目的 探讨亚硝酸盐预处理对骨肉瘤细胞多柔比星(ADM)化疗敏感性的影响。方法 四甲基偶氮唑蓝法分析多柔比星对骨肉瘤细胞MG-63的IC50并计算耐药指数，FITC-annexinⅤ/PI染色流式细胞术分析细胞凋亡，PI和hochest33258活细胞染色分析细胞死亡率，EGFP-LC3b细胞转染和透射电镜分析细胞自噬，细胞缺氧诱导因子-1α(HIF-1α)和微管相关蛋白1轻链3-β(LC3)蛋白表达水平用蛋白质印迹法分析。结果 用10~30 mg·L-1亚硝酸钠预处理MG-63细胞24 h，再用多柔比星 (0.31 μg·mL-1)处理24 h，与单纯多柔比星处理组相比，多柔比星对MG-63细胞的IC50值和耐药指数增加，这种现象可以被自噬抑制剂3-甲基腺嘌呤逆转。多柔比星可以诱发MG-63细胞自噬，自噬的细胞膜周围LC3-Ⅱ绿色荧光点分布，透射电镜下可见自噬体。用3-甲基腺嘌呤抑制自噬对细胞凋亡率和死亡率影响不明显。与单纯多柔比星处理组相比，亚硝酸钠(10 mg·L-1)预处理组自噬阳性细胞增多，用3-甲基腺嘌呤抑制自噬，细胞凋亡率和死亡率明显增加。亚硝酸钠预处理组的HIF-1α蛋白表达和LC3Ⅱ/ LC3Ⅰ蛋白比值明显比单纯多柔比星处理组增高。结论 亚硝酸钠预处理能够降低多柔比星对骨肉瘤细胞的化疗敏感性，机制可能与其促进细胞自噬和HIF-1α蛋白表达有关。
Abstract
OBJECTIVE To explore the effects of sodium nitrite pretreatment on the chemotherapeutic sensitivity to adriamycin (ADM) in MG63 cells. METHODS MTT assay was used to measure the IC50 of ADM and the resistance index to MG-63 cells. FITC-annexinⅤ/PI staining flow cytometry was used to analyze the apoptotic rate of cells. Cell death rate was assessed by PI and hochest33258 viable cell staining. The effects of autophagy in cells were investigated with EGFP-LC3b transfection and electron microscope. The expression of HIF-1α and LC3 in cells was detected by Western blotting. RESULTS Compared to ADM treatment alone, MG-63 cells was pretreated with sodium nitrite(10-30 mg·L-1) for 24 h and followed exposed to ADM (0.31 μg·mL-1) for 24 h,the IC50 of ADM and the resistance index to MG-63 increased significantly. These phenomenon could be reversed through down-regulation of autophagy with 3- methyladenine(3-MA). LC3-Ⅱ green dot fluorescence diffusely distributed near the cell membrane after treatment with ADM and the autophagosome could be found under the electronmicroscopy. ADM could induce autophagy and apoptosis, 3-MA could suppress autophagy, but had no significant effects on apoptosis. Compared to ADM treatment alone, MG-63 cells were pretreated with sodium nitrite (10 mg·L-1) for 24 h, and followed exposed to ADM, the autophagy-positive cells were increased significantly,3-MA administration following ADM could increase apoptosis and death rate in comparison to ADM treatment alone. In addition, western blotting showed that the expression of hypoxia-inducible factor 1α(HIF-1α) and LC3Ⅱ/ LC3Ⅰin the cells that pretreated with sodium nitrite and followed exposed to ADM were higher than ADM treatment alone group. CONCLUSION Sodium nitrite pretreatment could decrease the chemotherapeutic sensitivity to ADM in osteosarcoma cells and the mechanism may be related to promote autophagy and increase the expression of HIF-1α.
关键词
亚硝酸盐 /
骨肉瘤 /
多柔比星 /
化疗敏感性 /
自噬 /
缺氧诱导因子-1α
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Key words
nitrite /
osteosarcoma /
adriamycin /
chemotherapeutic sensitivity /
autophagy /
hypoxia-inducible factor 1α
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中图分类号：
R965
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参考文献
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脚注
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基金
国家重大环保公益项目专项(200809115)；省部共建河南大学科研项目(SBGJ090702)
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