Kalyanaraman Aparna, Gnanasampanthapandian Dhanavathy, Shanmughan Prasad, Kishore Puneet, Ramalingam Satish, Arunachalam Rathnaswami, Jayaraman Selvaraj, Kaliappan Ilango, Munuswamy-Ramanujam Ganesh, Ramachandran Ilangovan, Sambandam Yuvaraj, Anbalagan Muralidharan, Chandrakesan Parthasarathy, Palaniyandi Kanagaraj
Cancer Science Laboratory, Department of Biotechnology, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, Kancheepuram, India.
Department of Genetic Engineering, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, Kancheepuram, India.
Stem Cell Investig. 2020 Nov 3;7:20. doi: 10.21037/sci-2020-020. eCollection 2020.
To understand the mechanism underlying tamoxifen-induced multidrug resistance (MDR) and stem-like phenotypes in breast cancer cells, we treated the MCF-7 cells with 4-hydroxy-tamoxifen (TAM) for 6 months continuously and established MCF-7 tamoxifen resistance (TR) phenotypes.
In the present study, the following methods were used: cell viability assay, colony formation, cell cycle analysis, ALDEFLUOR assay, mammosphere formation assay, chromatin immunoprecipitation (ChIP) assay, PCR array, western blot analysis and quantitative reverse transcription polymerase chain reaction (QRT-PCR).
The expression of ERα was significantly higher in MCF7-TR cells when compared with parental MCF-7 cells. MCF7-TR cells exposed to TAM showed a significant increase in the proliferation and rate of colony formation. The number of cancer stem cells was higher in MCF7-TR cells as observed by the increase in the number of ALDH+ cells. Furthermore, the number of mammospheres formed from the FACS-sorted ALDH+ cells was higher in MCF7-TR cells. Using PCR array analysis, we were able to identify that the long-term exposure of TAM leads to alterations in the epigenetic and MDR stem cell marker genes. Furthermore, western blot analysis demonstrated elevated levels of Notch-1 expression in MCF-TR cells compared with MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay revealed that Notch-1 enhanced the cyclin D1 expression significantly in these cells. In addition, we observed that MCF7-TR cells were resistant to doxorubicin but not the MCF-7 cells.
In the present study, we conclude that the treatment with tamoxifen induces multiple epigenetic alterations that lead to the development of MDR and stem-like phenotypes in breast cancers. Therefore, our study provides better insights to develop novel treatment regime to control the progression of breast cancer.
为了解他莫昔芬诱导乳腺癌细胞多药耐药（MDR）及干细胞样表型的潜在机制，我们用4-羟基他莫昔芬（TAM）连续处理MCF-7细胞6个月，建立了MCF-7他莫昔芬耐药（TR）表型。
在本研究中，采用了以下方法：细胞活力测定、集落形成、细胞周期分析、ALDEFLUOR测定、乳腺球形成测定、染色质免疫沉淀（ChIP）测定、PCR阵列、蛋白质印迹分析和定量逆转录聚合酶链反应（QRT-PCR）。
与亲本MCF-7细胞相比，MCF7-TR细胞中ERα的表达显著更高。暴露于TAM的MCF7-TR细胞的增殖和集落形成率显著增加。通过ALDH+细胞数量的增加观察到，MCF7-TR细胞中的癌症干细胞数量更高。此外，MCF7-TR细胞中由FACS分选的ALDH+细胞形成的乳腺球数量更多。使用PCR阵列分析，我们能够确定TAM的长期暴露会导致表观遗传和MDR干细胞标记基因的改变。此外，蛋白质印迹分析表明，与MCF-7细胞相比，MCF-TR细胞中Notch-1的表达水平升高。染色质免疫沉淀（ChIP）测定显示，Notch-1在这些细胞中显著增强了细胞周期蛋白D1的表达。此外，我们观察到MCF7-TR细胞对阿霉素耐药，但MCF-7细胞不耐药。
在本研究中，我们得出结论，他莫昔芬治疗会诱导多种表观遗传改变，导致乳腺癌中MDR和干细胞样表型的发展。因此，我们的研究为开发控制乳腺癌进展的新治疗方案提供了更好的见解。
