Fabrice Matifat

The Antiestrogen Tamoxifen Activates BK Channels and Stimulates Proliferation of MCF-7 Breast Cancer Cells

In the present study, we investigated the effect of the antiestrogen compound tamoxifen on BK channels by the use of the patch-clamp technique. The perfusion of 10 nM tamoxifen significantly increased the magnitude of a voltage-dependent K+ current by 22.6 ± 10.6% (n = 23). The effect of tamoxifen was always obtained in the first minute, peaked at 5.9 ± 2.2 min (n = 23), and was abolished by the perfusion of tetraethylammonium (0.5 mM), charybdotoxin (50 nM), or iberiotoxin (100 nM). The stimulatory effect of 10 nM tamoxifen was the same at low (50 nM) and high (700 nM) internal calcium concentration and was not additive to that of 17-β-estradiol (E2) or its membrane-impermeant form, β-estradiol 6-(O-carboxymethyl)oxime:bovine serum albumin. Furthermore, the effect of tamoxifen was still recorded in the presence of the selective estrogen receptor antagonist faslodex (ICI-182,780; 1 μM). At the single-channel level, tamoxifen significantly increased the open probability of the BK channel by 46.2 ± 10.1% (n = 4) without changing its unitary conductance. Moreover, we show here that the stimulation of BK channel activity by tamoxifen is involved in MCF-7 cell proliferation. Taken together, these results permitted us to identify the BK channel as the molecular target of tamoxifen that probably acts at the same extracellular molecular level as E2. The site of action of tamoxifen is probably the channel itself or the auxiliary β subunits.

Inositol 1,4,5-trisphosphate-induced Ca2+ signalling is involved in estradiol-induced breast cancer epithelial cell growth

BackgroundCa2+ is a ubiquitous messenger that has been shown to be responsible for controlling numerous cellular processes including cell growth and cell death. Whereas the involvement of IP3-induced Ca2+ signalling (IICS) in the physiological activity of numerous cell types is well documented, the role of IICS in cancer cells is still largely unknown. Our purpose was to characterize the role of IICS in the control of growth of the estrogen-dependent human breast cancer epithelial cell line MCF-7 and its potential regulation by 17β-estradiol (E2).ResultsOur results show that the IP3 receptor (IP3R) inhibitors caffeine, 2-APB and xestospongin C (XeC) inhibited the growth of MCF-7 stimulated by 5% foetal calf serum or 10 nM E2. Furthermore, Ca2+ imaging experiments showed that serum and E2 were able to trigger, in a Ca2+-free medium, an elevation of internal Ca2+ in a 2-APB and XeC-sensitive manner. Moreover, the phospholipase C (PLC) inhibitor U-73122 was able to prevent intracellular Ca2+ elevation in response to serum, whereas the inactive analogue U-73343 was ineffective. Western-blotting experiments revealed that the 3 types of IP3Rs are expressed in MCF-7 cells and that a 48 hours treatment with 10 nM E2 elevated IP3R3 protein expression level in an ICI-182,780 (a specific estrogen receptor antagonist)-dependent manner. Furthermore, IP3R3 silencing by the use of specific small interfering RNA was responsible for a drastic modification of the temporal feature of IICS, independently of a modification of the sensitivity of the Ca2+ release process and acted to counteract the proliferative effect of 10 nM E2.ConclusionsAltogether, our results are in favour of a role of IICS in MCF-7 cell growth, and we hypothesize that the regulation of IP3R3 expression by E2 is involved in this effect.

17-β-Estradiol activates maxi-K channels through a non-genomic pathway in human breast cancer cells

We have investigated the acute effects of 17‐β‐estradiol (E2) on K+ channels in MCF‐7 breast epithelial cancer cells. E2 induced a rapid and irreversible augmentation of the K+ current for all membrane potentials superior to −25 mV. The effect of E2 was sensitive to Iberiotoxin, Charybdotoxin and TEA and can be elicited in the presence of the anti‐estrogen ICI 182 780 or be mimicked by the membrane impermeant form E2/BSA. Furthermore, E2/BSA was able to stimulate cell proliferation in a maxi‐K inhibitors‐sensitive manner. Thus, these results permit us to identify the maxi‐K channel as the molecular target of E2 that regulates cell proliferation independently of the estrogen receptor.

