下调HER2磷酸化水平对骨肉瘤细胞U2-OS体外增殖转移的抑制作用研究

摘要

Objective To investigate the effects of down-regulating phosphorylated human epidermal growth factor receptor 2 (HER2) on the proliferation and metastasis in human osteosarcoma cells (U2-OS) in vitro. Methods Various concentrations of HER2 phosphorylation inhibitor lapatinib ditosylate (5, 10, 20, 30 and 40 μmol/L) were adopted to deal with U2-OS. MTT assay was performed to evaluate the cell proliferation during various times (24, 48 and 72 h), and the IC50 value in 24 h was calculated. The value of 10μmol/L (IC50=22.15μmol/L) was chosen to deal with U2-OS cells. The expres-sion level of phosphorylated HER2 (p-HER2) was measured by Western blot assay. The cell migration and invasion abilities were detected by Wound healing and Transwell invasion assays. Results The cell proliferation of U2-OS was significantly inhibited by HER2 phosphorylation inhibitor lapatinib ditosylate in a concentration- and time-dependent manner. During 24 hours, the p-HER2 level was significantly lower in lapatinib ditosylate group than that of negative control group (0.093± 0.033 vs 0.306±0.033), the cell migration rate was significantly lower in lapatinib ditosylate group than that of negative con-trol group (32.70%±3.00%and 94.52%±4.76%), and the trans-membrane cells were significantly lower than those of nega-tive control group (37/HP±5/HP and 85/HP±10/HP), respectively. Conclusion The down-regulating p-HER2 in U2-OS could efficiently inhibit the cell proliferation, migration and invasion in vitro. HER2 has the potential to become a molecular target for anti-osteosarcoma metastasis.%目的：探讨下调人类表皮生长因子受体2（HER2）磷酸化水平对骨肉瘤细胞U2-OS体外增殖、侵袭转移能力的影响。方法采用不同浓度（5、10、20、30、40μmol/L）的HER2磷酸化抑制剂二甲苯磺酸拉帕替尼作用骨肉瘤细胞U2-OS。四甲基偶氮唑盐（MTT）检测作用不同时间（24、48、72 h）细胞的增殖能力，并计算出24 h药物的半数抑制浓度（IC50）。10μmol/L二甲苯磺酸拉帕替尼作用U2-OS细胞，Western blot检测磷酸化HER2（p-HER2）蛋白表达水平；Wound healing和Transwell invasion实验检测细胞迁徙、侵袭能力。结果二甲苯磺酸拉帕替尼显著抑制U2-OS细胞的增殖，并且呈时间、剂量依赖性；二甲苯磺酸拉帕替尼作用24 h后，细胞中p-HER2蛋白表达水平显著低于阴性对照组（0.093±0.033 vs 0.306±0.033）；细胞迁徙率低于阴性对照组（%：32.70±3.00 vs 94.52±4.76），穿膜细胞数低于阴性对照组（个/视野：37±5 vs 85±10）。结论体外下调U2-OS细胞中HER2磷酸化水平能显著抑制U2-OS细胞的增殖、迁徙和侵袭，HER2有望成为抗骨肉瘤侵袭转移的分子靶点。