Changes in the K+ current-density of MCF-7 cells during progression through the cell cycle: possible involvement of a h-ether.a-gogo K+ channel.
ABSTRACT MCF-7 cells express voltage-activated K+ channels. In the present study, we used the patch-clamp and RT-PCR techniques to investigate the involvement of these channels during the cell cycle progression. The outward rectifier current (IK) recorded during depolarization was almost completely suppressed by the classical K+ channel blocker tetraethylammonium (TEA) in MCF-7 cells. TEA also inhibited cell proliferation, as measured with 3H-thymidine incorporation. Moreover, profound changes were observed in both the resting membrane potential (RMP) and IK during the release from the G0/G1 phase of the cell cycle. MCF-7 cells arrested in G0/G1 were depolarized (-26.3 +/- 10 mV, n = 30) and IK-density was small (9.4 +/- 5.6 pA/pF, n = 60) compared to cells progressing in the G1 phase (RMP = -60 +/- 7.9 mV; n = 35 and IK-density = 30.2 +/- 8.5 pA/pF; n = 76). IK was highly sensitive to Mg2+, astemizole and TEA (10 mM). Extracellular perfusion of 5 mM Mg2+ dramatically slowed the activation and perfusion of 2 microM astemizole inhibited both IK (20 +/- 3%) and cell proliferation (23%). Moreover, the h-EAG mRNA expression was modulated during the cell cycle. Thus, these data suggested that h-EAG K+ channels play a role in controlling the proliferation and/or cell cycle.
Article: Astemizole Synergizes Calcitriol Antiproliferative Activity by Inhibiting CYP24A1 and Upregulating VDR: A Novel Approach for Breast Cancer Therapy.[show abstract] [hide abstract]
ABSTRACT: 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. 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. 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.PLoS ONE 01/2012; 7(9):e45063. · 4.09 Impact Factor
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ABSTRACT: K(V)10.1 is a voltage-gated potassium channel aberrantly expressed in many cases of cancer, and participates in cancer initiation and tumor progression. Its action as oncoprotein can be inhibited by a functional monoclonal antibody, and is therefore dependent, although not exclusively, on the presence of functional channels at the plasma membrane, which are the only ones accessible for the antibody. Cortactin is an actin-interacting protein implicated in cytoskeletal architecture, and is often amplified in several types of cancer. In this study, we describe a physical and functional interaction between cortactin and K(V)10.1. Binding of these two proteins occurs between the C-terminus of K(V)10.1 and the proline-rich domain of cortactin, both known to be substrate of many posttranslational modifications. This interaction is specific for K(V)10.1 and does not happen with K(V)10.2. Cortactin controls the abundance of K(V)10.1 at the plasma membrane, and is required for functional expression of K(V)10.1 channels.Journal of Biological Chemistry 11/2012; · 4.77 Impact Factor
Article: p38 MAPK regulates the expression of ether à go-go potassium channel in human osteosarcoma cells.[show abstract] [hide abstract]
ABSTRACT: The ether à go-go (Eag) channel has been shown to be overexpressed in a variety of cancers. However, the expression and function of Eag in osteosarcoma are poorly understood. In addition, the molecular mechanisms responsible for Eag overexpression in cancer cells remain unclear. The expression of Eag in human osteosarcoma cell line MG-63 was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The effect of Eag inhibition on MG-63 cell proliferation was assessed . The effect of short hairpin RNA (shRNA) mediated knockdown of Eag on osteosarcoma growth was evaluated in xenograft model . The activation of mitogen-activated protein kinase (MAPK) pathway and p53 in MG-63 cells was detected by Western blot analysis. Eag was overexpressed in MG-63 cells. Imipramine or Eag shRNA significantly suppressed the proliferation of MG-63 cells and . MG-63 cell proliferation was specifically inhibited by p38 MAPK inhibitor SB203580 or small interference RNA (siRNA). The inhibition of p38 MAPK activation by SB203580 or siRNA reduced Eag protein level but increased p53 protein level. Moreover, the activation of p53 by nutlin-3 induced cell growth arrest in MG-63 cells and reduced Eag protein level, while the inactivation of p53 by pifithrin-alpha (PFT-α) promoted MG-63 cell growth and increased Eag protein expression. Eag channel functions as an oncogene to promote the proliferation of human osteosarocma cells. Furthermore, the high expression of Eag in osteosarcoma cells is regulated by p38 MAPK/p53 pathway.Radiology and Oncology 03/2013; 47(1):42-9. · 0.91 Impact Factor