tetrodotoxin and Breast-Neoplasms

Functional expression of voltage-gated Na

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; NAV1.5 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; RNA, Small Interfering; Tetrodotoxin

Voltage-gated sodium channels (VGSC) are a well-established drug target for anti-epileptic, anti-arrhythmic and pain medications due to their presence and the important roles that they play in excitable cells. Recently, their presence has been recognized in non-excitable cells such as cancer cells and their overexpression has been shown to be associated with metastatic behavior in a variety of human cancers. The neonatal isoform of the VGSC subtype, Na

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Drug Design; Humans; NAV1.5 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; Neoplasm Metastasis; Quantitative Structure-Activity Relationship; Voltage-Gated Sodium Channel Blockers

Voltage-gated Na+ channels (VGSCs) are membrane proteins which are normally expressed in excitable cells but have also been detected in cancer cells, where they are thought to be involved in malignancy progression. In this study we examined the ion current and expression profile of VGSC (Nav1.5) in estrogen receptor (ER)-positive (MCF-7) and silenced (pII) breast cancer cells and its possible influence on their proliferation, motility and invasion. VGSC currents were analysed by whole cell patch clamp recording. Nav1.5 expression and localization, in response to EGF stimulation, was examined by western blotting and immunofluorescence respectively. Cell invasion (under-agarose and Matrigel assays), motility (wound healing assay) and proliferation (MTT assay) were assessed in pII cells in response to VGSC blockers, phenytoin (PHT) and tetrodotoxin (TTX), or by siRNA knockdown of Nav1.5. The effect of PHT and TTX on modulating EGF-induced phosphorylation of Akt and ERK1/2 was determined by western blotting. Total matrix metalloproteinase (MMP) was determined using a fluorometric-based activity assay. The level of various human proteases was detected by using proteome profiler array kit. VGSC currents were detected in pII cells, but were absent in MCF-7. Nav1.5 showed cytoplasmic and perinuclear expression in both MCF-7 and pII cells, with enhanced expression upon EGF stimulation. Treatment of pII cells with PHT, TTX or siRNA significantly reduced invasion towards serum components and EGF, in part through reduction of P-ERK1/2 and proteases such as cathepsin E, kallikrein-10 and MMP-7, as well as total MMP activity. At high concentrations, PHT inhibited motility while TTX reduced cell proliferation. Pharmacological or genetic blockade of Nav1.5 may serve as a potential anti-metastatic therapy for breast cancer.

Topics: Breast Neoplasms; Cell Movement; Cell Proliferation; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; NAV1.5 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; Patch-Clamp Techniques; Phenytoin; Tetrodotoxin

Voltage-gated Na(+) channels (VGSCs) are expressed in excitable cells (e.g. neurons and muscles), as well as in some classically 'non-excitable' cells (e.g. fibroblasts), and in carcinomas. In general, functional expression of VGSCs in plasma membrane (PM) is hierarchical and dynamic. Previously, we have shown that an activity-dependent positive feedback mechanism involving cAMP-dependent protein kinase A (PKA) plays a significant role in upregulation of VGSCs in strongly metastatic rat prostate cancer Mat-LyLu cells expressing Nav1.7. Here, we investigated the possible role of PKA in VGSC regulation and its functional consequences in strongly metastatic human breast cancer (BCa) MDA-MB-231 cells, where the neonatal splice form of Nav1.5 (nNav1.5) is the predominant VGSC present. Treatment with the PKA activator forskolin for 24h increased mRNA and PM protein levels of nNav1.5, without changing the total VGSC protein level. Opposite effects were obtained by application of the PKA inhibitor KT5720 or the highly specific VGSC blocker tetrodotoxin (TTX), the latter implying activity-dependent upregulation. We tested the possibility, therefore, that the activity dependence of VGSC (nNav1.5) expression involved PKA. Indeed, TTX pretreatment reduced the level of phosphorylated PKA and eliminated basal and PKA-stimulated cellular migration. These data suggested that activity-dependent positive feedback mediated by PKA plays an important role in the functional expression of nNav1.5 in BCa, and in turn, this enhances the cells' metastatic potential.

