1 Calcitriol and its analogues enhance the antiproliferative activity of 2 gefitinib in breast cancer cells Q1 3 Mariana Segovia-Mendoza a, b Q2 , Lorenza Díaz a , María Elena González-González a , 4 Isela Martínez-Reza a , Janice García-Quiroz a , Heriberto Prado-Garcia c , 5 María J. Ibarra-Sánchez d , José Esparza-López d , Fernando Larrea a , 6 Rocío García-Becerra a, * 7 a Departamento de Biología de la Reproducción, Q3 Vasco de Quiroga No. 15, Sección XVI, Tlalpan 14000, México, D.F., Mexico 8 b Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, Av. Cuidad Universitaria 3000, Coyoacán 04360, México, D.F, Mexico 9 c Departamento de Enfermedades Crónico-Degenerativas, Instituto Nacional de Enfermedades Respiratorias “Ismael Cosío Villegas”, Calzada de Tlalpan 4502, 10 Sección XVI, Tlalpan 14080, México, D.F., Mexico 11 d Unidad de Bioquímica, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, Sección XVI, Tlalpan 14000, México, 12 D.F., Mexico A R T I C L E I N F O Article history: Received 15 August 2014 Received in revised form 6 December 2014 Accepted 10 December 2014 Available online xxx Keywords: Gefitinib Calcitriol Vitamin D analogs Breast cancer HER2 EGFR A B S T R A C T Coexpression of EGFR and HER2 has been associated with poor disease outcome, high rates of metastasis and resistance to conventional treatments in breast cancer. Gefitinib, a tyrosine kinase inhibitor, reduces both cell proliferation and tumor growth of breast cancer cells expressing EGFR and/or HER2. On the other hand, calcitriol and some of its synthetic analogs are important antineoplastic agents in different breast cancer subtypes. Herein, we evaluated the effects of the combined treatment of gefitinib with calcitriol or its analogs on cell proliferation in breast cancer cells. The presence of EGFR, HER2 and vitamin D receptor were evaluated by Western blot in two established breast cancer cell lines: SUM-229PE, SKBR3 and a primary breast cancer-derived cell line. The antiproliferative effects of gefitinib alone or in combination with calcitriol and its analogs, calcipotriol and EB1089, were assessed by growth assay using a DNA content-based method. Inhibitory concentrations on cell proliferation were calculated by non-linear regression analysis using sigmoidal fitting of dose-response curves. Pharmacological effects of the drug combinations were calculated by the Chou–Talalay method. Phosphorylation of ERK1/2 MAPK was evaluated by Western blot. Gene expression of EGFR, HER2 and BIM was assessed by real time PCR. BIM protein levels were analyzed in cells by flow cytometry. The effects of the drugs alone or combinated on cell cycle phases were determined using propidium iodide. Apoptosis was evaluated by detection of subG1 peak and determination of active caspase 3 by flow cytometry. Gefitinib, calcitriol, calcipotriol and EB1089 inhibited cell proliferation in a dose dependent manner by reducing percentages of cells in G2/M phase. The combinations of gefitinib with calcitriol or its analogs were more effective to inhibit cell growth than each compound alone in all breast cancer cells studied. The gene expression of EGFR and HER2 was downregulated and not affected, respectively, by the combined treatment. Furthermore, phosphorylation of ERK 1/2 was inhibited a greater extent in co- treated cells than in the cells treated with alone compounds. The combination of gefitinib with calcitriol or their synthetic analogs induced apoptosis in SUM-229PE cells, this was shown by the significant upregulation of BIM protein levels, higher percentages of cells in subG1 peak and increase of caspase 3- positive cells. The combination of gefitinib with calcitriol or their synthetic analogs resulted in a greater antiproliferative effect than with either of the agents alone in EGFR and HER2 positive breast cancer Abbreviations: CK-7, cytokeratin 7; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FSC-A, forward scatter area; FSC-H, forward scatter height; GI, growth inhibitory; HER2, epidermal growth factor receptor type II; IC, inhibitory concentration; MFI, mean fluorescence intensity; TKI, tyrosine kinase inhibitors; VDR, vitamin D receptor. * Corresponding author at. Departamento de Biología de la Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, Sección XVI, Tlalpan 14000, México, D.F, Mexico. Tel.: +525 54 87 09 00x2417. E-mail address: rocio.garciab@quetzal.innsz.mx (R. García-Becerra). http://dx.doi.org/10.1016/j.jsbmb.2014.12.006 0960-0760/ ã 2014 Published by Elsevier Ltd. Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx G Model SBMB 4331 1–10 Please cite this article in press as: M. Segovia-Mendoza, et al., Calcitriol and its analogues enhance the antiproliferative activity of gefitinib in breast cancer cells, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.12.006 Contents lists available at ScienceDirect Journal of Steroid Biochemistry & Molecular Biology journal homepage: www.else vie r.com/locate /jsbmb