雷公藤红素通过激活内质网应激介导的细胞凋亡发挥抗ER^(+)乳腺癌的作用

Celastrol exerts anti-ER^(+) breast cancer by activating endoplasmic reticulum stress to induce apoptosis

目的探讨雷公藤红素(celastrol,Cel)通过激活内质网应激介导的细胞凋亡发挥抗雌激素受体阳性(estrogen receptor positive,ER^(+))乳腺癌的作用机制。方法以ER+人乳腺癌细胞系MCF-7与T47D为研究对象,采用MTT与细胞克隆实验检测Cel对细胞活力与增殖的影响;采用Western blot检测Cel对凋亡相关蛋白表达的影响;分别采用RT-qPCR和Western blot检测Cel对内质网应激相关基因和蛋白表达的影响;体内建立MCF-7细胞裸鼠原位移植瘤模型,观察Cel对移植瘤生长的影响。结果MTT与细胞克隆实验结果显示,Cel可呈剂量依赖性抑制MCF-7与T47D细胞活力与增殖(P<0.05);Western blot结果显示,Cel可上调凋亡相关蛋白Cleaved PARP与BAX表达,并下调BCL-2表达(P<0.05);RT-qPCR结果显示,Cel可上调内质网应激相关基因IRE1α、ATF6、PERK、ATF4、CHOP、BIP的表达水平(P<0.05);Cel可诱导内质网应激标志蛋白BIP表达升高,PERK通路相关蛋白p-PERK、p-eIF2α、ATF4、CHOP表达增加,IRE1α通路相关蛋白p-IRE1α、XBP1s表达增加,ATF6通路中的Cleaved ATF6水平增加;体内实验结果显示,Cel能显著降低裸鼠原位移植瘤的体积(P<0.05),且对裸鼠体质量无明显的影响。结论Cel具有一定的抑制ER^(+)乳腺癌MCF-7及T47D细胞增殖的作用,其机制可能与激活内质网应激信号通路从而诱导细胞凋亡有关。

Objective To investigate the mechanism of celastrol(Cel)against estrogen receptor positive(ER^(+))breast cancer by activating endoplasmic reticulum stress-mediated apoptosis.Methods ER^(+) human breast cancer cell lines MCF-7 and T47D were used as research objects.MTT assay was used to detect the effect of Cel on cell viability.Western blotting were employed to detect the expression of apoptosis-related proteins.RT-qPCR and Western blotting were applied to measure the expression of endoplasmic reticulum stress-related molecules at mRNA and protein levels.A nude mouse model of orthotopic transplantation of MCF-7 cells was established to observe the effect of Cel on the growth of transplanted tumors.Results MTT results showed that Cel inhibited the viability of MCF-7 and T47D cells in a dose-dependent manner(P0.05).Western blotting showed that Cel up-regulated the expression of apoptosis-related proteins,Cleaved PARP and BAX,and down-regulated the expression of BCL-2(P0.05).RT-qPCR indicated that Cel up-regulated the mRNA levels of endoplasmic reticulum stress-related genes IRE1α,ATF6,PERK,ATF4,CHOP and BIP(P0.05).Western blotting revealed that Cel treatment resulted in increased expression levels of endoplasmic reticulum stress marker BIP,of PERK pathway related proteins p-PERK,p-eIF2α,ATF4 and CHOP,of IRE1αpathway associated proteins p-IRE1αand XBP1s,and of ATF6 pathway associated protein Cleared ATF6.In vivo experiment results showed that Cel significantly reduced the volume of orthotopic transplanted tumor in nude mice(P0.05),and had no significant effect on their body weight.Conclusion Cel has a certain inhibitory effect on the proliferation of ER^(+) breast cancer MCF-7 and T47D cells,and its mechanism may be related to the activation of endoplasmic reticulum stress signaling pathway to induce apoptosis.