Mechanism of thalidomide to enhance cytotoxicity of temozolomide in U251-MG glioma cells in vitro
Mechanism of thalidomide to enhance cytotoxicity of temozolomide in U251-MG glioma cells in vitro
复制标题
沙利度胺增强替莫唑胺对U251-MG胶质瘤细胞体外细胞毒作用的机制
DOI：
10.3760/cma.j.issn.0366-6999.2009.11.005
复制
发表时间：
2009-06-05
影响因子：
6.1
通讯作者：
Pan Qiang
中科院分区：
文献类型：
--
作者：
Gao Song;Yang Xue-jun;Pan Qiang
背景胶质瘤是最常见的原发性脑肿瘤，预后差。替莫唑胺已与沙利度胺一起用于治疗神经胶质瘤。方法将体外培养的人恶性胶质瘤细胞U251-MG分为4组：替莫唑胺组（100 μ mol/L）、沙利度胺组（100 μ g/L）、替莫唑胺组（100 μ mol/L）+沙利度胺组（100 μ g/L）和对照组，分别给予不同浓度的药物处理3d。MTT法检测细胞活力。流式细胞仪分析细胞周期。电镜观察自噬体超微结构特征。采用吖啶橙子和单丹酰尸胺标记自噬体，流式细胞术定量自噬体。结果替莫唑胺联合沙利度胺治疗组肿瘤细胞增殖明显低于单药治疗组（P = 0.000）。联合处理诱导细胞周期停滞在G0/G1期。联合用药后细胞超微结构呈现典型的自噬特征。沙利度胺促进替莫唑胺诱导的自噬。与替莫唑胺单用相比，联合治疗更显著上调自噬相关蛋白-微管相关蛋白1轻链3（MAP1LC 3）和Beclin 1（MAP1LC 3，P = 0.000; Beclin 1，P = 0.004）。沙利度胺可提高通过抑制P13 K/Akt/mTOR信号通路促进自噬的第10号染色体缺失的磷酸酶和张力蛋白同源物（PTEN）的表达水平（沙利度胺组：P = 0.000;联合组：P = 0.002）。结论沙利度胺通过促进替莫唑胺诱导的自噬增强替莫唑胺的细胞毒作用，在体外联合应用时，自噬相关蛋白MAP1LC3和Beclin1的表达增强，可能与沙利度胺上调PTEN的表达有关。中华医学杂志2009，122（11）：1260 - 1266
Background Glioma is the most common primary brain tumor with poor prognosis. Temozolomide has been used with thalidomide to treat gliomas. We investigated the synergistic mechanism of these two drugs in vitro.Methods Human malignant glioma cells U251-MG were cultured and assigned to four groups with different treatments for 3 days: temozolomide group (100 mu mol/L), thalidomide group (100 mu g/L), temozolomide (100 mu mol/L) plus thalidomide group (100 mu g/L) and control group. MTT assay was applied to evaluate the cell viability. Cell cycle was analyzed by flow cytometry. The ultra-structural features of autophagosomes were observed with electron microscope. Acridine orange and monodansylcadaverine were adopted to label autophagosomes and flow cytometry was applied for quantification of autophagosomes. The expression of autophagy-associated protein was detected by Western blotting.Results Proliferation of tumor cell was obviously suppressed by temozolomide with thalidomide treatment than by either drug used alone (P=0.000 for each day). The combination treatment induced cell cycle arrest at G0/G1 phase. Typical autophagic ultra-structural character was found after the combined treatment. Thalidomide promoted the autophagy induced by temozolomide. The autophagy-associated proteins - microtubule associated protein 1 light chain 3 (MAP1LC3) and Beclin1 were more significantly up-regulated by the combined treatment than temozolomide used alone (MAP1LC3, P=0.000; Beclin1, P=0.004). The expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN), which promoted autophagy by suppressing Pl3K/Akt/mTOR signaling pathway, was elevated by thalidomide (thalidomide group: P=0.000; combined group: P=0.002). Conclusions Thalidomide enhances the cytotoxicity of temozolomide by promoting the autophagy induced by temozolomide.Contributing to the up-regulation of PTEN by thalidomide, the expression of autophagy associated protein-MAP1LC3 and Beclin1 was enhanced, which leads to a reinforced autophagy in the combined treatment of temozolomide and thalidomide in vitro. Chin Med J 2009,122(11):1260-1266