替莫唑胺联合粉防己碱对人脑胶质瘤U87细胞的抑制作用

Inhibitory effect of temozolomide combined with tetrandrine on human glioblastoma U87 cells

目的探讨粉防己碱与替莫唑胺联合用药对人脑胶质瘤细胞U87细胞存活、克隆形成、迁移能力及凋亡的影响。方法采用CCK-8法检测粉防己碱8～64μmol·L^-1,或替莫唑胺50～400μmol·L^-1,或替莫唑胺＋粉防己碱3.2和6.4μmol·L^-1作用24 h后细胞存活率。粉防己碱6.4μmol·L^-1、替莫唑胺100μmol·L^-1或替莫唑胺＋粉防己碱3.2和6.4μmol·L^-1作用24 h后,Giemsa染色检测细胞的克隆形成;Transwell小室检测细胞迁移能力;流式细胞术AnnexinⅤ/PI双染法检测细胞凋亡;Western蛋白质印迹法检测Bcl-XL、活化胱天蛋白酶3及活化聚ADP核糖聚合酶（PARP）蛋白表达。结果 CCK-8实验结果显示,单用粉防己碱和替莫唑胺均能抑制U87细胞的生长,且具有浓度依赖性（r=0.903,P〈0.05）,替莫唑胺100μmol·L^-1＋粉防己碱3.2和6.4μmol·L^-1抑制率高于两药单用,经分析两者作用为相加。替莫唑胺可抑制U87细胞的克隆形成和迁移能力,两药联用时比单用替莫唑胺抑制作用更明显（P〈0.05）;与单用替莫唑胺相比,两药联用时抗凋亡蛋白Bcl-XL明显减少,执行凋亡的活化的剪切胱天蛋白酶3蛋白及剪切PARP明显增多。结论粉防己碱联用替莫唑胺能明显抑制人脑胶质瘤U87细胞的生长、克隆形成及迁移能力,并诱导激活胱天蛋白酶3依赖的细胞凋亡。

OBJECTIVE To observe the effect of temozolomide（ TMZ） in combination with tetrandrine（ TET） on cell viability,colony formation,migration and cell apoptosis of human glioblastoma U87 cells. METHODS The viability of U87 cells treated with TET（ 8- 64 μmol·L^-1）,TMZ（ 50-400 μmol·L^-1） and TMZ combined with TET（ 3.2,6.4 μmol·L^-1） was detected by cytotoxicity assays with Cell Counting Kit-8（ CCK-8）,the colony formation was detected by Giemsa staining,cell migration ability was detected by Transwell migration assay,cell apoptosis was assayed by flow cytometry using Annexin Ⅴ / PI double staining,and the expression of apoptosis-related proteins expression was detected by Western blotting. RESULTS The data of CCK-8 showed that TET（ r = 0. 903,P〈0. 05） or TMZ（ r =0.995,P〈0.05） could inhibit U87 cell viability alone in a concentration-dependent manner. The cell viability inhibition rate of U87 cells by TMZ combined with TET was higher than by TMZ or TET alone. Data showed that the effect of TMZ combined with TET was additive. TMZ 100 μmol·L^-1inhibited U87 cell colony formation and migration ablility compared with normal control. The inhibition rate of U87 cells by TMZ 100 μmol·L^-1combined with TET（ 3. 2 and 6. 4 μmol·L^-1） was more significant than by TMZ alone（ P〈0. 05）. Compared with TMZ alone,TMZ combined with TET（ 3. 2 and 6. 4 μmol·L^-1） significantly down-regulated the expression of anti-apoptotic protein Bcl-XL,but significantly up-regulated the expression of cleaved caspase 3 protein and cleaved poly（ ADP-ribose） polymerase. CONCLUSION TET combined with TMZ can inhibit U87 cell viability,colony formation and migration by activating caspase-dependent apoptotic pathway,resulting in apoptosis.