title: Calcitriol and its analogues enhance the antiproliferative activity of gefitinib in breast cancer cells.
abstract: 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. 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 cells. The mechanistic explanation for these results includes downregulation of MAPK signaling pathway, decrease of cells in G2/M phase and induction of apoptosis mediated by upregulation of BIM and activation of caspase 3. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.
authors.affiliation: 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; Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, Av. Cuidad Universitaria 3000, Coyoacán 04360, México, D.F, Mexico.
authors.forename: Mariana
authors.initials: M
authors.lastname: Segovia-Mendoza
authors.affiliation: 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.
authors.forename: Lorenza
authors.initials: L
authors.lastname: Díaz
authors.affiliation: 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.
authors.forename: María Elena
authors.initials: ME
authors.lastname: González-González
authors.affiliation: 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.
authors.forename: Isela
authors.initials: I
authors.lastname: Martínez-Reza
authors.affiliation: 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.
authors.forename: Janice
authors.initials: J
authors.lastname: García-Quiroz
authors.affiliation: Departamento de Enfermedades Crónico-Degenerativas, Instituto Nacional de Enfermedades Respiratorias "Ismael Cosío Villegas", Calzada de Tlalpan 4502, Sección XVI, Tlalpan 14080, México, D.F., Mexico.
authors.forename: Heriberto
authors.initials: H
authors.lastname: Prado-Garcia
authors.affiliation: Unidad de Bioquímica, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, Sección XVI, Tlalpan 14000, México, D.F., Mexico.
authors.forename: María J
authors.initials: MJ
authors.lastname: Ibarra-Sánchez
authors.affiliation: Unidad de Bioquímica, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, Sección XVI, Tlalpan 14000, México, D.F., Mexico.
authors.forename: José
authors.initials: J
authors.lastname: Esparza-López
authors.affiliation: 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.
authors.forename: Fernando
authors.initials: F
authors.lastname: Larrea
authors.affiliation: 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. Electronic address: rocio.garciab@quetzal.innsz.mx.
authors.forename: Rocío
authors.initials: R
authors.lastname: García-Becerra
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D000072224:Bcl-2-Like Protein 11; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000473:pathology; D002117:Calcitriol / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D004357:Drug Synergism; D005260:Female; D000077156:Gefitinib; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D011799:Quinazolines / Q000008:administration & dosage; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D014815:Vitamins / Q000494:pharmacology*
chemical_list: D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D011799:Quinazolines; D012333:RNA, Messenger; D014815:Vitamins; D053148:Caspase 3; D002117:Calcitriol; C078903:seocalcitol; D000077156:Gefitinib
keywords: Breast cancer; Calcitriol; EGFR; Gefitinib; HER2; Vitamin D analogs