替莫唑胺酯对脑胶质瘤的作用及其机制研究

Effects of temozolomide easter on glioma cells and its possible mechanisms

目的探讨替莫唑胺酯在体外抑制脑胶质瘤LN229细胞生长并诱导凋亡的作用及可能的机制,同时检测替莫唑胺酯是否能够克服替莫唑胺耐药。方法用四甲基偶氮唑蓝比色法比较相同浓度梯度(62.5,125,250,500,1000μmol·L-1)的替莫唑胺和替莫唑胺酯对LN229细胞增殖的影响。用流式细胞仪检测细胞凋亡率及细胞周期。用Western blot法检测替莫唑胺和替莫唑胺酯对凋亡蛋白Bax和Bcl-2的影响。构建过表达MGMT耐药细胞系LN229/MGMT,用四甲基偶氮唑蓝法检测替莫唑胺酯对耐药细胞系的增殖抑制作用。结果替莫唑胺酯较替莫唑胺明显抑制LN229细胞的生长,呈浓度和时间依赖性。替莫唑胺酯诱导LN229细胞发生凋亡率高于替莫唑胺。替莫唑胺酯能够导致LN229细胞发生S和G2期阻滞。替莫唑胺酯能够增加Bax/Bcl-2的比值,诱导发生凋亡。替莫唑胺酯能够克服替莫唑胺MGMT阳性的耐药。结论替莫唑胺酯对脑胶质瘤LN229细胞的增殖抑制及诱导凋亡的作用优于替莫唑胺,且能够克服替莫唑胺耐药。

Objective To investigate the contribution of temozolomide easter（TMZ-HE） to the proliferation and apoptosis of brain glioma LN229 cells in vitro compared with temozolomide（TMZ）,and then test whether TMZ-HE could overcome TMZ resistance.Methods Methyl thiazolyl tetrazolium assay was used to measure the effects on the proliferation of LN229 cells cultured with graded concentrations（62.5,125,250,500,1000 μmol·L-1） of TMZ and TMZ-HE.The Annexin V and propidium iodide（PI） assay by flow cytometer were applied to detect the apoptosis induced by TMZ and TMZ-HE.The changes of cell cycle were detected by flow cytometer.Western blot detected the expressing of Bax and Bcl-2 in LN229 after treatment with TMZ and TMZ-HE.Constructed resistant cell lines LN229 / MGMT which MGMT in LN229 / MGMT was over-expressed,then the effect of TMZ-HE on resistant cells was detected by MTT assay.Results TMZ-HE obviously suppressed the proliferation of LN229 glioma cells in a concentration-and time-dependent manner compared with TMZ,and the half inhibitory concentration（IC50） value of TMZ was much higher than TMZ-HE.TMZ-HE induced more cellular apoptosis of the LN229 cells in a concentration-dependent manner.Furthermore,high concentration of TMZ-HE induced a distinct cell cycle arrest with accumulation of cells in S phase that is not observed for TMZ.Then TMZ-HE inducedapoptosis by increasing the expression of Bax and down-regulating the level of Bcl-2.The study presented here also demonstrates that TMZ-HE is able to reverse TMZ resistance especially with high MGMT activity.Conclusion TMZ-HE inhibits more proliferation and induced more apoptosis than TMZ and TMZ-HE reversed TMZ resistance.These findings indicated that TMZ-HE may be developed as a possible therapeutic agent for the management of glioblastoma multiform.