7-ketocholesterol and 27-hydroxycholesterol decreased doxorubicin sensitivity in breast cancer cells: estrogenic activity and mTOR pathway

// Chun-Wei Wang 1, 2 , Chiung-Chiao Huang 1 , Pei-Hsin Chou 4 , Yu-Ping Chang 1 , Shouzuo Wei 5 , Frederick Peter Guengerich 6 , Yueh-Ching Chou 3, 7, 9 , Sheng-Fan Wang 1, 7 , Ping-Shan Lai 8 , Pavel Soucek 11 and Yune-Fang Ueng 1, 2, 3, 10 1 National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan, R.O.C 2 Institute of Biopharmaceutical Sciences, School of Pharmacy, National Yang-Ming University, Taipei, Taiwan, R.O.C 3 Department of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan, R.O.C 4 Department of Environmental Engineering, National Chung-Kung University, Tainan, Taiwan, R.O.C 5 Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA 6 Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA 7 Department of Pharmacy, Taipei Veterans General Hospital, Taipei, Taiwan, R.O.C 8 Department of Chemistry, College of Science, National Chung-Hsin University, Taichung, Taiwan, R.O.C 9 Department of Pharmacy, Taipei Medical University, Taipei, Taiwan, R.O.C 10 Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan, R.O.C 11 Department of Toxicogenomics, National Institute of Public Health, Prague, Czech Republic Correspondence to: Yune-Fang Ueng, email: ueng@nricm.edu.tw Keywords: 7-ketocholesterol, 27-hydroxycholesterol, doxorubicin, P-glycoprotein, estrogen receptor Received: March 13, 2017      Accepted: June 27, 2017      Published: August 02, 2017 ABSTRACT Hypercholesterolemia is one of the risk factors for poor outcome in breast cancer therapy. To elucidate the influence of the main circulating oxysterols, cholesterol oxidation products, on the cell-killing effect of doxorubicin, cells were exposed to oxysterols at a subtoxic concentration. When cells were exposed to oxysterols in fetal bovine serum-supplemented medium, 7-ketocholesterol (7-KC), but not 27-hydroxycholesterol (27-HC), decreased the cytotoxicity of doxorubicin in MCF-7 (high estrogen receptor (ER)α/ERβ ratio) cells and the decreased cytotoxicity was restored by the P-glycoprotein inhibitor verapamil. 7-KC stimulated the efflux function of P-glycoprotein and reduced intracellular doxorubicin accumulation in MCF-7 but not in ERα(-) MDA-MB-231 and the resistant MCF-7/ADR cells. In MCF-7 cells, 7-KC increased the mRNA and protein levels of P-glycoprotein. The 7-KC-suppressed doxorubicin accumulation was restored by the fluvestrant and ERα knockdown. In a yeast reporter assay, the ERα activation by 7-KC was more potent than 27-HC. 7-KC, but not 27-HC, stimulated the expression of an ER target, Trefoil factor 1 in MCF-7 cells. When charcoal-stripped fetal bovine serum was used, both 7-KC and 27-HC induced Trefoil factor 1 expression and reduced doxorubicin accumulation in MCF-7 cells. 7-KC-reduced doxorubicin accumulation could be reversed by inhibitors of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin (mTOR). These findings demonstrate that 7-KC decreases the cytotoxicity of doxorubicin through the up-regulation of P-glycoprotein in an ERα- and mTOR-dependent pathway. The 7-KC- and 27-HC-elicited estrogenic effects are crucial in the P-glycoprotein induction in breast cancer cells.