Biomedical subjects

Mustafa B A Djamgoz

Publications and source records attributed to Mustafa B A Djamgoz.

At least 19 recordsLinked to original sources

PubMed2007

Promoter profiling and coexpression data analysis identifies 24 novel genes that are coregulated with AMPA receptor genes, GRIAs.

We identified a set of transcriptional elements that are conserved and overrepresented within the promoters of human, mouse, and rat GRIAs by comparing these promoters against a collection of 10,741 gene promoters. Cells regulate functional groups of genes by coordinating the transcriptional and/or posttranscriptional mRNA levels of interacting genes. As such, it is expected that functional groups of genes share the same transcriptional features within their promoters. We found 47 genes whose promoters contain the same combination of transcriptional elements that are overrepresented within the promoters of the GRIA gene family. Coexpressed genes may be transcriptionally coregulated, which in turn suggests that these genes may play complementary roles within a particular functional context. Using microarray expression data, we found 24 (of the 47) genes that share not only a similar promoter profile with GRIAs but also a well-correlated gene expression profile and, thus, we believe these to be coregulated with GRIAs.

Animals↗

PubMed2007

Nerve growth factor enhances voltage-gated Na+ channel activity and Transwell migration in Mat-LyLu rat prostate cancer cell line.

The highly dynamic nature of voltage-gated Na+ channel (VGSC) expression and its controlling mechanism(s) are not well understood. In this study, we investigated the possible involvement of nerve growth factor (NGF) in regulating VGSC activity in the strongly metastatic Mat-LyLu cell model of rat prostate cancer (PCa). NGF increased peak VGSC current density in a time- and dose-dependent manner. NGF also shifted voltage to peak and the half-activation voltage to more positive potentials, and produced currents with faster kinetics of activation; sensitivity to the VGSC blocker tetrodotoxin (TTX) was not affected. The NGF-induced increase in peak VGSC current density was suppressed by both the pan-trk antagonist K252a, and the protein kinase A (PKA) inhibitor KT5720. NGF did not affect the Nav1.7 mRNA level, but the total VGSC alpha-subunit protein level was upregulated. NGF potentiated the cells' migration in Transwell assays, and this was not affected by TTX. We concluded that NGF upregulated functional VGSC expression in Mat-LyLu cells, with PKA as a signaling intermediate, but enhancement of migration by NGF was independent of VGSC activity.

Animals↗

PubMed2006

Docosahexaenoic acid (omega-3) blocks voltage-gated sodium channel activity and migration of MDA-MB-231 human breast cancer cells.

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.

Breast Neoplasms↗

PubMed2006

The neonatal splice variant of Nav1.5 potentiates in vitro invasive behaviour of MDA-MB-231 human breast cancer cells.

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.

Blotting, Western↗

PubMed2006

Citrate enhances in vitro metastatic behaviours of PC-3M human prostate cancer cells: status of endogenous citrate and dependence on aconitase and fatty acid synthase.

Prostate is a unique organ that produces and releases large amounts of citrate. This is reduced significantly in cancer and it is possible that citrate is (re)taken up and used as a metabolite to enhance cellular activity. The main purpose of this study was to determine how cytosolic citrate might affect in vitro metastatic cell behaviours (lateral motility, endocytosis and adhesion). Normal (PNT2-C2) and metastatic (PC-3M) human prostate cancer cells were used in a comparative approach. As regards intermediary metabolic enzymes, aconitase and fatty acid synthase, already implicated in prostate cancer, were evaluated. The level of intracellular citrate was significantly higher in PNT2-C2 cells under both control conditions and following preincubation in extracellular citrate. Supply of exogenous citrate enhanced endocytosis, lateral motility, decreased cell adhesion of PC-3M cells but failed to produce any effect on normal cells. Real-time PCR measurements showed that the mRNA levels of mitochondrial and cytosolic aconitases and fatty acid synthase were significantly higher in PC-3M cells. Correspondingly, aconitase activity was also higher in PC-3M cells. Using cerulenin (an inhibitor of fatty acid synthase), oxalomalate and fluorocitrate (inhibiting aconitases), we investigated the dependence of citrate-induced down-regulation of cellular adhesion on aconitase and fatty acid synthase activities. It was concluded: (1) that strongly metastatic PC-3M cells stored less/utilised more cytosolic citrate than the normal PNT2-C2 cells and (2) that cancer cells could metabolise cytoplasmic citrate via aconitase and fatty acid synthase to enhance their metastatic behaviour.

