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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

张志宏,龙新华,刘志礼,王恒,汪逃芳,朱粮博   

  1. 南昌大学第一附属医院
  • 收稿日期:2013-07-11 修回日期:2013-09-17 出版日期:2014-01-15 发布日期:2014-01-15
  • 通讯作者: 刘志礼

The inhibitory effect study of down-regulating phosphorylated HER2 on osteosarcoma cells U2-OS proliferation and metastasis in vitro.

  • Received:2013-07-11 Revised:2013-09-17 Published:2014-01-15 Online:2014-01-15
  • Contact: Zhi-Li LIU

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摘要： [摘要] 【摘要】 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、 40 μmol/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 ）。10 μmol/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。

关键词: 受体, 表皮生长因子, 氧化磷酸化, 骨肉瘤, 细胞增殖, 肿瘤侵润, 二甲苯磺酸拉帕替尼

Abstract: [Abstract] Objective: To investigate the effect of down-regulating phosphorylated human epidermal growth factor receptor 2 (HER2) on osteosarcoma cells U2-OS proliferation and metastasis in vitro. Methods: Various concentration of HER2 phosphorylating inhibitor Lapatinib Ditosylate (5,10,20,30,40μmol/L) were adopted to deal with human osteosarcoma cells U2-OS. MTT assay was performed to evaluate the cell proliferation during various time（24, 48, 72 h), and the IC50 value in 24h was calculated. A concentrtion value of 10umol/L below IC50 (IC50=22.15umol/L) was chosen to deal with U2-OS cells. The expression level of phosphorylated HER2 (p-HER2) was measured by western blot; Wound healing and Transwell invasion assay were performed to evaluated the cell migration and invasion abilities. Results: The HER2 phosphorylating inhibitor Lapatinib Ditosylate inhibited U2-OS cell proliferation dramatically, in which the inhibitory effect depend on the medicine concentration and duration. During 24 hours, the p-HER2 level in Lapatinib management group was lower than negative group (deal with PBS), the cell migration rate in Lapatinib management group and nagative group were (32.70±3.00)% and (94.52±4.76)% and the trans-membrane cells in two groups were 37±5/HP（×400）and 85±10/HP(×400), respectively. The differences in two groups were dramatically (P<0.05). Conclusions: 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.

Key words: receptor, epidermal growth factor, oxidative phosphorylation, osteosarcoma, cell proliferation, neoplasm invasiveness, lapatinib ditosylate