Human ether à‐gogo K+ channel 1 (hEag1) regulates MDA‐MB‐231 breast cancer cell migration through Orai1‐dependent calcium entry

Breast cancer (BC) has a poor prognosis due to its strong metastatic ability. Accumulating data present ether à go‐go (hEag1) K+ channels as relevant player in controlling cell cycle and proliferation of non‐invasive BC cells. However, the role of hEag1 in invasive BC cells migration is still unknown. In this study, we studied both the functional expression and the involvement in cell migration of hEag1 in the highly metastatic MDA‐MB‐231 human BC cells. We showed that hEag1 mRNA and proteins were expressed in human invasive ductal carcinoma tissues and BC cell lines. Functional activity of hEag1 channels in MDA‐MB‐231 cells was confirmed using astemizole, a hEag1 blocker, or siRNA. Blocking or silencing hEag1 depolarized the membrane potential and reduced both Ca2+ entry and MDA‐MB‐231 cell migration without affecting cell proliferation. Recent studies have reported that Ca2+ entry through Orai1 channels is required for MDA‐MB‐231 cell migration. Down‐regulation of hEag1 or Orai1 reduced Ca2+ influx and cell migration with similar efficiency. Interestingly, no additive effects on Ca2+ influx or cell migration were observed in cells co‐transfected with sihEag1 and siOrai1. Finally, both Orai1 and hEag1 are expressed in invasive breast adenocarcinoma tissues and invaded metastatic lymph node samples (LNM+). In conclusion, this study is the first to demonstrate that hEag1 channels are involved in the serum‐induced migration of BC cells by controlling the Ca2+ entry through Orai1 channels. hEag1 may therefore represent a potential target for the suppression of BC cell migration, and thus prevention of metastasis development. J. Cell. Physiol. 227: 3837–3846, 2012.

DNA methylation of channel-related genes in cancers.

DNA methylation at CpG sites is an epigenetic mechanism that regulates cellular gene expression. In cancer cells, aberrant methylation is correlated with the abnormalities in expression of genes that are known to be involved in the particular characteristics of cancer cells such as proliferation, apoptosis, migration or invasion. During the past 30 years, accumulating data have definitely convinced the scientific community that ion channels are involved in cancerogenesis and cancer properties. As they are situated at the cell surface, they might be prime targets in the development of new therapeutic strategies besides their potential use as prognostic factors. Despite the progress in our understanding of the remodeling of ion channels in cancer cells, the molecular mechanisms underlying their over- or down-expression remained enigmatic. In this review, we aimed to summarize the available data on gene promoter methylation of ion channels and to investigate their clinical significance as novel biomarkers in cancer. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

Regulation by protein kinase-C of putative P-type Ca channels expressed in Xenopus oocytes from cerebellar mRNA.

Xenopus oocytes injected with rat cerebellar mRNA expressed functional voltage‐dependent Ca channels detected as an inward Ba current (I Ba). The pharmacological resistance to dihydropyridines and ω‐conotoxin together with the blockade obtained with Agelenopsis aperta venom suggest that these channels could be somehow assimilated to P‐type Ca channels. The precise nature of the transplanted Ca channels was assessed by hybrid‐arrest experiments using a specific oligonucleotide antisense‐derivated from the recently cloned α1‐subunit of P channels (BI‐1 clone). In addition, we demonstrate that exogenous Ca channel activity was enhanced by two different PKC activators (a phorbol ester and a structural analog to diacylglycerol). The general electrophysiological and pharmacological properties of the stimulated Ca channels remain unchanged. This potentiation induced by PKC activators is antagonized by a PKC inhibitor (staurosporine) and by a monoclonal antibody directed against PKC. It is concluded that P‐type Ca channels are potentially regulated by PKC phosphorylation and the functional relevance of this intracellular pathway is discussed.

Extraction of lignans from flaxseed and evaluation of their biological effects on breast cancer MCF-7 and MDA-MB-231 cell lines.