Topics: Animals; Breast Neoplasms; Carbazoles; Cell Line, Tumor; Cell Movement; Colforsin; Cyclic AMP-Dependent Protein Kinases; Feedback, Physiological; Gene Expression Regulation, Neoplastic; Humans; Infant, Newborn; Muscle Proteins; NAV1.5 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; Phosphorylation; Pyrroles; Rats; Sodium Channels; Stromal Cells; Tetrodotoxin; Up-Regulation

Although voltage-gated sodium channel (VGSC) activity, upregulated significantly in strongly metastatic human breast cancer cells, has been found to potentiate a variety of in vitro metastatic cell behaviors, the mechanism(s) regulating channel expression/activity is not clear. As a step toward identifying possible serum factors that might be responsible for this, we tested whether medium in which fetal bovine serum (FBS) was substituted with a commercial serum replacement agent (SR-2), comprising insulin and bovine serum albumin, would influence the VGSC-dependent in vitro metastatic cell behaviors. Human breast cancer MDA-MB-231 cells were used as a model. Measurements of lateral motility, transverse migration and adhesion showed consistently that the channel's involvement in metastatic cell behaviors depended on the extracellular biochemical conditions. In normal medium (5% FBS), tetrodotoxin (TTX), a highly specific blocker of VGSCs, suppressed these cellular behaviors, as reported before. In contrast, in SR-2 medium, TTX had opposite effects. However, blocking endogenous insulin/insulin-like growth factor receptor signaling with AG1024 eliminated or reversed the anomalous effects of TTX. Insulin added to serum-free medium increased migration, and TTX increased it further. In conclusion, (1) the biochemical constitution of the extracellular medium had a significant impact upon breast cancer cells' in vitro metastatic behaviors and (2) insulin, in particular, controlled the mode of the functional association between cells' VGSC activity and metastatic machinery.

Topics: Blotting, Western; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Culture Media; Humans; Immunohistochemistry; Insulin; Ion Channel Gating; Microscopy, Confocal; Models, Biological; Receptors, Somatomedin; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Tyrphostins

Upregulation of functional voltage-gated Na+ channels (VGSCs) occurs in metastatic human breast cancer (BCa) in vitro and in vivo. The present study aimed to ascertain the specific involvement of the "neonatal" splice variant of Nav1.5 (nNav1.5), thought to be predominant, in the VGSC-dependent invasive behaviour of MDA-MB-231 cells. Functional activity of nNav1.5 was suppressed by two different methods targeting nNav1.5: (i) small interfering RNA (siRNA), and (ii) a polyclonal antibody (NESO-pAb); effects upon migration and invasion were determined. nNav1.5 mRNA, protein and signalling were measured using real-time PCR, Western blotting, and patch clamp recording, respectively. Treatment with the siRNA rapidly reduced (by approximately 90%) the level of nNav1.5 (but not adult Nav1.5) mRNA, but the protein reduction was much smaller (approximately 30%), even after 13 days. Nevertheless, the siRNA reduced peak VGSC current density by 33%, and significantly increased the cells' sensitivity to nanomolar tetrodotoxin (TTX). Importantly, the siRNA suppressed in vitro migration by 43%, and eliminated the normally inhibitory effect of TTX. Migrated MDA-MB-231 cells expressed more nNav1.5 protein at the plasma membrane than non-migrated cells. Furthermore, NESO-pAb reduced migration by up to 42%, in a dose-dependent manner. NESO-pAb also reduced Matrigel invasion without affecting proliferation. TTX had no effect on cells already treated with NESO-pAb. It was concluded that nNav1.5 is primarily responsible for the VGSC-dependent enhancement of invasive behaviour in MDA-MB-231 cells. Accordingly, targeting nNav1.5 expression/activity may be useful in clinical management of metastatic BCa.

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; In Vitro Techniques; Infant, Newborn; Microscopy, Confocal; Muscle Proteins; NAV1.5 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; Patch-Clamp Techniques; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

