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Ether à go-go (EAG) potassium channels display oncogenic properties. In normal tissues, EAG mRNA is almost exclusively expressed in brain, but it is expressed in several somatic cancer cell lines, including HeLa, from cervix. Antisense experiments against eag reduce cell proliferation in some cancer cell lines, and inhibition of EAG-mediated currents has been suggested to decrease cell proliferation in a melanoma cell line. Because of the potential clinical relevance of EAG, we investigated EAG mRNA expression in the following fresh samples from human uterine cervix: 5 primary cultures obtained from cancerous biopsies, 1 cancerous fresh tissue, and 12 biopsies of control normal tissue. All of the control cervical samples came from patients with negative pap smears. Reverse transcription-PCR and Southern-blot experiments revealed eag expression in 100% of the cancerous samples and in 33% of the normal biopsies. Immunochemistry experiments showed the presence of EAG channel protein in cells from the primary cultures and in cervical cancer biopsies sections from the same patients. In addition, we looked for EAG-mediated currents in the cultures from cervical cancer cells. Here we show for the first time EAG channel activity in human tumors. Patch-clamp recordings showed typical EAG-mediated currents modulated by magnesium and displaying a pronounced Cole-Moore shift. Because EAG expression and channel activity have been suggested to be important in cell proliferation, our findings strongly support the idea of considering EAG as a tumor marker as well as a potential membrane therapeutic target for cervical cancer.

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Vitamin D has garnered a great deal of attention in recent years due to a global prevalence of vitamin D deficiency associated with an increased risk of a variety of human diseases. Specifically, hypovitaminosis D in pregnant women is highly common and has important implications for the mother and lifelong health of the child, since it has been linked to maternal and child infections, small-for-gestational age, preterm delivery, preeclampsia, gestational diabetes, as well as imprinting on the infant for life chronic diseases. Therefore, factors that regulate vitamin D metabolism are of main importance, especially during pregnancy. The hormonal form and most active metabolite of vitamin D is calcitriol. This hormone mediates its biological effects through a specific nuclear receptor, which is found in many tissues including the placenta. Calcitriol synthesis and degradation depend on the expression and activity of CYP27B1 and CYP24A1 cytochromes, respectively, for which regulation is tissue specific. Among the factors that modify these cytochromes expression and/or activity are calcitriol itself, parathyroid hormone, fibroblast growth factor 23, cytokines, calcium and phosphate. This review provides a current overview on the regulation of vitamin D metabolism, focusing on vitamin D deficiency during gestation and its impact on pregnancy outcomes.

Ether-à-go-go-1 (Eag1) potassium channels are potential tools for detection and therapy of numerous cancers. Here, we show human Eag1 (hEag1) regulation by cancer-associated factors. We studied hEag1 gene expression and its regulation by estradiol, antiestrogens, and human papillomavirus (HPV) oncogenes (E6/E7). Primary cultures from normal placentas and cervical cancer tissues; tumor cell lines from cervix, choriocarcinoma, keratinocytes, and lung; and normal cell lines from vascular endothelium, keratinocytes, and lung were used. Reverse transcription-PCR (RT-PCR) experiments and Southern blot analysis showed Eag1 expression in all of the cancer cell types, normal trophoblasts, and vascular endothelium, in contrast to normal keratinocytes and lung cells. Estradiol and antiestrogens regulated Eag1 in a cell type-dependent manner. Real-time RT-PCR experiments in HeLa cells showed that Eag1 estrogenic regulation was strongly associated with the expression of estrogen receptor-A. Eag1 protein was detected by monoclonal antibodies in normal placenta and placental blood vessels. Patch-clamp recordings in normal trophoblasts treated with estradiol exhibited potassium currents resembling Eag1 channel activity. Eag1 gene expression in keratinocytes depended either on cellular immortalization or the presence of HPV oncogenes. Eag1 protein was found in keratinocytes transfected with E6/E7 HPV oncogenes. Cell proliferation of E6/E7 keratinocytes was decreased by Eag1 antibodies inhibiting channel activity and by the nonspecific Eag1 inhibitors imipramine and astemizole; the latter also increased apoptosis. Our results propose novel oncogenic mechanisms of estrogen/ antiestrogen use and HPV infection. We also suggest Eag1 as an early indicator of cell proliferation leading to malignancies and a therapeutic target at early stages of cellular hyperproliferation. [Cancer Res 2009;69(8):3300-7]

Background: In pregnancy, maternal serum concentrations of calcitriol significantly rise as a result of increased renal and placental contribution in order to assure calcium supply for the developing fetus. Considering that placenta is a site for vitamin D activation, and the versatility and potency of calcitriol, it is feasible that this hormone participates in fetal/placental development and physiology. In the present work we studied calcitriol actions upon human chorionic gonadotropin (hCG) secretion and expression in cultured trophoblasts, as well as vitamin D receptor (VDR) and CYP27B1 immunolocalization in placental villi.

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BackgroundCalcitriol 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 FindingsMolecular 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 IC20 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/SignificanceAstemizole 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.