Departamento de Biología de la Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México, DF, México.
PLoS One. 2012;7(9):e45063. doi: 10.1371/journal.pone.0045063. Epub 2012 Sep 12.
Calcitriol antiproliferative effects include inhibition of the oncogenic ether-à-go-go-1 potassium channel (Eag1) expression, which is necessary for cell cycle progression and tumorigenesis. Astemizole, a new promising antineoplastic drug, targets Eag1 by blocking ion currents. Herein, we characterized the interaction between calcitriol and astemizole as well as their conjoint antiproliferative action in SUM-229PE, T-47D and primary tumor-derived breast cancer cells.
METHODOLOGY/PRINCIPAL FINDINGS: Molecular markers were studied by immunocytochemistry, Western blot and real time PCR. Inhibitory concentrations were determined by dose-response curves and metabolic activity assays. At clinically achievable drug concentrations, synergistic antiproliferative interaction was observed between calcitriol and astemizole, as calculated by combination index analysis (CI <1). Astemizole significantly enhanced calcitriol's growth-inhibitory effects (3-11 folds, P<0.01). Mean IC(20) values were 1.82 ± 2.41 nM and 1.62 ± 0.75 µM; for calcitriol (in estrogen receptor negative cells) and astemizole, respectively. Real time PCR showed that both drugs alone downregulated, while simultaneous treatment further reduced Ki-67 and Eag1 gene expression (P<0.05). Astemizole inhibited basal and calcitriol-induced CYP24A1 and CYP3A4 mRNA expression (cytochromes involved in calcitriol and astemizole degradation) in breast and hepatoma cancer cells, respectively, while upregulated vitamin D receptor (VDR) expression.
CONCLUSIONS/SIGNIFICANCE: Astemizole synergized calcitriol antiproliferative effects by downregulating CYP24A1, upregulating VDR and targeting Eag1. This study provides insight into the molecular mechanisms involved in astemizole-calcitriol combined antineoplastic effect, offering scientific support to test both compounds in combination in further preclinical and clinical studies of neoplasms expressing VDR and Eag1. VDR-negative tumors might also be sensitized to calcitriol antineoplastic effects by the use of astemizole. Herein we suggest a novel combined adjuvant therapy for the management of VDR/Eag1-expressing breast cancer tumors. Since astemizole improves calcitriol bioavailability and activity, decreased calcitriol dosing is advised for conjoint administration.
钙三醇的抗增殖作用包括抑制致癌性醚-去甲肾上腺素-1 钾通道（Eag1）的表达，这对于细胞周期进展和肿瘤发生是必要的。阿替美唑是一种新的有前途的抗肿瘤药物，通过阻断离子流来靶向 Eag1。在此，我们描述了钙三醇和阿替美唑之间的相互作用及其在 SUM-229PE、T-47D 和原发性乳腺癌细胞中的联合抗增殖作用。
方法/主要发现：通过免疫细胞化学、Western blot 和实时 PCR 研究了分子标记物。通过剂量反应曲线和代谢活性测定确定抑制浓度。在临床可达到的药物浓度下，通过组合指数分析（CI<1）计算，观察到钙三醇和阿替美唑之间具有协同的抗增殖相互作用。阿替美唑显著增强了钙三醇的生长抑制作用（3-11 倍，P<0.01）。钙三醇和阿替美唑的平均 IC20 值分别为 1.82±2.41 nM 和 1.62±0.75 µM。实时 PCR 显示，两种药物单独处理时均下调 Ki-67 和 Eag1 基因表达，而同时处理时进一步降低（P<0.05）。阿替美唑抑制了乳腺癌和肝癌细胞中 CYP24A1 和 CYP3A4 mRNA 的基础表达和钙三醇诱导的表达（与钙三醇和阿替美唑降解相关的细胞色素），同时上调了维生素 D 受体（VDR）的表达。
结论/意义：阿替美唑通过下调 CYP24A1、上调 VDR 和靶向 Eag1 增强了钙三醇的抗增殖作用。本研究深入了解了阿替美唑-钙三醇联合抗肿瘤作用的分子机制，为进一步在 VDR 和 Eag1 表达的肿瘤的临床前和临床研究中测试这两种化合物的联合应用提供了科学依据。VDR 阴性肿瘤也可能通过使用阿替美唑对钙三醇的抗肿瘤作用敏感。在此，我们建议将这种新型联合辅助疗法用于管理表达 VDR/Eag1 的乳腺癌肿瘤。由于阿替美唑提高了钙三醇的生物利用度和活性，因此建议在联合给药时减少钙三醇的剂量。