Aconitate Hydratase↗

PubMed2006

Activity-dependent regulation of voltage-gated Na+ channel expression in Mat-LyLu rat prostate cancer cell line.

We have shown previously that voltage-gated Na(+) channels (VGSCs) are up-regulated in human metastatic disease (prostate, breast and small-cell lung cancers), and that VGSC activity potentiates metastatic cell behaviours. However, the mechanism(s) regulating functional VGSC expression in cancer cells remains unknown. We investigated the possibility of activity-dependent (auto)regulation of VGSC functional expression in the strongly metastatic Mat-LyLu model of rat prostate cancer. Pretreatment with tetrodotoxin (TTX) for 24-72 h subsequently suppressed peak VGSC current density without affecting voltage dependence. The hypothesis was tested that the VGSC auto-regulation occurred via VGSC-mediated Na(+) influx and subsequent activation of protein kinase A (PKA). Indeed, TTX pretreatment reduced the level of phosphorylated PKA, and the PKA inhibitor KT5720 decreased, whilst the adenylate cyclase activator forskolin and the Na(+) ionophore monensin both increased the peak VGSC current density. TTX reduced the mRNA level of Nav1.7, predominant in these cells, and VGSC protein expression at the plasma membrane, although the total VGSC protein level remained unchanged. TTX pretreatment eliminated the VGSC-dependent component of the cells' migration in Transwell assays. We concluded that the VGSC activity in Mat-LyLu rat prostate cancer cells was up-regulated in steady-state via a positive feedback mechanism involving PKA, and this enhanced the cells' migratory potential.

Animals↗

PubMed2006

An acetylcholinesterase-derived peptide inhibits endocytic membrane activity in a human metastatic breast cancer cell line.

Acetylcholinesterase (AChE) is well established as having non-cholinergic functions and is also expressed in breast tumours where its function(s) is not known. Recently, a candidate peptide sequence towards the C-terminal of the AChE molecule has been identified, as the salient site remote from normal catalysis in neurons, and possibly other cells. The main aim of this study was to explore the possibility that 'AChE-peptide' might also affect human breast cancer cells. Uptake of the non-cytotoxic tracer horseradish peroxidase (HRP) was used as an index of endocytosis, a key component of the metastatic cascade, representing exocytosis/secretory membrane activity and/or plasma membrane protein turnover. AChE-peptide had no affect on the weakly metastatic MCF-7 human breast cancer cell line. By contrast, application of AChE-peptide to the strongly metastatic MDA-MB-231 cells resulted in a dose-dependent inhibition of HRP uptake; treatment with a scrambled variant of the peptide of comparable amino acid length was ineffective. The action of AChE-peptide was suppressed by lowering the extracellular Ca2+ concentration and co-applying a selective antagonist of alpha7, but not alpha4/beta2, nicotinic receptor. The results suggest that AChE-peptide has a novel, selective bioactivity on breast cancer cells and can potentiate metastatic cell behaviour.

Acetylcholinesterase↗

PubMed2005

A novel polyclonal antibody specific for the Na(v)1.5 voltage-gated Na(+) channel 'neonatal' splice form.

Voltage-gated Na(+) channel (VGSC) diversity is achieved through a number of mechanisms: multiple subunits, multiple genes encoding the pore-forming VGSC alpha-subunit and multiple gene isoforms generated by alternative splicing. A major type of VGSCalpha alternative splicing is in D1:S3, which has been proposed to be developmentally regulated. We recently reported a D1:S3 spliced form of Na(v)1.5 in human metastatic breast cancer cells. This novel 'neonatal' isoform differs from the counterpart 'adult' form at seven amino acids (in the extracellular loop between S3-S4 of D1). Here, we generated an anti-peptide polyclonal antibody, named NESOpAb, which specifically recognised 'neonatal' but not 'adult' Na(v)1.5 when tested on cells specifically over-expressing one or other of these Na(v)1.5 spliced forms. The antibody was used to investigate developmental expression of 'neonatal' Na(v)1.5 (nNa(v)1.5) in a range of mouse tissues by immunohistochemistry. Overall, the results were consistent with nNa(v)1.5 protein being more abundantly expressed in selected tissues (particularly heart and brain) from neonate as compared to adult animals. Importantly, NESOpAb blocked functional nNa(v)1.5 ion conductance when applied extracellularly at concentrations as low as 0.05 ng/ml. Possible biological and clinical applications of NESOpAb are discussed.

