| 摘要: |
| 目的 了解雌激素受体α (estrogen receptorα,ERα)通路代偿性激活在乳腺癌细胞BT474的拉帕替尼获得性耐药发生过程中的作用和可能机制。 方法 利用real-time PCR和蛋白质印迹检测BT474被拉帕替尼作用后人表皮生长因子受体2(HER2)通路和ERα通路活性的改变；利用拉帕替尼浓度递增、持续培养的方法建立乳腺癌细胞BT474的拉帕替尼获得性耐药细胞(rBT474)模型；采用流式细胞术检测拉帕替尼对rBT474细胞凋亡的影响,进一步以蛋白质印迹法分析BT474和rBT474在HER2通路和ER通路上的差别；应用MTT法检测rBT474在拉帕替尼和氟维司群作用下的生长情况；利用克隆形成实验观察双靶点治疗对预防拉帕替尼获得性耐药的可能性。 结果 Real-time PCR和蛋白质印迹检测显示拉帕替尼抑制BT474细胞HER2磷酸化,同时诱导叉头蛋白3A (FOXO3a)和孕激素受体(PR)的表达升高；成功获得的耐药细胞株rBT474在含有5 μmol/L拉帕替尼的培养液中仍可持续生长；蛋白质印迹结果显示,rBT474细胞与BT474细胞相比,其磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/AKT)通路受抑制,而促分裂素原活化蛋白激酶(MAPK)通路激活,ER通路的活化更加明显；MTT法检测结果显示,与单用拉帕替尼相比,联合使用拉帕替尼和氟维司群可抑制rBT474细胞活力(P<0.01)；克隆形成实验结果表明,与二甲亚砜组、拉帕替尼组和氟维司群组相比,拉帕替尼和氟维司群联合用药组可抑制BT474细胞克隆形成(P<0.01),具有预防获得性耐药发生的可能。 结论 ERα通路的代偿性激活可能是导致HER2(+)/ERα(+)的乳腺癌细胞对拉帕替尼产生获得性耐药的机制之一,而PI3K/AKT抑制和MAPK激活可能是ERα代偿激活的主要原因。 |
| 关键词: 拉帕替尼；乳腺肿瘤；肿瘤抗药性；雌激素受体α erbB-2受体 |
| DOI：10.3724/SP.J.1008.2013.00616 |
| 投稿时间：2013-01-17修订日期：2013-02-22 |
| 基金项目:国家自然科学基金(51003078),上海市科学技术委员会资助课题(12140902302),上海市卫生局资助科研课题(2008133). |
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| Compensatory activation of estrogen receptor α signaling in acquired resistance to lapatinib of HER2-overexpressing/ERα-positive breast cancer cells |
| LI Zhe1*,CHANG Tao1,SHI Lin-xiang1,FANG Lin1,YANG Sheng-sheng2*,FANG Guo-en3 |
(1. Department of Thyroid and Breast Surgery, the Tenth Hospital, Tongji University School of Medicine, Shanghai 200072, China
2. Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China
3. Department of General Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
*Corresponding authors.) |
| Abstract: |
| Objective To investigate the role of compensatory activation of estrogen receptor α (ERα) signaling in acquired resistance to lapatinib of breast cancer cells BT474 and the related mechanism. Methods Real-time PCR and Western blotting analysis were used to determine the changes of HER2 and ERα pathways in BT474 cells treated by lapatinib. Acquired resistant model of rBT474 cells was induced with increasing concentrations of lapatinib (from 0.25 μmol/L to 5 μmol/L); the apoptosis in rBT474 cells was determined by flow cytometry. Western blotting analysis was used to evaluate the differences between BT474 and rBT474 in the HER2 and ER pathways. The growth of rBT474 cells treated by lapatinib and/or fulvestrant was detected by MTT assay, and colony formation was used to observe the possibility of preventing acquired resistance to lapatinib in BT474 cells by double targets therapy. Results The results of real-time PCR and Western blotting analysis showed that Lapatinib inhibited phosphorylation of HER2 and induced expression of forkhead-box protein O 3A (FOXO3a) and progesterone receptor (PR) in BT474 cells. Acquired resistance cell model of rBT474 was established in a 5 μmol/L lapatinib condition. Western blotting analysis showed that the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway was inhibited in rBT474 cells compared with that in the BT474 cells, while the mitogen-activated protein kinase (MAPK) pathways, especially the ER pathway, were activated in the BT474 cells. MTT results showed that, Lapatinib combined with fulvestrant had a significantly greater inhibition on rBT474 cell vitality compared with lapatinib alone (P<0.01). Colony formation results also showed that combination of lapatinib and fulvestrant had a significantly greater inhibition effect against colon formation of BT474 cells compared with each drug alone and DMSO (P<0.01), showing a possible prevention ability against acquired resistance.Conclusion Compensatory activation of estrogen receptor α signaling might be one of the mechanisms of acquired resistance to lapatinib in HER2-overexpressing/ERα-positive breast cancer cells, and inhibition of PI3K/AKT and activation of MAPK might be the main reason for compensatory activation of ERα. |
| Key words: lapatinib breast neoplasms neoplasm drug resistance estrogen receptor alpha erbB-2 receptor |
