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目的研究海洋新型蒽环类化合物D19对人乳腺癌细胞MDA-MB-435的增殖和细胞周期的影响及其相关的分子机制。方法以0.5μmol/L的D19对MDA-MB-435经不同时间(0、1、2、3、4 d)处理后,以不加D19的细胞作为对照组,用MTT方法(四甲基偶氮唑蓝比色实验)检测不同孔的吸光率,分析D19对乳腺癌细胞增殖的影响。采用western blotting检测以0.5μmol/L的D19作用不同时间(0、16、24、48 h)后,细胞周期调节相关蛋白浓度及磷酸化水平的变化。进一步采用western blotting检测0.5μmol/L的D19作用不同时间(0、4、8、16、24、36、48 h)对与乳腺癌和细胞周期都密切相关的蛋白ATM的磷酸化水平的变化。结果 MTT实验显示D19能抑制MDA-MB-435细胞增殖。并且western blotting检测发现,随着D19作用时间的延长,Cyclin D1和CDK4表达水平逐步降低,Rb蛋白的磷酸化水平逐步降低,呈时间依赖性,且ATM磷酸化水平也随时间增加而增高。结论 D19能抑制MDA-MB-435细胞增殖,可阻滞MDA-MB-435细胞周期,其作用机制可能是通过激活ATM,从而抑制其下游的细胞周期调控蛋白Cyclin D1、CDK4的表达以及Rb的磷酸化来实现的。
Objective To study the effects of novel marine anthracycline D19 on the proliferation and cell cycle of human breast cancer cell line MDA-MB-435 and its related molecular mechanisms. Methods MDA-MB-435 cells were treated with 0.5 micromol / L D19 for different times (0, 1, 2, 3, 4 d) Azitrazyl blue colorimetric assay) to detect the absorbance of different wells, analysis of D19 breast cancer cell proliferation. Western blotting was used to detect the changes of cell cycle related proteins and phosphorylation levels after treated with 0.5 μmol / L D19 for different time (0, 16, 24, 48 h). Western blotting was used to detect the changes of phosphorylation of ATM at different times (0, 4, 8, 16, 24, 36, 48 h) with breast cancer cells and cell cycle at the D19 concentration of 0.5 μmol / L. Results MTT assay showed that D19 could inhibit the proliferation of MDA-MB-435 cells. Western blotting showed that the expression of Cyclin D1 and CDK4 decreased gradually with the prolongation of D19, and the phosphorylation of Rb decreased gradually and in a time-dependent manner. The phosphorylation of ATM increased with time. Conclusions D19 can inhibit the proliferation of MDA-MB-435 cells and block the cell cycle of MDA-MB-435 cells. Its mechanism may be through the activation of ATM, thereby inhibiting the expression of cyclin D1, CDK4 and Rb Phosphorylation to achieve.