Scott P. Fraser

Voltage-Gated Sodium Channel Expression and Potentiation of Human Breast Cancer Metastasis

Purpose: 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. Experimental Design: 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 α-subunit(s) expressed in vitro was achieved using reverse transcription-PCR, immunocytochemistry, and Western blot techniques and used to investigate VGSCα expression and its association with metastasis in vivo. Results: 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 Nav1.5, 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 Nav1.5 protein in breast cancer cells and tissues. Furthermore, a strong correlation was found between neonatal Nav1.5 expression and clinically assessed lymph node metastasis. Conclusions: Up-regulation of neonatal Nav1.5 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.

Differential expression of voltage-activated Na+ currents in two prostatic tumour cell lines: contribution to invasiveness in vitro

The voltage‐gated ionic currents of two rodent prostatic cancer cell lines were investigated using the whole‐cell patch clamp technique. The highly metastatic Mat‐Ly‐Lu cells expressed a transient, inward Na+ current (blocked by 600 nM tetrodotoxin), which was not found in any of the weakly metastatic AT‐2 cells. Although both cell lines expressed a sustained, outward K+ current, this occurred at a significantly higher density in the AT‐2 than in the Mat‐Ly‐Lu cells. Incubation of the Mat‐Ly‐Lu cell line with 600 nM tetrodotoxin significantly reduced the invasive capacity of the cells in vitro. Under identical conditions, tetrodotoxin had no effect on the invasiveness of the AT‐2 cells.

Sodium channel protein expression enhances the invasiveness of rat and human prostate cancer cells

Expression of Na+ channel protein was analysed in established cell lines of rat and human prostatic carcinoma origin by flow cytometry using a fluorescein‐labelled polyclonal antibody. In many cell lines examined, the obtained frequency distribution profiles were bimodal and identified a subpopulation of cells which expressed high levels of Na+ channel protein. A significant positive correlation was demonstrated between the proportion of channel‐expressing cells and the functional ability of individual cell lines to invade a basement membrane matrix in vitro. In addition, two transfectant cell lines containing rat prostate cancer genomic DNA were found to express significantly elevated levels of Na+ channel protein when compared with the original benign recipient cell line. Enhanced Na+ channel expression by two metastatic derivatives of these transfectant cells directly correlated with increased invasiveness in vitro. These studies strongly support the hypothesis that expression of Na+ channel protein and the metastatic behaviour of prostatic carcinoma cells are functionally related, either by endowing the membranes of these cells with specialised electrophysiological properties (e.g. enhancing their motility and/or secretory activities) and/or by perturbing endogenous mechanisms regulating ionic homeostasis within the cells.

Contribution of functional voltage-gated Na+ channel expression to cell behaviors involved in the metastatic cascade in rat prostate cancer: I. lateral motility

Previous work suggested that functional voltage‐gated Na+ channels (VGSCs) are expressed specifically in strongly metastatic cells of rat and human prostate cancer (PCa), thereby raising the possibility that VGSC activity could be involved in cellular behavior(s) related to the metastatic cascade. In the present study, the possible role of VGSCs in the lateral motility of rat PCa cells was investigated in vitro by testing the effect of modulators that either block or enhance VGSC activity. Two rat PCa cell lines of markedly different metastatic ability were used in a comparative approach: the strongly metastatic MAT‐LyLu and the weakly metastatic AT‐2 cell line, only the former being known to express functional VGSCs. Using both electrophysiological recording and a motility assay, the effects of two VGSC blockers (tetrodotoxin and phenytoin) and four potential openers (veratridine, aconitine, ATX II, and brevetoxin) were monitored on (a) Na+ channel activity and (b) cell motility over 48 h. Tetrodotoxin (at 1 μM) and phenytoin (at 50 μM) both decreased the motility index of the MAT‐LyLu cell line by 47 and 11%, respectively. Veratridine (at 20 μM) and brevetoxin (at 10 nM) had no effect on the motility of either cell line, whilst aconitine (at 100 μM) and ATX II (at 25 pM) significantly increased the motility of the MAT‐LyLu cell line by 15 and 9%, respectively. Importantly, at the concentrations used, none of these drugs had effects on the proliferation or viability of either cell line. The results, taken together, would suggest strongly that functional VGSC expression enhances cellular motility of PCa cells. The relevance of these findings to the metastatic process in PCa is discussed.

Ionic effects of the Alzheimer's disease β-amyloid precursor protein and its metabolic fragments

Alzheimers disease is a progressive dementia characterized in part by deposition of proteinaceous plaques in various areas of the brain. The main plaque protein component is beta-amyloid, a metabolic product of the beta-amyloid precursor protein. Substantial evidence has implicated beta-amyloid (and other amyloidogenic fragments of the precursor protein) with the neurodegeneration observed in Alzheimers disease. Recently, beta-amyloid precursor protein and its amyloidogenic metabolic fragments have been shown to alter cellular ionic activity, either through interaction with existing channels or by de novo channel formation. Such alteration in ionic homeostasis has also been linked with cellular toxicity and might provide a molecular mechanism underlying the neurodegeneration seen in Alzheimers disease.

