Sigma-1 receptors modulate neonatal Nav1.5 ion channels in breast cancer cell lines.

The main aim of this study was to investigate a possible functional connection between sigma-1 receptors and voltage-gated sodium channels (VGSCs) in human breast cancer cells. The hypothesis was that sigma-1 drugs could alter the metastatic properties of breast cancer cells via the VGSC. Evidence was found for expression of sigma-1 receptor and neonatal Nav1.5 (nNav1.5) expression in both MDA-MB-231 and MDA-MB-468 cells. Sigma-1 drugs (SKF10047 and dimethyltryptamine) did not affect cell proliferation or migration but significantly reduced adhesion to the substrate. Silencing sigma-1 receptor expression by siRNA similarly reduced the adhesion. Blocking nNav1.5 activity with a polyclonal antibody (NESOpAb) targeting an extracellular region of nNav1.5 also reduced the adhesion in both cell lines. Importantly, the results of combined treatments with NESOpAb and a sigma-1 drug or sigma-1 siRNA suggested that both treatments targeted the same mechanism. The possibility was tested, therefore, that the sigma-1 receptor and the nNav1.5 channel formed a physical, functional complex. This suggestion was supported by the results of co-immunoprecipitation experiments. Furthermore, application of sigma-1 drugs to the cells reduced the surface expression of nNav1.5 protein, which could explain how sigma-1 receptor activation could alter the metastatic behaviour of breast cancer cells. Overall, these results are consistent with the idea of a sigma-1 protein behaving like either a "chaperone" or a regulatory subunit associated with nNav1.5.

Polycystic kidney disease channel and synaptotagmin homologues play roles in schizosaccharomyces pombe cell wall synthesis/repair and membrane protein trafficking.

Eukaryotic cells can sense a wide variety of environmental stresses, including changes in temperature, pH, osmolarity and nutrient availability. They respond to these changes through a variety of signal-transduction mechanisms, including activation of Ca(2+)-dependent signaling pathways. This research has discovered important implications in the function(s) of polycystic kidney disease (PKD) channels and the mechanisms through which they act in the control of cell growth and cell polarity in Schizosaccharomyces pombe by ion channel-mediated Ca(2+) signaling. Pkd2 was expressed maximally during the exponential growth phase. At the cell surface pkd2 was localized at the cell tip during the G(2) phase of the cell cycle, although following cell wall damage, the cell surface-expressed protein relocalized to the whole plasma membrane. Pkd2 depletion affected Golgi trafficking, resulting in a buildup of vesicles at the cell poles, and strongly affected plasma membrane protein delivery. Surface-localized pkd2 was present in the plasma membrane for a very short time and was rapidly internalized. Internalization was dependent on Ca(2+), enhanced by amphipaths and inhibited by gadolinium. The pkd2 protein was in a complex with a yeast synaptotagmin homologue and myosin V. Depletion of pkd2 severely affected the localization of glucan synthase. A role for pkd2 in a cell polarity and cell wall synthesis signaling complex with a synaptotagmin homologue, myosin V and glucan synthase is proposed.

Abnormal expression, localization and interaction of canonical transient receptor potential ion channels in human breast cancer cell lines and tissues: a potential target for breast cancer diagnosis and therapy.

BACKGROUND: Ca2+ is known to be involved in a number of metastatic processes including motility and proliferation which can result in store-depletion of Ca2+. Up regulation of genes which contribute to store operated channel (SOC) activity may plausibly be necessary for these processes to take place efficiently. TRPC proteins constitute a family of conserved Ca2+-permeable channels that have been shown to contribute to SOC activity. RESULTS: In breast cancer biopsy tissues, TRPC3 and TRPC6 were the predominant TRPC genes expressed with TRPC3 and TRPC6 being significantly up regulated compared to normal breast tissue. In the lowly metastatic breast cancer cell line MCF-7, TRPC6 was the chief TRPC gene expressed while in the highly metastatic breast cancer cell line MDA-MB-231 both TRPC3 and TRPC6 were the predominant TRPC genes expressed. Western blotting, immunoconfocal analysis and immunoprecipitation experiments confirmed that the MDA-MB-231 cell line expressed both TRPC3 and TRPC6 protein with the majority of protein being intracellular. TRPC3 and TRPC6 were found to be in an immunoprecipitatble complex and co-localize within the cell. To demonstrate the potential of targeting TRP channels in breast cancer, hyperforin reportably a specific activator of TRPC6 significantly reduced the growth and viability of the breast cancer cell lines but had no effect on the non-cancerous breast cell line. Silencing of TRPC6 in MDA-MB-231 cells resulted in a significant reduction in cell growth but not viability. CONCLUSION: TRPC channels may be potential future targets for breast cancer diagnosis and therapy and deserve further investigation to evaluate their role in cancer cell physiology.

