纺锤丝毒性药物诱导的多倍体肿瘤细胞对常见化疗药物的敏感性

Sensitivity to chemotherapeutic drugs of polyploid tumor cells induced by a spindle poison nocodazole

目的探讨纺锤丝毒性药物诱导的多倍体肿瘤细胞对化疗药物的敏感性及其可能的分子机制。方法以100ng／ml纺锤丝毒性药物诺考达唑处理人乳腺癌MDA-MB-231细胞，流式细胞仪分选获得多倍体肿瘤细胞T-MDA-MB．231，观察T-MDA-MB-231细胞的形态学变化和增殖分裂情况。采用四甲基偶氮唑蓝（Mrrr）法检测紫杉醇、多西他赛、长春新碱、表阿霉素、氟尿嘧啶、依托泊甙和奥沙利铂对T—MDA—MB．231和MDA．MB-231细胞的增殖抑制作用，并计算出不同药物的半数抑制浓度（IC50）。以siRNA沉默T—MDA—MB-231细胞中Bcl-2mRNA的表达，采用MT＇F法检测siRNA干扰后不同化疗药物对T—MDA—MB-231细胞的增殖抑制作用，并计算Ic50。结果T—MDA—MB-231细胞在体外能稳定生长，较MDA—MB-231细胞体积增大，其增殖分裂速度明显低于MDA—MB-231细胞（P〈0．05）。紫杉醇、多西他赛、长春新碱、奥沙利铂、氟尿嘧啶和表阿霉素对T—MDA—MB-231细胞的IC∞分另0为（6．37±0．07）μmol／L、（32．98±1．48）μmol／L、（35．28±1．66）μmol／L、（19．07±0．45）μmol／L、（85．49±3．21）μmol／L和（0．53±0．06）μmol／L，均明显高于MDA-MB-231细胞（均P〈0．05）；依托泊甙对T-MDA—MB-231细胞的IC50为（2．85±0．50）μmol／L，明显低于MDA-MB-231细胞（P〈0．05）。以siRNA沉默T-MDA—MB-231细胞中Bcl-2mRNA的表达后，多西他赛对T-MDA-MB-231细胞的I‰[（21．52±0．68）μmol／L]明显降低（P〈0．05），依托泊甙对T-MDA-MB-231细胞的IC50[（19．59±0．48）μmol／L]明显增高（P〈0．05）。结论纺锤丝毒性药物诱导的多倍体肿瘤细胞T-MDA．MB-231对多数化疗药物更加耐药。下调Bcl-2mRNA的表达可增加多倍体肿瘤细胞对多西他赛的敏感性，Bcl-2的高表达可能为多倍体肿瘤细胞耐药的机制之一。多倍体肿瘤细胞对依托泊甙相对敏感，依托泊甙可能成为治疗多倍体肿瘤的有效药物。

Objective To investigate the changes of drug sensitivity of spindle poison-induced polyploid tumor cells to chemotherapeutic agents and its possible mechanism. Methods Nocodazole in a dose of 100 ng/ml was used to induce polyploidization in a breast cancer cell line MDA-MB-231 cells. The polyploid cells （T-MDA-MB-231） were sorted by flow cytometry. The morphological changes and proliferation of T-MDA-MB-231 cells were compared with that of MDA-MB-231 cells. The cell growth inhibition was assessed by MTF assay. The cells were treated with paclitaxel, docetaxel, vincristine, epirubicin, 5-Fu, VP16 and oxaliplatin, respectivelhy. Those cells were labeled with annexin V-FITC/PI and analyzed by flow cytometry. Bcl-2 was knocked down in T-MDA-MB-231 cells using SiRNA and their growth inhibition was evaluated by MTT assay to evaluate the reversing effect of Bcl-2-silencing on drug resistance. Results The polyploid T-MDA-MB-231 cells grew in vitro continuously and maintained constant DNA content. They had a larger cell size, and grew more slowly than MDA-MB-231 cells. The IC50） of T- MDA-MB-231 cells were significantly higher than that of the MDA-MB-231 cells ： paclitaxel ： （ 6.37 ±0.07 ）vs. （2.05 ±0. 83 ） μ mol/L; docetaxel： （ 32.98 ±1.48 ） vs. （ 11.95±0. 98 ） μmol/L; vincristine： （ 35.28 ± 1.66） vs. （ 14.58 ± 0.94 ） μmol/L; oxaliplatin： （ 19.07±0.45 ） vs. （ 9.75 ± 1.05 ） μmol/L; 5 - Fu： （85.49 ±3.21） vs. （31.35 ± 1.51 ） μmol/L; and epirubicin： （0. 53 ±0.06） vs. （0.15 ±0. 01 ） μmol/L, （all P 〈 0. 05）. The IC50.） of VP16 in T-MDA-MB-231 cells was （2. 85 ± 0. 50）μmol/L, significantly lower than the （ 12. 20± 1.55 ） μmol/L in MDA-MB-231 cells （ P 〈 0. 05 ）, and that of T- MDA-MB-231 cells after Bcl-2-knocked down by siRNA was （ 19.59 ±0. 48 ） μmol/L, significantly higher than the （12.20 ±1.55） μmol/L in the MDA-MB-231 cells （P 〈0. 05）. The IC50（s） of docetaxel of T- MDA-MB-231 cells after Bcl-2-knocked down by siRNA was （ 21. 52±0. 68 ） μ mol/L, significantly decreased and lower than that before Bcl-2 silencing （ 32.98±1.48 ） μmol/L. Conclusions Our results indicate that polyploid tumor cells induced by spindle poison Nocedazole are more resistant to most of chemotherapeutic drugs. Downregulation of Bel-2 increases the sensitivity of polyploid cells to docetaxel. The high expression of Bcl-2 may be one of the drug resistance mechanisms of polyploid tumor cells. The polyploid tumor cells are relatively sensitive to VP16, suggesting that VP16 might be an effective candidate drug for treatment of chemoresistant polyploid tumors.