柳氮磺吡啶诱导乳腺癌ZR-75-1细胞发生铁死亡及其机制研究

Sulfasalazine induces ferroptosis of breast cancer ZR-75-1 cells and its mechanism

目的:观察柳氮磺吡啶(sulfasalazine)对乳腺癌ZR-75-1细胞铁死亡(ferroptosis)的激活,并探讨其可能的机制。方法:采用不同浓度的柳氮磺吡啶(0、0.5、1.0和2.0 mmol/L)处理乳腺癌ZR-75-1细胞。随后,在光学显微镜下观察细胞形态的变化;CCK-8法检测细胞的增殖抑制率;蛋白质印迹法检测谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)及胱氨酸-谷氨酸反向转运体亚基xCT蛋白的表达水平;采用荧光探针标记不同浓度柳氮磺吡啶处理后的ZR-75-1细胞,通过流式细胞仪检测细胞内的荧光强度以监测活性氧(reactive oxygen specie,ROS)含量的变化;实时荧光定量PCR法检测二价金属离子转运体1(divalent metal transporter 1,DMT1)mRNA的表达水平。结果 :柳氮磺吡啶可引起ZR-75-1细胞形态学变化和死亡。低浓度柳氮磺吡啶对ZR-75-1细胞增殖的影响不大(P> 0.05),中浓度和高浓度组的柳氮磺吡啶对细胞增殖有明显的抑制作用(P值均<0.05)。较高浓度的柳氮磺吡啶可以抑制细胞内GPX4和xCT蛋白的表达水平(P值均<0.05),也可以促进ZR-75-1细胞内ROS的累积(P值均<0.05)。柳氮磺吡啶能提高ZR-75-1细胞内DMT1 mRNA的表达水平(P <0.05)。结论 :柳氮磺吡啶通过抑制xCT和GPX4蛋白表达使ZR-75-1细胞中ROS大量累积,细胞发生铁死亡,DMT1的激活可能是其机制之一。

Objective： To observe the activation effect of sulfasalazine on ferroptosis of breast cancer ZR-75-1 cells, and to explore its possible mechanism.Methods： The different concentrations of sulfasalazine（0, 1.0, 0.5, 1.0, and 2.0 mmol/L） were used to treat ZR-75-1 cells. The morphological change of ZR-75-1 cells was observed under an inverted microscope. The proliferation inhibitory rate of ZR-75-1 cells was detected by CCK-8 method. The expression levels of glutathione peroxidase 4（GPX4） and cysteine-glutamate antiporter subunit（xCT） proteins in ZR-75-1 cells were detected by Western blotting. The fluorescent probe was used to label the ZR-75-1 cells treated with different concentrations of sulfasalazine, then the change of reactive oxygen specie（ROS） level（as indicated by fluorescence intensity） was detected by FCM method. The expression level of divalent metal transporter 1（DMT1） mRNA was detected by real-time fluorescent quantitative PCR.Results： Sulfasalazine caused the morphological changes and death of ZR-75-1 cells. The low doses of sulfasalazine had little effect on the proliferation of ZR-75-1 cells（P〉 0.05）, but the high concentrations of sulfasalazine inhibited cell proliferation significantly（P〈 0.05）. The high concentrations of sulfasalazine inhibited the expressions of GPX4 and xCT proteins in ZR-75-1 cells（both P〈 0.05）, and also promoted the accumulation of intracellular ROS（P〈 0.05）. Sulfasalazine increased the expression of DMT1 mRNA in ZR-75-1 cells（P〈 0.05）.Conclusion： Sulfasalazine can increase the accumulation of ROS by inhibiting the expressions of GPX4 and xCT proteins to induce the ferroptosis of ZR-75-1 cells. In which, the activation of DMT1 expression may be one of the mechanism.