Voltage-gated Na + channels: multiplicity of expression, plasticity, functional implications and pathophysiological aspects

Voltage-gated Na+ channels (VGSCs) are well known for mediating regenerative cell membrane depolarization and conduction of electrical signalling in nerves and muscles. However, VGSCs may also be expressed in traditionally “non-excitable” cell types, including lymphocytes, glia, fibroblasts and metastatic cancer cells of epithelial origin. Both the diversity and modulation of VGSC expression are far more complex than was initially apparent. There are at least 10 different genes that encode the α-subunits of VGSCs. Since VGSCs can contribute to a range of human disease conditions, it is important to understand both the control and consequences of VGSC functioning and how these aspects may be altered under pathophysiological conditions. Such mechanisms can be at the transcriptional, pre-translational or post-translational levels. This article reviews recent literature that has contributed to our understanding of how individual VGSC subtypes can generate their unique physiological signatures within different cell types. We also highlight emerging areas of interest, in particular, the finding of multiple expression of individual VGSC subtypes within single cells, the generation of alternative splice variants and the increasingly complex set of mechanisms of plasticity through which individual VGSC subtypes may be subtly controlled, including intracellular trafficking of VGSC protein.

T-lymphocyte invasiveness: control by voltage-gated Na+ channel activity

Whole‐cell patch‐clamp recordings showed that a sub‐population (10%) of Jurkat cells, a model of human T‐cells, expressed a functional voltage‐gated sodium channel, which was tetrodotoxin (TTX)‐resistant. Expression of voltage‐gated sodium channel protein was confirmed by western blots. Semi‐quantitative PCR analysis revealed that mRNAs for the α‐subunits of multiple voltage‐gated sodium channel subtypes were present but indicated that Nav1.5 was the predominant subtype, consistent with the TTX‐resistant nature of the recorded currents. Importantly, 10 μM TTX reduced the number of Jurkat cells invading a Matrigel basement membrane by 93.0 ± 5.5%. Since similar sodium channels have also been detected in normal human T‐lymphocytes, it is concluded that the activity of voltage‐gated sodium channels could represent a novel mechanism potentiating the invasive capacity of these cells.

Effects of voltage-gated ion channel modulators on rat prostatic cancer cell proliferation: comparison of strongly and weakly metastatic cell lines.

The strongly metastatic MAT‐LyLu and the weakly metastatic AT‐2 rat prostatic cancer cell lines have been shown to express voltage‐gated ion channels differentially. In the present study, the possible contribution of voltage‐gated ion channel activity to the proliferation of these cell lines was investigated, in a comparative approach.

Angiogenic Functions of Voltage-gated Na+ Channels in Human Endothelial Cells MODULATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) SIGNALING

Voltage-gated sodium channel (VGSC) activity has previously been reported in endothelial cells (ECs). However, the exact isoforms of VGSCs present, their mode(s) of action, and potential role(s) in angiogenesis have not been investigated. The main aims of this study were to determine the role of VGSC activity in angiogenic functions and to elucidate the potentially associated signaling mechanisms using human umbilical vein endothelial cells (HUVECs) as a model system. Real-time PCR showed that the primary functional VGSC α- and β-subunit isoforms in HUVECs were Nav1.5, Nav1.7, VGSCβ1, and VGSCβ3. Western blots verified that VGSCα proteins were expressed in HUVECs, and immunohistochemistry revealed VGSCα expression in mouse aortic ECs in vivo. Electrophysiological recordings showed that the channels were functional and suppressed by tetrodotoxin (TTX). VGSC activity modulated the following angiogenic properties of HUVECs: VEGF-induced proliferation or chemotaxis, tubular differentiation, and substrate adhesion. Interestingly, different aspects of angiogenesis were controlled by the different VGSC isoforms based on TTX sensitivity and effects of siRNA-mediated gene silencing. Additionally, we show for the first time that TTX-resistant (TTX-R) VGSCs (Nav1.5) potentiate VEGF-induced ERK1/2 activation through the PKCα-B-RAF signaling axis. We postulate that this potentiation occurs through modulation of VEGF-induced HUVEC depolarization and [Ca2+]i. We conclude that VGSCs regulate multiple angiogenic functions and VEGF signaling in HUVECs. Our results imply that targeting VGSC expression/activity could be a novel strategy for controlling angiogenesis.

Alternative splicing of Nav1.5: An electrophysiological comparison of ‘neonatal’ and ‘adult’ isoforms and critical involvement of a lysine residue

In developmentally regulated D1:S3 splicing of Nav1.5, there are 31 nucleotide differences between the 5′‐exon (‘neonatal’) and the 3′‐exon (‘adult’) forms, resulting in 7 amino acid differences in D1:S3‐S3/S4 linker. In particular, splicing replaces a conserved negative aspartate residue in the ‘adult’ with a positive lysine. Here, ‘neonatal’ and ‘adult’ Nav1.5 α‐subunit splice variants were stably transfected into EBNA‐293 cells and their electrophysiological properties investigated by whole‐cell patch‐clamp recording. Compared with the ‘adult’ isoform, the ‘neonatal’ channel exhibited (1) a depolarized threshold of activation and voltage at which the current peaked; (2) much slower kinetics of activation and inactivation; (3) 50% greater transient charge (Na+) influx; (4) a stronger voltage dependence of time to peak; and (5) a slower recovery from inactivation. Tetrodotoxin sensitivity and VGSCβ1‐4 mRNA expression levels did not change. The significance of the charge‐reversing aspartate to lysine substitution was investigated by mutating the lysine in the ‘neonatal’ channel back to aspartate. In this ‘neonatal K211D’ mutant, the electrophysiological parameters studied strongly shifted back towards the ‘adult’, that is the lysine residue was primarily responsible for the electrophysiological effects of Nav1.5 D1:S3 splicing. Taken together, these data suggest that the charge reversal in ‘neonatal’ Nav1.5 would (1) modify the channel kinetics and (2) prolong the resultant current, allowing greater intracellular Na+ influx. Developmental and pathophysiological consequences of such differences are discussed. J. Cell. Physiol. 216: 716–726, 2008,