Effect of Sigma-1 Receptors on Voltage-Gated Sodium Ion Channels in Colon Cancer Cell Line SW620.

Background: Voltage-gated sodium channels (VGSCs) play pivotal roles in the metastatic process in several cancers, including breast and colon cancers. Sigma-1 receptors are known to interact and form complexes with a number of ion channels aiding the delivery of the channel protein to the plasma membrane. Drugs that bind the Sigma-1 receptor are hypothesized to affect this process and reduce the delivery of the channel protein to the plasma membrane, in turn reducing the metastatic potential of the cells. Methods: Human colon cancer cell line SW620 was utilized as a model to investigate the interaction between the neonatal VGSC (nNav1.5) and the Sigma-1 receptor. This was accomplished using drugs that bind the Sigma-1 receptor, Sigma-1 receptor silencing, and antibodies that bind and block the nNav1.5 channel. Results: Sigma-1 receptor drugs SKF10047 and dimethyl tryptamine were found to alter (reduce) the adhesion of these cells by 46-54% at a 20 µM drug concentration. In a similar manner, gene silencing of the Sigma-1 receptor had a similar effect in reducing the adhesion of these cells to collagen-coated plates by 30%. The Sigma-1 receptor was found to be in a complex with nNav1.5 in SW620 cells, and Sigma-1 drugs or gene silencing of the Sigma-1 receptor results in a reduction of the surface expression of nNav1.5 by ∼50%. Culture of SW620 cells under hypoxic conditions resulted in upregulation of the Sigma-1 receptor and nNav1.5. In addition, surface expression of nNav1.5 protein increased under hypoxic culture conditions and this was inhibited by the application of SKF10047. Conclusions: It is proposed that in colon cancer cells, upregulated Sigma-1 receptor expression in hypoxia led to increased nNav1.5 protein expression at the plasma membrane and resulted in the cells switching to a more invasive state.

The sigma receptor as a ligand-regulated auxiliary potassium channel subunit.

The sigma receptor is a novel protein that mediates the modulation of ion channels by psychotropic drugs through a unique transduction mechanism depending neither on G proteins nor protein phosphorylation. The present study investigated sigma receptor signal transduction by reconstituting responses in Xenopus oocytes. Sigma receptors modulated voltage-gated K+ channels (Kv1.4 or Kv1.5) in different ways in the presence and absence of ligands. Association between Kv1.4 channels and sigma receptors was demonstrated by coimmunoprecipitation. These results indicate a novel mechanism of signal transduction dependent on protein-protein interactions. Domain accessibility experiments suggested a structure for the sigma receptor with two cytoplasmic termini and two membrane-spanning segments. The ligand-independent effects on channels suggest that sigma receptors serve as auxiliary subunits to voltage-gated K+ channels with distinct functional interactions, depending on the presence or absence of ligand.

The expression and functional characterization of sigma (sigma) 1 receptors in breast cancer cell lines.

