反馈激活STAT3调控HER2阳性乳腺癌细胞拉帕替尼耐药的机制

胡晓红; 柏华; 段娟娟; 雷秀; 张启芳

doi:10.3971/j.issn.1000-8578.2019.18.1410

反馈激活STAT3调控HER2阳性乳腺癌细胞拉帕替尼耐药的机制

Feedback Activation of STAT3 Confers Resistance of HER2-positive Breast Cancer Cells to Lapatinib

摘要

摘要:
目的探讨白介素-6（IL-6）在HER2阳性BT-474乳腺癌细胞拉帕替尼耐药中的作用及其相关分子机制。
方法建立BT-474耐拉帕替尼细胞株（BT-474^R）。Western blot检测耐药标志蛋白P-gp的表达，MTT法检测BT-474^R细胞的IC₅₀。Caspase-Glo^® 3/7法检测拉帕替尼BT-474^R与BT-474细胞的Caspase3/7酶活性的变化。Western blot法检测IL-6、p-STAT3、STAT3、Cleaved Caspase 3的蛋白表达水平；RNAi法沉默STAT3基因表达；Caspase-Glo^® 3/7法和Annexin V-FITC/PI染色检测沉默后细胞凋亡程度。
结果 BT-474^R组P-gp表达水平显著高于BT-474组（P < 0.05）。拉帕替尼增强STAT3活性，沉默STAT3基因可恢复BT-474^R细胞对拉帕替尼的敏感度、增加cleaved caspase 3蛋白表达水平和Caspase 3/7酶的活性（P < 0.01）。沉默STAT3增强了拉帕替尼诱导的耐药细胞凋亡（P < 0.01）。拉帕替尼诱导的IL-6表达和分泌激活STAT3，用IL-6抗体抑制拉帕替尼诱导的IL-6，可以阻止拉帕替尼刺激STAT3活性。
结论拉帕替尼通过诱导BT-474细胞分泌IL-6激活STAT3信号通路，增加HER2阳性乳腺癌细胞对拉帕替尼的耐药性。

Abstract:
Objective To explore the role of interleukin-6 (IL-6) in lapatinib-resistant HER2-positive breast cancer cells BT-474.
Methods Lapatinib-resistant BT-474 (BT-474^R) cells were established. Western blot was used to test P-gp expression. The growth inhibition rate(IC₅₀) was tested by MTT assay, and Caspase 3/7 enzymatic activity levels in parental BT474 and BT-474^R cells were investigated by Caspase-Glo^® 3/7 assay kit. The apoptotic rate of BT-474^R cells infected by siRNA STAT3 was tested by flow cytometry. The protein expression levels of IL-6, P-STAT3, STAT3 and Cleaved Caspase 3 were assayed by Western blot. Caspase 3/7 Glo assay and Annexin V-FITC/PI were used to assay cellular apoptosis.
Results BT-474^R cells displayed significantly elevated P-gp expression when compared with BT-474 cells. Lapatinib treatment stimulated signal transducer and activator of transcription 3 (STAT3) activity, and STAT3 activity was also upregulated in BT-474^R cells. STAT3 silencing not only remarkably restored sensitivity to lapatinib and enhanced lapatinib-induced cleaved caspase 3 and cspase3/7 enzymatic activity, but also increased the apoptosis of BT-474^R cells (P < 0.01). Furthermore, lapatinib-mediated IL-6 secretion regulated STAT3 activity. Blocking IL-6 using anti-IL-6 antibody abrogated lapatinib-induced STAT3 activity.
Conclusion Lapatinib-induced IL-6 secretion enhances the resistance through STAT3 signaling in HER2-positive breast cancer cells.

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