Omega-3 polyunsaturated fatty acids have been suggested to play an important role in cancer prevention/progression, on the one hand, and in modulation of membrane ion channels on the other. We investigated whether docosahexaenoic acid would influence the in vitro migration of MDA-MB-231 human breast cancer cells. An important follow-up question was whether any effect would involve voltage-gated Na(+) channels, shown previously to occur in human breast cancer in vitro and in vivo and to correlate with metastatic potential. Short-term (acute) and long-term (24-72 h) application of docosahexaenoic acid suppressed the activity of the channel activity in a dose-dependent manner. At the working concentrations of docosahexaenoic acid used (0.05-0.5 microM), there was no effect on proliferation. Long-term treatment with docosahexaenoic acid down-regulated mRNA and protein (total and plasma membrane) levels of neonatal Nav1.5 voltage-gated Na(+) channel, known to be predominant in these cells. Docosahexaenoic acid suppressed migration of the MDA-MB-231 cells to the same extent as tetrodotoxin, a highly specific blocker of voltage-gated Na(+) channels, but the two effects were not additive. It was concluded that the docosahexaenoic acid-induced suppression of cellular migration occurred primarily via down-regulation of voltage-gated Na(+) channel (neonatal Nav1.5) mRNA and functional protein expression.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Docosahexaenoic Acids; Electrophysiology; Gene Expression Regulation, Neoplastic; Humans; Infant, Newborn; Ion Channel Gating; Muscle Proteins; NAV1.5 Voltage-Gated Sodium Channel; RNA, Messenger; Sodium Channels; Tetrodotoxin

Ion channel activity is involved in several basic cellular behaviors that are integral to metastasis (e.g., proliferation, motility, secretion, and invasion), although their contribution to cancer progression has largely been ignored. The purpose of this study was to investigate voltage-gated Na(+) channel (VGSC) expression and its possible role in human breast cancer.. Functional VGSC expression was investigated in human breast cancer cell lines by patch clamp recording. The contribution of VGSC activity to directional motility, endocytosis, and invasion was evaluated by in vitro assays. Subsequent identification of the VGSC alpha-subunit(s) expressed in vitro was achieved using reverse transcription-PCR, immunocytochemistry, and Western blot techniques and used to investigate VGSCalpha expression and its association with metastasis in vivo.. VGSC expression was significantly up-regulated in metastatic human breast cancer cells and tissues, and VGSC activity potentiated cellular directional motility, endocytosis, and invasion. Reverse transcription-PCR revealed that Na(v)1.5, in its newly identified "neonatal" splice form, was specifically associated with strong metastatic potential in vitro and breast cancer progression in vivo. An antibody specific for this form confirmed up-regulation of neonatal Na(v)1.5 protein in breast cancer cells and tissues. Furthermore, a strong correlation was found between neonatal Na(v)1.5 expression and clinically assessed lymph node metastasis.. Up-regulation of neonatal Na(v)1.5 occurs as an integral part of the metastatic process in human breast cancer and could serve both as a novel marker of the metastatic phenotype and a therapeutic target.

Topics: Amino Acid Sequence; Biopsy; Blotting, Western; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Dose-Response Relationship, Drug; Electrophysiology; Endocytosis; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; In Vitro Techniques; Ions; Lymphatic Metastasis; Molecular Sequence Data; NAV1.5 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; Neoplasm Metastasis; Patch-Clamp Techniques; Phenotype; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; Sodium Channels; Tetrodotoxin; Up-Regulation

1. A voltage-dependent sodium current has been described in the highly invasive breast cancer cell line MDA-MB-231. Its activity is associated with the invasive properties of the cells. The aim of our study was to test whether this current (I(Na)) is sensitive to three representative calcium channel blockers: verapamil, diltiazem and nifedipine. I(Na) was studied in patch-clamp conditions. 2. I(Na) was sensitive to verapamil (IC(50)=37.6+/-2.5 microM) and diltiazem (53.2+/-3.6 microM), while it was weakly sensitive to nifedipine. 3. The tetrodotoxin (TTX) concentration, which fully blocks I(Na) (30 microM), did not affect cell proliferation. Diltiazem and verapamil, at concentrations that do not fully block I(Na), strongly reduced cell proliferation, suggesting, regarding proliferation, that these molecules act on targets distinct from sodium channels. These targets are probably not other ionic channels, since the current measured at the end of a 500 ms long pulse in the voltage range between -60 and +40 mV was unaffected by verapamil and diltiazem. 4. We conclude that the sodium channel expressed in MDA-MB-231 cells is sensitive to several calcium channel blockers. The present study also underlines the danger of concluding to the possible involvement of membrane channel proteins in any phenomenon on the sole basis of pharmacology, and without an electrophysiological confirmation.

