替莫唑胺-尼莫地平-O^6-苄基鸟嘌呤联用对胶质瘤细胞株U251抑制作用的研究

THE INHIBITION OF COMBINED CHEMOTHERAPY OF TEMOZOLOMIDE,NIMODIPINE AND O^6 BENZYL-GUANINE ON GLIOMA CELL LINE U251

目的观察替莫唑胺(TMZ)、尼莫地平(NIM)、O6-苄基鸟嘌呤(O6-BG)联用对脑胶质瘤细胞株的抑制作用。探讨胶质瘤耐药发生的机制,寻找有效的多药耐药逆转剂,克服耐药性的产生,提高化疗效果,增加胶质瘤细胞对化疗药物的敏感性,最大限度地抑制胶质瘤细胞增殖,延缓肿瘤复发。方法用免疫细胞化学法检测胶质瘤细胞中MGMT蛋白表达,观察比较MGMT在用O6-BG处理前后表达的差异。分别以尼莫地平和O6-BG作为耐药逆转剂,与替莫唑胺单独或联合应用于体外培养的脑胶质瘤细胞U251。用MTT法检测细胞抑制率;观察TMZ、NIM、O6-BG联用对脑胶质瘤细胞的抑制作用。结果与对照组相比,MGMT在用O6-BG处理后表达明显减少。MTT比色法分别检测TMZ、TMZ+NIM、TMZ+O6-BG组及TMZ+NIM+O6-BG组对胶质瘤细胞U251的抑制作用,结果显示各组对胶质瘤细胞的抑制作用为TMZ+NIM+O6-BG联合组>TMZ+O6-BG>TMZ+NIM>TMZ。与TMZ组比较,TMZ+NIM组、TMZ+O6-BG组对U251细胞株的抑制率显著升高,差异有统计学意义(P<0.01);同样,与TMZ组比较,TMZ+NIM+O6-BG组对U251细胞株的抑制率也显著升高,差异有统计学意义(P<0.05)。结论 O6-BG能够有效抑制MGMT表达,从而能逆转人脑胶质瘤细胞株U251对替莫唑胺的耐药性。尼莫地平、O6-BG二者均可增强替莫唑胺对脑胶质瘤细胞株U251的细胞毒作用,对于替莫唑胺的耐药有较好的逆转效果,因而可望作为临床化疗增敏剂与替莫唑胺联合使用。替莫唑胺、尼莫地平、O6-BG三者联用对胶质瘤细胞株U251有更好的抑制作用,其效果优于二者联合应用,强于单独应用替莫唑胺。

Objective To investigate the inhibition of temozolomide {TMZ）, nimodipine （NIM）, 06 - Ben- zyl- guanine {O6 - BG} on glioma cell lines in vitro, to investigate the mechanism of resistance and search for effective multi- drug resistance reversal agents for overcoming the resistance of produce and to enhance the effect of chemotherapy and increase glioma cell sensitivity to chemotherapy drugs for the maximum in- hibiting glioma cells proliferation and delaying tumor recurrence. Methods The MGMT protein expression in glioma cells in were observed by immunocytochemical detection, and then compared with MGMT 06 - Benzyl- guanine expression differences before and after treatment. Respectively nimodipine and 06 - Ben- zyl-guanine as a resistance reversal agent, temozolomide alone or in combination with the application in vitro U251 glioma cells. Cell inhibition rate was detected By MTT assay; the inhibitions of temozolomide, nimodipine, combined with 06 - Benzyl- guanine were observed in the glioma cells. Results Compared with the control group, MGMT expression was significantly reduced after treatment by using 6 - Benzyl -guanine. MTT assay was used to detect the inhibition of TMZ, TMZ ＋ NIM, TMZ ＋ 06 - BG group and NIM ＋ TMZ ＋ 06 - BG group to U251 glioma cells, the results showed that the group of glioma cells in vitro as TMZ ＋ NIM ＋ 06 - BG ioint group〉 TMZ ＋ 06 - BG〉 TMZ ＋ NIM〉TMZ. Compared with TMZ, the inhibited rates of TMZ ＋ NIM group, TMZ ＋ 06 - BG group on U251 cells were significantly increased （P^0.01） ; Similarly, TMZ group, the NIM＋ TMZ ＋ Os - BG group U251 cells was also in- hibited significantly increased compared with TMZ group, the difference was significantly different （P% 0.05）. Conclusions 06 - Benzyl- guanine can effectively inhibit the expression of MGMT, which can re- verse human glioma cell line U251 to temozolomide resistance. Nimodipine, 06 - Benzyl- guanine may in- crease temozolomide cytotoxic effect on glioma cell line U251, has good resistance reversal effect for temo- zolomide, which is expected as a clinical chemotherapy sensitizer and combination with temozolomide. The combination of temozolomide, nimodipine, and O 6 - BG has a better inhibitory effect than every both com- bined, and also stronger than temozolomide alone.