Sigma (sigma) receptors have been implicated in cancer. However, to date there is little molecular data demonstrating the role of sigma1 receptors in cancer. Expression of sigma1 receptors in various human cancer cell lines in comparison to non-cancerous cell lines was investigated, using real time RT-PCR and by western blotting with a sigma1 receptor specific antibody. Our results indicate that cancer cells express higher levels of sigma1 receptors than corresponding non-cancerous cells. Localization of the sigma1 receptor was investigated in MDA-MB-231 cells by immunocytochemistry and confocal microscopy, expression was visualized predominantly at the cell periphery. We have tested the effect of sigma1 and sigma2 drugs and a sigma1 receptor silencing construct on various aspects of the metastatic process on two breast cell lines of different metastatic potential and a normal breast cell line. Both sigma1 and sigma2 drugs and the sigma1 receptor silencing construct had effects on proliferation and adhesion for breast cancer cell lines, compared to a non-cancerous breast cell line. This data suggests sigma1 receptor plays a role in proliferation and adhesion of breast cancer cells. Therefore, it is likely to be a potential target for the diagnosis and therapy of breast cancer.

A microbial TRP-like polycystic-kidney-disease-related ion channel gene.

Ion channel genes have been discovered in many microbial organisms. We have investigated a microbial TRP (transient receptor potential) ion channel gene which has most similarity to polycystic-kidney-disease-related ion channel genes. We have shown that this gene (pkd2) is essential for cellular viability, and is involved in cell growth and cell wall synthesis. Expression of this gene increases following damage to the cell wall. This fission yeast pkd2 gene, orthologues of which are found in all eukaryotic cells, appears to be a key signalling component in the regulation of cell shape and cell wall synthesis in yeast through an interaction with a Rho1-GTPase. A model for the mode of action of this Schizosaccharomyces pombe protein in a Ca2+ signalling pathway is hypothesized.

Sigma receptors and cancer: possible involvement of ion channels.

The sigma (sigma) receptor and its agonists have been implicated in a myriad of cellular functions, biological processes and diseases. Whereas the precise molecular mechanism(s) of sigma receptors and their involvement in cancer cell biology have not been elucidated, recent work has started to shed some light on these issues. A molecular model has been proposed for the cloned sigma1 receptor; the precise molecular nature of the sigma2 receptor remains unknown. sigma receptors have been found to be frequently up-regulated in human cancer cells and tissues. sigma2 receptor drugs particularly have been shown to have antiproliferative effects. An interesting possibility is that sigma and/or sigma1 drugs could produce anticancerous effects by modulating ion channels. As well as proliferation, a variety of other metastatic cellular behaviors such as adhesion, motility, and secretion may also be affected. Other mechanisms of sigma receptor action may involve interaction with ankyrin and modulation of intracellular Ca(2+) and sphingolipid levels. Although more research is needed to further define the molecular physiology of sigma receptors, their involvement in the cellular pathophysiology of cancer raises the possibility that sigma drugs could be useful as novel therapeutic agents.

Systems biology of monovalent cation homeostasis in yeast: the translucent contribution.

Maintenance of monovalent cation homeostasis (mainly K(+) and Na(+)) is vital for cell survival, and cation toxicity is at the basis of a myriad of relevant phenomena, such as salt stress in crops and diverse human diseases. Full understanding of the importance of monovalent cations in the biology of the cell can only be achieved from a systemic perspective. Translucent is a multinational project developed within the context of the SysMO (System Biology of Microorganisms) initiative and focussed in the study of cation homeostasis using the well-known yeast Saccharomyces cerevisiae as a model. The present review summarize how the combination of biochemical, genetic, genomic and computational approaches has boosted our knowledge in this field, providing the basis for a more comprehensive and coherent vision of the role of monovalent cations in the biology of the cell.

Single cell adhesion measuring apparatus (SCAMA): application to cancer cell lines of different metastatic potential and voltage-gated Na+ channel expression.

