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目的肝癌是常见的恶性肿瘤之一,寻找其低毒有效的化疗药物具有重要的临床价值。本研究旨在探讨一种临床常用的骨吸收抑制剂帕米膦酸二钠对人肝癌HepG2及Bel-7402细胞体外增殖和凋亡的影响及其机制。方法采用MTS法检测不同浓度的帕米膦酸二钠对HepG2和Bel-7402细胞体外增殖的影响;以TUNEL末端标记法和Annexin-Ⅴ-FITC双标记流式法检测帕米膦酸二钠对细胞凋亡的影响采用蛋白质印迹法检测帕米膦酸二钠对凋亡相关蛋白:聚腺苷二磷酸-核糖聚合酶(poly ADP-ribose polymerase,PARP)、Caspase-9、Caspase-3、active-Caspase-3、Bax及Bcl-2表达的影响。结果 MTS检测显示,帕米膦酸二钠对HepG2及Bel-7402细胞均具有明显抑制作用,并呈剂量依赖性,药物的半数抑制浓度(50%inhibitory concentration,IC_(50))分别为46.12和51.35μmol/L。流式细胞术检测发现,帕米膦酸二钠可诱导肝癌细胞剂量依赖性凋亡,20、40和60μmol/L作用HepG2细胞24h凋亡率分别为(25.70±6.00)%、(43.53±4.06)%和(74.73±2.90)%,与对照组的(3.43±1.07)%相比差异有统计学意义,F=175.5,P<0.001;20、40和60μmol/L作用Bel-7402细胞24h凋亡率分别为(25.50±3.00)%、(48.30±1.02)%和(68.97±4.90)%,与对照组(3.33±0.05)%相比差异有统计学意义,F=81.97,P<0.001。蛋白质印迹法检测结果显示,帕米膦酸二钠可诱导细胞内凋亡相关蛋白Caspase-9、Caspase-3及PARP的活化,促凋亡蛋白Bax的上调,抑凋亡蛋白Bcl-2的下调。结论帕米膦酸二钠可以明显抑制人肝癌HepG2及Bel-7402细胞的体外增殖,其机制可能与线粒体凋亡途径的激活而诱导癌细胞发生凋亡相关,帕米膦酸二钠可能成为潜在的肝癌治疗药物。
Purpose Liver cancer is one of the most common malignant tumors. It is of great clinical value to search for its low toxicity and effective chemotherapeutic drugs. The aim of this study was to investigate the effects and mechanisms of pamidronate, a commonly used bone resorption inhibitor, on proliferation and apoptosis of human hepatoma HepG2 and Bel-7402 cells in vitro. Methods The effect of different concentrations of pamidronate disodium on the proliferation of HepG2 and Bel-7402 cells was detected by MTS assay. The effect of pamidronate disodium on the proliferation of HepG2 and Bel-7402 cells was detected by TUNEL terminal labeling and Annexin-V-FITC double labeling flow cytometry Effects of pamidronate on apoptosis-related proteins: poly-ADP-ribose polymerase (PARP), Caspase-9, Caspase-3, - Caspase-3, Bax and Bcl-2 expression. Results MTS assay showed that pamidronate disodium had a significant inhibitory effect on HepG2 and Bel-7402 cells in a dose-dependent manner. The 50% inhibitory concentration (IC 50) of the drug was 46.12 and 51.35 μmol / L. Flow cytometry showed that pamidronate disodium could induce dose-dependent apoptosis in HepG2 cells. The apoptosis rates of HepG2 cells treated with 20, 40 and 60 μmol / L for 24 h were (25.70 ± 6.00)%, (43.53 ± 4.06) ) And% (74.73 ± 2.90)%, respectively, compared with that of the control group (3.43 ± 1.07)%, F = 175.5, P <0.001; The mortality rates were (25.50 ± 3.00)%, (48.30 ± 1.02)% and (68.97 ± 4.90)%, respectively, which were significantly different from the control group (3.33 ± 0.05)%, F = 81.97, P <0.001. Western blotting showed that pamidronate disodium could induce activation of caspase-9, Caspase-3 and PARP, upregulate proapoptotic protein Bax and downregulate proapoptotic protein Bcl-2 . Conclusion Pamidronate disodium could significantly inhibit the proliferation of human hepatoma HepG2 and Bel-7402 cells in vitro. The mechanism may be related to the activation of mitochondrial apoptotic pathway and the induction of apoptosis in cancer cells. Pamidronate disodium may be potential Liver cancer treatment drugs.