Over the last decade, there has been an increasing interest in using flaxseed (Linum usitatissimum) in diet in order to improve nutritional and health status. Lignans are major components of flaxseed. Therefore an extraction procedure for lignans from flaxseed has been optimized. The influence of some parameters was investigated: first the preliminary extraction step with alcoholic solvent, and then the solvent polarity and pH of the extract. All these conditions affected the total lignan content, but the most critical variables were preliminary extraction and solvent polarity. The optimized procedure, consisting of a direct hydrolysis in hydrochloric acid (1 M) at 100 degrees C for 1 hour followed by an extraction with a mixture of ethyl acetate/hexane (90:10 vol/vol), was applied to 340 g of defatted flaxseed and resulted in the isolation of secoisolariciresinol and anhydrosecoisolariciresinol with a purity of 97% and 98%, respectively, as determined by high-performance liquid chromatography. The ability of these two compounds and that of secoisolariciresinol diglucoside to modulate the growth of human breast cancer MCF-7 and MDA-MB-231 cell lines was assessed. Our results show that lignans modulate development of breast cancer cells. The most intense effect was observed for anhydrosecoisolariciresinol, which significantly decreased cell growth at 50 and 100 microM.

The carcinogen Cd2+ activates InsP3-mediated Ca2+ release through a specific metal ions receptor in Xenopus oocyte

The effects of the carcinogen Cd(2+) on Xenopus oocyte were evaluated by Inositol (1,4,5)-trisphosphate (InsP(3)) assays and electrophysiological experiments. The stimulation of the Ca(2+)-dependent Cl(-) current by Cd(2+) is clearly linked to InsP(3) formation since the effects of the metal are antagonized by neomycin, heparin and caffeine. A similar inhibition of the Cd(2+) effects is observed when the oocytes are pretreated with thapsigargin. Moreover, the use of sulfhydryl groups reductors such as 2-mercaptoethanol or N-ethylmaleimide strongly suggests that the Cd(2+) response is mediated by an extracellular receptor. Finally, measurements of InsP(3) production demonstrate that Cd(2+) superfusion actually leads to a PIP(2) breakdown. We conclude that extracellular Cd(2+) evokes an increase in [Ca(2+)](i) by stimulating the emptying of the InsP(3)-sensitive Ca(2+) stores, and that it may do so by interacting with a specific cell-surface ion receptor. This putative ion receptor may be important in allowing oocytes to respond to heavy metals.

Caffeine exerts a dual effect on capacitative calcium entry in Xenopus oocytes.

Caffeine increases the amplitude of the Cl- currents evoked by capacitative Ca2+ entry (CCE) on thapsigargin-treated Xenopus oocytes. The caffeine-induced potentiation of the CCE process appears to rest on two distinct and additive components. The first component involves the cAMP second messenger system since it can be mimicked by either IBMX perfusion or cAMP microinjection into the oocyte and inhibited by the PKA inhibitory peptide i-PKA. The second component, although activatory, is dynamically related to the caffeine-evoked inhibition of InsP3-mediated Ca+ release and may arise from an interaction between caffeine and the InsP3 receptor in the context of a conformational coupling between the InsP, receptor and the channels responsible for CCE.

The inositol (1,4,5)-trisphosphate 3-kinase of Xenopus oocyte is activated by CaMKII and involved in the regulation of InsP3-mediated Ca2+ release

The effect of Ca2+ on inositol (1,4,5)‐trisphosphate 3‐kinase (3‐kinase) activity was measured on Xenopus oocyte cytosolic extracts. The Ca2+‐evoked elevation in 3‐kinase activity appeared to be mediated by calmodulin (CaM) and the calmodulin‐dependent protein kinase II (CaMKII). The results observed in vitro were totally retrieved in intact oocytes and tend to demonstrate the involvement of a CaMKII‐mediated phosphorylation in the regulation of 3‐kinase activity. Finally, electrophysiological recordings of InsP3‐elicited chloride current transients in the presence of CaM/CaMKII inhibitors allowed to postulate an involvement of 3‐kinase activity in the regulation of InsP3‐mediated Ca2+ release.