追毒方调控c-JUN抑制糖基化异常逆转三阴性乳腺癌耐药的机制

Mechanism of Zhuidu Formula Regulating c-JUN,Inhibiting Abnormal Glycosylation,and Reversing Drug Resistance of Triple Negative Breast Cancer

目的从c-JUN调控的糖基化角度探究追毒方逆转三阴性乳腺癌(TNBC)耐药的机制。方法MTT检测细胞增殖,采用Western blot检测c-JUN、β3GnT8和ppGalNAc-T1/2、CD147、耐药蛋白BCRP和MDR1的表达。采用转染c-JUN质粒建立过表达c-JUN的MDA-MB-231/ADR细胞。体内实验采用耐药TNBC原位移植裸鼠模型。结果追毒方可显著抑制MDA-MB-231/ADR细胞的增殖(P<0.01),具有时效和量效关系;可明显抑制耐药TNBC原位移植裸鼠的肿瘤质量(P<0.01);降低耐药蛋白BCRP和MDR1表达(P<0.01),并同时明显降低c-JUN、β3GnT8和ppGalNAc-T1/2、CD147蛋白的表达(P<0.05)。过表达c-JUN后,与空白质粒对照组比较,β3GnT8、ppGalNAc T1/2、CD147、BCRP和MDR1的表达均显示增加(P<0.05,P<0.01)。而空白质粒对上述蛋白的表达无影响。追毒方可以显著降低过表达c-JUN后的上述蛋白的表达,但其表达水平还是显著高于空白质粒加药组(P<0.05,P<0.01)。结论追毒方通过作用于调控因子c-JUN,下调β3GnT8和ppGalNAc-T1/2的激活,从而抑制CD147的糖基化修饰,导致耐药蛋白BCRP和MDR1下调而逆转TNBC耐药。

OBJECTIVE To investigate the mechanism of reversal of drug resistance in triple negative breast cancer(TNBC)by Zhuidu Formula from the perspective of c-JUN-regulated glycosylation.METHODS MTT was used to detect cell proliferation.Western blot was used to detect the expression of c-JUN,β3GnT8,ppGalNAc-T1/2,CD147,and the drug resistance proteins BCRP and MDR1.MDA-MB-231/ADR cells overexpressing c-JUN were established using transfected c-JUN plasmids.In vivo experiments were performed by in situ transplantation of drug-resistant TNBC into nude mice.RESULTS Zhuidu Formula can significantly inhibit the proliferation of MDA-MB-231/ADR cells(P<0.01),with a time-effect and dose-effect relationship.It also significantly inhibited the tumor weight of drug-resistant TNBC transplanted nude mice in situ(P<0.01),reduced the expression of drug-resistant proteins BCRP and MDR1(P<0.01),and significantly decreased the expression of c-JUN,β3GnT8,ppGalNAc-T1/2,CD147 proteins(P<0.01).After overexpression of c-JUN,the expression ofβ3GnT8,ppGalNAc T1/2,CD147,BCRP and MDR1 all showed an increase compared with the blank plasmid control group(P<0.05,P<0.01).The blank plasmid had no effect on the expression of the above proteins(compared with the blank control group,P>0.05).Zhuidu Formula can significantly reduce the expression of the above proteins after overexpressing c-JUN,but the expression levels were still significantly higher than those in the blank plasmid group(P<0.05,P<0.01).CONCLUSION By acting on the regulatory factor c-JUN,Zhuidu Formula down-regulated the activation ofβ3GnT8 and ppGalNAc-T1/2,thereby inhibiting the glycosylation modification of CD147 and leading to the down-regulation of drug-resistant proteins BCRP and MDR1 to reverse TNBC drug resistance.