We have developed a simple yet effective apparatus, based upon negative pressure directed to the tip of a micro-pipette, to measure the adhesiveness of single cells. The "single cell adhesion measuring apparatus" (SCAMA) could differentiate between the adhesion of strongly versus weakly metastatic cancer cells as well as normal cells. Adhesion was quantified as "detachment negative pressure" (DNP) or "DNP relative to cell size" (DNPR) where a noticeable difference in cell size was apparent. Thus, for rat and human prostate and human breast cancer cell lines, adhesiveness (DNPR values) decreased in line with increased metastatic potential. Using the SCAMA, we investigated the effect of tetrodotoxin (TTX), a specific blocker of voltage-gated Na(+) channels (VGSCs), on the adhesion of rat and human prostate cancer cell lines of markedly different metastatic potential. Following pretreatment with TTX (48 h with 1 microM), the adhesion values for the Mat-LyLu cells increased significantly 4.3-fold; there was no effect on the AT-2 cells. For the strongly metastatic PC-3M cells, TTX treatment caused a significant (approximately 30%) increase in adhesion. The adhesion of PNT2-C2 ("normal") cells was not affected by the TTX pretreatment. The TTX-induced increase in the adhesiveness of the strongly metastatic cells was consistent with the functional VGSC expression in these cells and the proposed role of VGSC activity in metastatic cell behaviour. In conclusion, the SCAMA, which can be constructed easily and cheaply, offers a simple and effective method to characterise single-cell adhesion and its modulation.

Sigma-1 receptors bind cholesterol and remodel lipid rafts in breast cancer cell lines.

Lipid rafts are membrane platforms that spatially organize molecules for specific signaling pathways that regulate various cellular functions. Cholesterol is critical for liquid-ordered raft formation by serving as a spacer between the hydrocarbon chains of sphingolipids, and alterations in the cholesterol contents of the plasma membrane causes disruption of rafts. The role that sigma receptors play in cancer is not clear, although it is frequently up-regulated in human cancer cells and tissues and sigma receptors inhibit proliferation in carcinoma and melanoma cell lines, induce apoptosis in colon and mammary carcinoma cell lines, and reduce cellular adhesion in mammary carcinoma cell lines. In this study, we provide molecular and functional evidence for the involvement of the enigmatic sigma 1 receptors in lipid raft modeling by sigma 1 receptor-mediated cholesterol alteration of lipid rafts in breast cancer cell lines. Cholesterol binds to cholesterol recognition domains in the COOH terminus of the sigma 1 receptor. This binding is blocked by sigma receptor drugs because the cholesterol-binding domains form part of the sigma receptor drug-binding site, mutations of which abolish cholesterol binding. Furthermore, we outline a hypothetical functional model to explain the myriad of biological processes, including cancer, in which these mysterious receptors are involved. The findings of this study provide a biological basis for the potential therapeutic applications of lipid raft cholesterol regulation in cancer therapy using sigma receptor drugs.

Expression and functional characterization of the human ether-à-go-go-related gene (HERG) K+ channel cardiac splice variant in Xenopus laevis oocytes.

HERG C(Cardiac), a C-terminal splice variant of the human ether-à-go-go-related gene (HERG A), was identified and found to be 100% homologous to HERG(USO). Real-time polymerase chain reaction data indicated that in the human heart HERG C(Cardiac )mRNA was expressed eight times more than HERG A, whereas in human ventricular tissue it was expressed six times more than HERG A. A HERG C(Cardiac)-green fluorescence protein (GFP) construct was heterologously expressed in Xenopus oocytes. Confocal micrographs revealed that HERG C(Cardiac )was mainly expressed in the plasma membrane. HERG C(Cardiac) channel expressed in oocytes produced slower inactivating outward currents and faster deactivating tail currents than those of HERG A channel. Equal amounts of HERG A and HERG C(Cardiac) cRNA coinjected into oocytes formed intermediate HERG A + HERG C(Cardiac) heteromultimers, which was reconfirmed by immunoprecipitation experiments with a HERG A N-terminal antibody. These heteromultimers had different inactivation, deactivation and activation kinetics from those of HERG A and HERG C(Cardiac) channels. HERG A + HERG C(Cardiac) heteromultimers significantly reduced the model action potential mean amplitude and increased the fast and slow inactivation tau values of the action potential repolarization phase, suggesting involvement of HERG A and HERG C(Cardiac) heteromultimers in modulation of the refractory interval.