Aparna Kalyanaraman1, Dhanavathy Gnanasampanthapandian1, Prasad Shanmughan1, Puneet Kishore1, Satish Ramalingam2, Rathnaswami Arunachalam3, Selvaraj Jayaraman4, Ilango Kaliappan5, Ganesh Munuswamy-Ramanujam6, Ilangovan Ramachandran7, Yuvaraj Sambandam7, Muralidharan Anbalagan8, Parthasarathy Chandrakesan9, Kanagaraj Palaniyandi1. 1. Cancer Science Laboratory, Department of Biotechnology, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, Kancheepuram, India. 2. Department of Genetic Engineering, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, Kancheepuram, India. 3. Department of Surgical Gastroenterology, SRM Medical College Hospital and Research Center, Kattankulathur, Kancheepuram, India. 4. Department of Biochemistry, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Velappanchavadi, Velappanchavadi, Chennai, India. 5. Departmemt of Pharmaceutical Chemistry, SRM College of Pharmacy, SRM Institute of Science and Technology, Kattankulathur, India. 6. Interdisciplinary Institute of Indian System of Medicine, SRM Institute of Science and Technology, Kattankulathur, India. 7. Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani campus, Chennai, India. 8. Department of Structural and Cellular Biology, Tulane University, New Orleans, LA, USA. 9. Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
Abstract
BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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. 2020 Stem Cell Investigation. All rights reserved.
BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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. 2020 Stem Cell Investigation. All rights reserved.
Entities:
Keywords:
Notch-1; Tamoxifen resistance (TR); breast cancer stem cells; epigenetics; multidrug resistance (MDR)
Authors: Yanrong Su; Nathan R Hopfinger; Theresa D Nguyen; Thomas J Pogash; Julia Santucci-Pereira; Jose Russo Journal: J Exp Clin Cancer Res Date: 2018-12-14
Authors: J Yun; A Pannuti; I Espinoza; H Zhu; C Hicks; X Zhu; M Caskey; P Rizzo; G D'Souza; K Backus; M F Denning; J Coon; M Sun; E H Bresnick; C Osipo; J Wu; P R Strack; D A Tonetti; L Miele Journal: Oncogenesis Date: 2013-08-05 Impact factor: 7.485