唑来膦酸对骨肉瘤细胞增殖凋亡和VEGF表达的影响

Effect of zoledronic acid on proliferation,apoptosis and expression of VEGF of osteosarcoma cell line LM8

背景与目的:双膦酸盐是一类强有力的骨吸收抑制剂,被广泛的应用于治疗骨质疏松症、变形性骨炎和恶性肿瘤引起的高钙血症。越来越多实验表明双膦酸盐具有直接的抗肿瘤作用。本研究探讨第三代双膦酸盐:唑来膦酸诱导骨肉瘤LM8细胞凋亡的机制和对VEGF表达的影响。方法:CCK-8法、Hoechst 33258染色形态学观察及Annexin V-FITC/PI双染法证实细胞凋亡,流式细胞仪检测活化caspase-3的表达,免疫组化SABC法检测VEGF的表达。结果:唑来膦酸对骨肉瘤细胞株LM8的增殖抑制呈浓度依赖性,50μmol/L唑来膦酸作用48 h能明显抑制LM8细胞增殖,抑制率为(54±3)%,形态学观察可见明显胞核凝聚、固缩;流式细胞仪检测可见细胞凋亡率为(31.7±2.9)%和caspase-3活化;免疫组化SABC显示VEGF的表达明显被抑制。结论:唑来膦酸可能通过激活caspase-3诱导LM8细胞凋亡,并抑制LM8细胞VEGF的表达。

Background and purpose： Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption, and have been widely used as a therapeutic agent for osteoporosis, Paget＇ s disease, and hypercalcemia associated with cancer. Moreover, there is increasing preclinical evidence that bisphosphonates have direct antitumor effects on a variety of human cancer cells. The purpose of our study was to investigate the mechanism of apoptosis of osteosarcoma cells induced by zoledronic acid, a newly developed third-generation bisphosphonate, and its effect on expression of VEGF. Methods： Cell line LM8（osteosarcoma cells） were cuhured and treated with zoledronic acid. Cell Counting Kit-8 assay and flow cytometry were used to detect the proliferation inhibition and apoptosis. Hoechst 33258 staining war used to observe the morphology of the cells. The expression rate of activated caspase-3 and VEGF of LM8 cells were determined by flow eytometry and immuniohistochemical staining respectively. Results： Zoledronic acid could significantly inhibit the growth of LM8 cells in a dosedependent manner. After being treated with 50 μmol/L zoledronic acid for 48 hours, the proliferation of LM8 cells was significantly inhibited, the inhibition rate was （54 ±3）% and LM8 cells was observed to present typical apoptosis changes. FCM showed that apoptosis rate was （31.7 ± 2.9）% 48 hours after treatment of 50 μmol/L concentrations of zoledronic acid and the expression rate of activated caspase-3 was up-regulated significantly. The inhibition of expression of VEGF was detected by immuniohistochemical staining. Conclusions： The mechanism of zoledronic acid to induce apoptosis of osteosarcoma cell line LM8 is associated with activation of caspase-3, zoledronic acid and down-regulation of VEGF expression of LM8 cells.