CCND1表达沉默促进胶质母细胞瘤细胞株对替莫唑胺的敏感性

目的 利用已构建的CCND1表达沉默及过表达的人源性胶质母细胞瘤细胞株SHG-44，筛选能够与CCND1协同抑制胶质母细胞瘤细胞增殖的化学治疗药物。方法 用蛋白质印迹法检测CCND1表达沉默及过表达的人源性胶质母细胞瘤细胞株SHG-44中多药耐药基因MDR1的表达产物P-糖蛋白（P-gp）及凋亡因子Bcl-2、Caspase-3的表达。使用卡莫司汀（BCNU）、洛莫司汀（CCNU）、替莫唑胺（TMZ）3种化学治疗药物分别处理CCND1过表达或表达沉默的SHG-44细胞，筛选能够与CCND1表达沉默协同抑制肿瘤细胞生长的化学治疗药物，并在人源性胶质母细胞瘤细胞株系U251中进一步验证。结果 蛋白质印迹结果示CCND1表达沉默能够下调P-gp、Bcl-2的表达，上调Caspase-3的表达（P均<0.01）。BCNU （0.05、0.25 μg/mL）、CCNU （20、80 μg/mL）处理CCND1过表达或表达沉默的SHG-44细胞的第2、3、4、5天时，细胞生长曲线的差异均无统计学意义；而在药物处理后细胞培养的第4、5天，CCND1表达沉默SHG-44细胞的生长受到TMZ （9.1 μg/mL）的抑制作用较亲代SHG-44细胞强（P<0.05）。U251细胞实验证实CCND1表达沉默促进TMZ的化学治疗敏感性。结论 CCND1表达沉默联合TMZ较两者单独作用能更有效地抑制人源性胶质母细胞瘤细胞株SHG-44的增殖，提示CCND1可能参与TMZ化学治疗耐药机制。
Objective To screen chemotherapeutic drugs that can synergistically inhibit the proliferation of glioblastoma cells with CCND1 by constructing CCND1-silenced and overexpressed human glioblastoma cell lines SHG-44. Methods SHG-44 cells with CCND1 silenced or overexpressed were constructed, and the expression of P-glycoprotein (P-gp, expression product of multidrug resistance gene MDR1) and apoptotic factors (Bcl-2, Caspase-3) in the cells were detected by Western blotting. The SHG-44 cells with CCND1 silenced or overexpressed were cultured with carmustine (BCNU), lomustine (CCNU) and temozolomide (TMZ), respectively; and then the available chemotherapeutic drugs were screened, which could synergistically inhibit the proliferation of tumor cells with CCND1 sliencing. Human glioblastoma cell lines U251 were used to verify the findings in SHG-44 cells. Results Western blotting analysis showed that CCND1 silencing significantly down-regulated the expressions of MDR1 and Bcl-2, and up-regulated the expression of Caspase-3 (all P<0.01). There were no significant differences in cell growth curves between the CCND1-silenced cells treating with BCNU (0.05 μg/mL, 0.25 μg/mL) and CCNU (20 μg/mL, 80 μg/mL) for 2, 3, 4, and 5 days; However, the proliferation of CCND1-silenced SHG-44 cells was significantly inhibited by TMZ (9.1 μg/mL) compared with parent SHG-44 cells on the 4th and 5th day after treatment (P<0.05). The findings that CCND1 silencing promoted chemosensitivity of TMZ were confirmed by U251 cell experiments. Conclusion CCND1 silencing combined with TMZ is more effective in inhibiting the proliferation of human glioblastoma cell lines SHG-44 than CCND1 silencing or TMZ alone, suggesting that CCND1 may be involved in chemotherapeutic resistance of glioblastoma cells to TMZ