M.B.A. Djamgoz

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.

Neurobiology of retinal dopamine in relation to degenerative states of the tissue

Neurobiology of retinal dopamine is reviewed and discussed in relation to degenerative states of the tissue. The Introduction deals with the basic physiological actions of dopamine on the different neurons in vertebrate retinae with an emphasis upon mammals. The intimate relationship between the dopamine and melatonin systems is also covered. Recent advances in the molecular biology of dopamine receptors is reviewed in some detail. As degenerative states of the retina, three examples are highlighted: Parkinsons disease; ageing; and retinal dystrophy (retinitis pigmentosa). As visual functions controlled, at least in part, by dopamine, absolute sensitivity, spatial contrast sensitivity, temporal (including flicker) sensitivity and colour vision are reviewed. Possible cellular and synaptic bases of the visual dysfunctions observed during retinal degenerations are discussed in relation to dopaminergic control. It is concluded that impairment of the dopamine system during retinal degenerations could give rise to many of the visual abnormalities observed. In particular, the involvement of dopamine in controlling the coupling of horizontal and amacrine cell lateral systems appears to be central to the visual defects seen.

Use-Dependent Effects of Amyloidogenic Fragments of β-Amyloid Precursor Protein on Synaptic Plasticity in Rat Hippocampus In Vivo

The Alzheimers disease-related β-amyloid precursor protein (β-APP) is metabolized to a number of potentially amyloidogenic peptides that are believed to be pathogenic. Application of relatively low concentrations of the soluble forms of these peptides has previously been shown to block high-frequency stimulation-induced long-term potentiation (LTP) of glutamatergic transmission in the hippocampus. The present experiments examined how these peptides affect low-frequency stimulation-induced long-term depression (LTD) and the reversal of LTP (depotentiation). We discovered that β-amyloid peptide (Aβ1–42) and the Aβ-containing C -terminus of β-APP (CT) facilitate the induction of LTD in the CA1 area of the intact rat hippocampus. The LTD was frequency- and NMDA receptor-dependent. Thus, although low-frequency stimulation alone was ineffective, after intracerebroventricular injection of Aβ1–42, it induced an LTD that was blocked byd-(−)-2-amino-5-phosphonopentanoic acid. Furthermore, an NMDA receptor-dependent depotentiation was induced in a time-dependent manner, being evoked by injection of CT 10 min, but not 1 hr, after LTP induction. These use- and time-dependent effects of the amyloidogenic peptides on synaptic plasticity promote long-lasting reductions in synaptic strength and oppose activity-dependent strengthening of transmission in the hippocampus. This will result in a profound disruption of information processing dependent on hippocampal synaptic plasticity.

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.

A potential novel marker for human prostate cancer: voltage-gated sodium channel expression in vivo.

Functional expression of voltage-gated sodium channel α-subunits (VGSCαs), specifically Nav1.7, is associated with strong metastatic potential in prostate cancer (CaP) in vitro. Furthermore, VGSC activity in vitro directly potentiates processes integral to metastasis. To investigate VGSCα expression in CaP in vivo, immunohistochemistry and real-time PCR were performed on human prostate biopsies (n>20). VGSCα immunostaining was evident in prostatic tissues and markedly stronger in CaP vs non-CaP patients. Importantly, RT-PCRs identified Nav1.7 as the VGSCα most strikingly upregulated (∼20-fold) in CaP, and the resultant receiver-operating characteristics curve demonstrated high diagnostic efficacy for the disease. It is concluded that VGSCα expression increases significantly in CaP in vivo and that Nav1.7 is a potential functional diagnostic marker.

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.

Sigma Receptors and Cancer Possible Involvement of Ion Channels

The sigma (σ) receptor and its agonists have been implicated in a myriad of cellular functions, biological processes and diseases. Whereas the precise molecular mechanism(s) of σ 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 σ1 receptor; the precise molecular nature of the σ2 receptor remains unknown. σ receptors have been found to be frequently up-regulated in human cancer cells and tissues. σ2 receptor drugs particularly have been shown to have antiproliferative effects. An interesting possibility is that σ and/or σ1 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 σ receptor action may involve interaction with ankyrin and modulation of intracellular Ca2+ and sphingolipid levels. Although more research is needed to further define the molecular physiology of σ receptors, their involvement in the cellular pathophysiology of cancer raises the possibility that σ drugs could be useful as novel therapeutic agents.

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.