中华眼镜蛇膜毒素12对膀胱癌细胞增殖、凋亡及自噬的影响*

Effects of membrane toxin 12 on proliferation, apoptosis, and autophagy of bladder cancer cells*

摘要:

目的 探讨中华眼镜蛇膜毒素12（MT-12）对膀胱癌细胞的增殖、凋亡及自噬的影响。方法 ①采用CCK-8法检测不同浓度（0.10、0.25和0.50?μg/ml）的MT-12处理膀胱癌RT4、T24细胞0、24、48、72、96和120?h后，对膀胱癌RT4、T24细胞增殖能力的影响；②采用流式细胞术检测MT-12处理膀胱癌RT4、T24细胞6及24?h后，对膀胱癌RT4、T24细胞凋亡的影响，主要观察细胞凋亡率的改变；③Pan-Caspase抑制剂和MT-12处理膀胱癌细胞后通过MTT法检测细胞活力；④通过GFP-LC3转染，观察自噬小体形成，通过Western blotting检测自噬标志物的表达情况。结果 CCK-8检测结果显示，不同浓度的MT-12处理膀胱癌RT4、T24细胞0、24、48、72、96和120?h后，MT-12能抑制膀胱癌RT4、T24的细胞增殖。流式细胞术检测细胞凋亡的结果显示，MT-12处理膀胱癌RT4、T24细胞6及24?h后，实验组可以增加凋亡细胞的比例。Pan-Caspase抑制剂V-ZAD-FMK对2株膀胱癌细胞系进行处理后，MT-12并未完全失去对膀胱癌的抑制效果。Western blotting和GFP-LC3转染检测结果显示，LC3-Ⅱ/LC3-Ⅰ比值增大，GFP-LC3转染结果自噬体（绿色荧光点）增多。结论 MT-12以浓度-时间依赖的方式抑制膀胱癌RT4、T24细胞的增殖能力，并诱导其调亡；MT-12能够引起细胞的自噬，并且自噬性的死亡可能发生在MT-12对膀胱癌细胞的抑制作用过程中。

Abstract:

Objective To investigate the effects of membrane toxin 12 (MT-12), as one of the extracts from Naja naja atra venom, on the proliferation, apoptosis and autophagy of bladder cancer cells. Methods We Used CCK8 method to detect the proliferation of bladder cancer RT4 and T24 cells after 0, 24, 48, 72, 96 and 120?h treatment of different concentrations of MT-12 (0.10, 0.25 and 0.50?μg/ml). The effect of MT-12 treatment of bladder cancer RT4 and T24 cells (after 6 or 24?h) on the apoptosis was determined by flow cytometry. The cell viability was detected by MTT assay after bladder cancer cells were treated with pan-caspase inhibitor and MT-12. Autophagic body formation was observed after GFP-LC3 transfection and the expression of autophagic markers was detected by Western blotting. Results The results of CCK8 showed that MT-12 could inhibit proliferation ability of bladder cancer RT4 and T24 cells after 0, 24, 48, 72, 96 and 120?h treatment of different concentrations of MT-12. The results of flow cytometry showed that the experimental group can increase the apoptotic rates of bladder cancer RT4 and T24 cells after 6-and 24-hour treatment of MT-12. After treatment of pan-caspase inhibitor V-ZAD-FMK, MT-12 did not completely lose its inhibitory effect on bladder cancer. Western blotting and GFP-LC3 transfection assay results showed LC3-II/LC3-I ratio increased and autophagosome transfected by GFP-LC3 (green fluorescence point) increased. Conclusions MT-12 can inhibit the proliferation of bladder cancer RT4 and T24 cell lines in a concentration-time-dependent manner and induce apoptosis. MT-12 can induce autophagy and autophagic death may occur in cells in the process of bladder cancer inhibited by MT-12.