 含量在联合用药时增加也最为明显( P <0.05) 。Caspase- 3 活性在丝裂霉素 C 与 C2- 神经酰胺单独及联合应用时均较对照组升高, 联合用药时升高最为明显( P <0.05) 。结论丝裂霉素 C 与 C2- 神经酰胺联合应用可以通过共同诱导细胞凋亡, 协同抑制膀胱癌细胞生长。线粒体细胞色素 C释放和 Caspase- 3 活性变化可能发挥重要作用。

Research about synergetic effect and its mechanism of combining mitomycin C with C2_ceramide in inhibiting growth of human bladder cancer cells

[Objective] To evaluate the therapeutic efficacy and its mechanism of combining mitomycin C with C2-ceramide in human bladder cancer cells.[Methods] Different concentrations of mitomycin C and C2-cer were applied, individually or simultaneously, to human bladder cancer BIU-87 cells. MTT method was used to detect the cytotoxicity and then assayed the synergetic effect by calculating combination index (CI). Flow cytometry (FCM) and fluorescent staining with acridine orange (AO) were used to detect cell apoptosis. Western blot was used to detect the distribution changes of intracellular cytochrome C. At the same time, the changes of Caspase-3 activity were detected. [Results] The IC50 of mitomycin C and C2-ceramide were 159 μmol/L and 28 μmol/L when they were applied alone, while the numbers were 55 μmol/L and 11 μmol/L when they were combined. Combination index (CI) was 0.74. Cell apoptosis may be induced no matter mitomycin C and C2-ceramide were applied alone or together, while the apoptosis rates when these two drugs were combined were higher than that when they were alone (P＜0.05). The amounts of cytochrome C in mitochondria were decreased whether or not mitomycin C and C2-ceramide were applied together, while the phenomenon was the most obvious when they were combined. The amounts of cytochrome C in cytosol were also increased most obviously when two drugs were combined together (P＜0.05). The Caspase-3 activity of BIU-87 cells was the highest when mitomycin C and C2-ceramide were used together although Caspase-3 activities increased either these two drugs were applied alone or together.[Conclusions] Combination of mitomycin C with C2-ceramide can promote cell apoptosis, inhibit the growth of bladder cancer cells synergistically. The release of cytochrome C from mitochondria to cytosol and the changes of Caspase-3 activity play critical roles during this process.