MCL-1和FBW7蛋白在纺锤丝毒性药物诱导的乳腺癌多倍体中的表达及临床意义

Expression and clinical significance of MCL-1 and FBW7 proteins in breast cancer polyploid induced by spindle poisons

目的探讨MCL-1和FBW7蛋白在纺锤丝毒性药物诱导的乳腺癌多倍体中的表达及临床意义。方法 (1)以纺锤丝毒性药物Nocodazole处理人乳腺癌MDA-MB-231细胞,显微镜下观察细胞形态学变化,并于0、6、12、24、48及72 h收获细胞,流式细胞术检测细胞周期和染色体倍体变化,Western blot检测细胞中FBW7和MCL-1蛋白的表达。(2)用多激酶抑制剂索拉非尼(Sorafenib)分别与Nocodazole、紫杉醇(Taxol)联合或单独处理细胞,Westernblot检测处理48 h后各组细胞中MCL-1蛋白的表达,流式细胞术检测处理48 h细胞周期和染色体倍体变化,MTT法检测处理48、72 h细胞增殖情况。结果 (1)Nocodazole处理后,细胞出现体积增大、核大的多倍体形态学改变,0、6、12、24、48及72 h八倍体细胞所占百分比分别为(0.8±0.2)%、(8.5±2.3)%、(7.8±2.0)%、(9.9±0.9)%、(28.2±0.8)%及(35.1±4.9)%,逐渐增加(P<0.001),48 h后多倍体(四倍体和八倍体)细胞数高达(97.6±0.7)%;随时间延长,FBW7蛋白表达量减少,MCL-1蛋白表达量增加。(2)处理细胞48 h后,Nocodazole+Sorafenib组较Nocodazole组、Taxol+Sorafenib组较Taxol组MCL-1蛋白表达量均明显减少,多倍体细胞均减少,细胞生长增殖率下降(P<0.05)。结论 FBW7蛋白低表达,MCL-1蛋白高表达与乳腺癌多倍体细胞的形成密切相关;Sorafenib抑制MCL-1蛋白表达,可能减少多倍体肿瘤形成。

Objective To investigate the expression and clinical significance of myeloid cell leukemia-1 （MCL-1） and F-box and WD repeat domain-containing 7 （FBW7） in breast cancer polyploid induced by spindle poisons. Methods （1） Nocodazole spindle poison was used to treat breast cancer cell MDA-MB-231. The morphological changes of cells were observed under microscope, and cells were harvested in 0, 6, 12, 24, 48 and 72 h. The cell cycle and DNA-ploidy changes were examined by flow cytometry. The expressions of FBW7 and MCL-1 proteins were detected by Western blot assay. （2） A multikinase inhibitor （Sorafenib） with Nocodazole or Taxol was used to treat MDA-MB-231 cells. MCL-1 protein expression was detected by Western blot assay after 48 h treatment. The cell cycle and DNA-ploidy changes were examined by flow cytometry after 48 h treatment. MTT method was used to observe cell proliferation after 48 and 72 h treatment. Results （1）After treatment by Nocodazole, polyploid characteristics of large cell size and nucleus were appeared. The percentages of octaploid were （0.8±0.2）%, （8.5±2.3）%, （7.8±2.0）%, （9.9±0.9）%, （28.2±0.8）%and （35.1±4.9）%after 0, 6, 12, 24, 48 and 72 h treatment, showing the increasing trend in turn （P〈0.001）. The number of polyploidy （tetraploid and octaploid） cells was as high as （97.6±0.7）%after 48 h treatment. The expression level of FBW7 protein was decreased significantly but the expression of MCL-1 protein was increased significantly after 48 h treatment. （2） After 48 h treatment, the expression level of MCL-1 protein, polyploidy percentage and cell proliferation decreased significantly in Nocodazole＋Sorafenib group and Taxol＋Sorafenib group compared with those of Nocodazole group and Taxol group （P〈0.05）. Conclusion The lower expression of FBW7 protein and over-expression of MCL-1 protein are correlated with the formation of breast cancer polyploidy. Sorafenib can reduce polyploid tumor cells by inhibiting MCL-1 protein expression.