Topics: Breast Neoplasms; Calcium Channel Blockers; Cell Division; Cell Line, Tumor; Cell Survival; Diltiazem; Electrophysiology; Female; Humans; Nifedipine; Potassium Channel Blockers; Sodium Channels; Tetraethylammonium; Tetrodotoxin; Verapamil

Pulsatile secretion of gonadotropin-releasing hormone-1 (GnRH-1) is essential for reproduction. GnRH-1 induces gonadotropin release and is regulated by 17beta-estradiol (E2). Although a subpopulation of GnRH-1 neurons expresses estrogen receptor (ER) beta, it is unclear whether E2 acts directly on GnRH-1 neurons or indirectly through interneuronal connections. To test the hypothesis that E2 acts directly on GnRH-1 neurons to regulate neuronal activity, we used calcium imaging to monitor intracellular calcium oscillations in GnRH-1 neurons maintained in nasal explants. TTX was used to minimize synaptic input from other cells. Consistent with previous studies, TTX reduced the activity of individual GnRH-1 neurons to a basal level, while the population of cells maintained synchronized calcium oscillations. Exposure of GnRH-1 cells to TTX plus E2 increased the number of calcium peaks/cell, percentage of cells with > or =10 peaks, mean peak amplitude, and percentage of cells that contributed to each calcium pulse in explants maintained in vitro for 7 d (7 div) compared with TTX alone. These effects were induced within 30 min and were not mimicked by 17alpha-estradiol, E2 conjugated to BSA (which does not cross the plasma membrane), or seen at 21 div, when the percentage of GnRH-1 cells expressing ERbeta transcripts declines. In addition, these effects were inhibited by the ER antagonist ICI 182,780 and prevented by inhibition of gene transcription. These data suggest that, via ERbeta, E2 can rapidly act as a hormone-activated transcription complex and are the first to show that E2 directly increases GnRH-1 neuronal activity and synchronization.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Calcium; Calcium Signaling; Estradiol; Estrogen Receptor beta; Estrogen Receptor Modulators; Female; Fluorescent Dyes; Fulvestrant; Gonadotropin-Releasing Hormone; Humans; Mice; Nasal Mucosa; Neurons; Organ Culture Techniques; Organic Chemicals; Secretory Rate; Sodium Channel Blockers; Tetrodotoxin; Transcription, Genetic

This work reports the finding of a unique fast inward sodium current (I(Na)) in MDA-MB-231 cells which is missing in MDA-MB-468 cells and in MCF-7 cells. This current is high-voltage-activated and displays a window current at the membrane potential of MDA-MB-231 cells. This current is blocked by high concentrations of tetrodotoxin (TTX). In MDA-MB-231 cells, which are the most invasive cells among the three cell lines tested, proliferation and migration were not sensitive to TTX while invasion was reduced by approximately 30%. These experiments suggest that I(Na) is involved in the invasion process, probably through its participation to the regulation of the intracellular sodium homeostasis.

Topics: Breast Neoplasms; Female; Humans; Neoplasm Invasiveness; Sodium; Sodium Channels; Tetrodotoxin; Tumor Cells, Cultured

Increasing evidence that ion channels play a key role in the modulation of cellular mitogenesis led us to investigate the membranes of T47D human breast cancer cells to identify the ion currents present. We report here the results of voltage-clamp studies in the whole-cell configuration on isolated, non-synchronized single cells obtained from a ductal breast carcinoma. In these studies we identified an outward rectifying potassium current and a chloride current. The potassium current activated at potentials more positive than -40 mV, reached an average value of 1.4 nA, and did not inactivate with time. This current was sensitive to block by extracellular tetraethylammonium chloride (TEA, IC50 = 1 micro M), was insensitive to charybdotoxin (CTX, IC50 = 7.8 micro M), and was not diminished by repetitive pulses separated by 1 s. Rapid voltage-dependent inactivation of the current was demonstrated by tail current analysis. The current appeared calcium-insensitive. Application of hyperpolarizing pulses did not elicit an inward potassium rectifier current. Treatment with tetrodotoxin did not reveal the presence of an inward sodium current. The potassium current was increased by the presence of aspartate in place of chloride and in the presence of the chloride channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We conclude that currents present in T47D breast cancer cells include a chloride current and a voltage-gated potassium outward rectifier. We suggest that the potassium current, either alone or in conjunction with potassium currents reported in different human breast cancer cell lines by others, may play a role in the modulation of the cell cycle.

Topics: Breast Neoplasms; Calcium Channels; Carcinoma, Ductal, Breast; Charybdotoxin; Chloride Channels; Chlorides; Female; Humans; Patch-Clamp Techniques; Potassium; Potassium Channels; Signal Transduction; Sodium; Sodium Channels; Tetraethylammonium; Tetraethylammonium Compounds; Tetrodotoxin; Tumor Cells, Cultured