Alternative Splicing↗

PubMed2005

Endogenous and exogenous citrate transport and release in prostatic preparations: semi-polarized two-dimensional cultures of human PNT2-C2 cells and isolated tubules and segments of rat prostate.

BACKGROUND: Electrophysiological characterization of normal human prostate epithelial cells showed exogenous trivalent citrate transport (release) to be K(+)-dependent. METHODS: (1) Ussing chamber recordings of short circuit current (SCC) were used to study citrate transport in the same (PNT2-C2) cell line grown on micro-pore filters as a monolayer. (2) Release of endogenous citrate from confluent cultures and tubules and segments of rat prostate was measured using a fluorescence technique. (3) Enzyme-spectrophotometry was employed to detect citrate release from segments of rat prostate. RESULTS: Citrate transport across the PNT2-C2 monolayer was asymmetrical, consistent with release into the lumen-side. Fluorescence and/or enzyme-spectrophotometric measurements showed that time-dependent citrate release (endogeneous and preabsorbed) occurred from rat prostate (tubules and segments), but not kidney or lung. The release was dependent on extracellular K(+) but not Na(+). CONCLUSIONS: Citrate release from prostatic cells and tissues (rat and human) was K(+)-dependent, consistent with the previous electrophysiological data.

Animals↗

PubMed2005

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 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. 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 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. CONCLUSIONS: 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.

Amino Acid Sequence↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2005

Nitric oxide and metastatic cell behaviour.

Nitric oxide (NO) is a pleiotropic signalling molecule that subserves a wide variety of basic cellular functions and also manifests itself pathophysiologically. As regards cancer and its progression, however, the reported role of NO appears surprisingly inconsistent. In this review, we focus on metastasis, the process of cancer cell spread and secondary tumour formation. In a 'reductionist' approach, we consider the metastatic cascade to be made up of a series of basic cellular behaviours (such as proliferation, apoptosis, adhesion, secretion migration, invasion and angiogenesis). We evaluate how NO controls such behaviours, in comparison with normal cells. The available information suggests strongly that NO signalling would be expected to regulate these behaviours both positively and negatively and this probably leads to the observed apparent variability in the NO status of cancer cells and tissues. Thus, the role of NO in cancer is more complex than previously thought. A number of suggestions are made, including consideration of novel mechanisms, such as ion channels, in order to achieve a more consistent and integrated understanding of NO signalling in cancer and to realise its clinical potential.

Animals↗

PubMed2004

Expression of Na+-dependent citrate transport in a strongly metastatic human prostate cancer PC-3M cell line: regulation by voltage-gated Na+ channel activity.

Prostate is a unique organ which synthesizes and releases large amounts of citrate. It has been shown that in metastatic prostate cancer, the amount of citrate in prostatic fluid is significantly reduced, approaching the level normally found in blood. In our previous study, we characterized electrophysiologically the mechanism of citrate transport in a normal prostatic epithelial (PNT2-C2) cell line. It was concluded that the cells expressed a novel transporter carrying 1 citrate3- together with 4 K+, primarily out of cells. In the present study, we aimed similarly to characterize the mechanism(s) of citrate transport in a strongly metastatic human prostate cancer (PC-3M) cell line and to compare this with the previous data. Citrate transport in PC-3M cells was found to be both Na+ and K+ dependent. Intracellular application of citrate produced an outward current that was primarily K+ dependent whilst extracellular citrate elicited an inward current that was mainly Na+ dependent. The electrophysiological and pharmacological characteristics of the citrate outward current were similar to the K+-dependent citrate transporter found in the PNT2-C2 cells. On the other hand, the inward citrate current had a markedly different reversal potential, ionic characteristics, inhibitor profile and pH sensitivity. Preincubation of the PC-3M cells (24 or 48 h) with the voltage-gated Na+ channel (VGSC) blocker tetrodotoxin (TTX) significantly reduced the Na+ sensitivity of the citrate current, up-regulated VGSC mRNA expression but did not change the partial permeability of the membrane to Na+. It was concluded (a) that PC-3M cells express a K+-dependent transporter (carrying citrate outward), similar to that found in normal prostate epithelial cells, as well as (b) a Na+-dependent transporter (carrying citrate inward). The molecular nature of the latter was investigated by RT-PCR; the three known Na+-dependent citrate/dicarboxylate transporters could not be detected. VGSC activity, which itself has been associated with metastatic prostate cancer, had a differential effect on the two citrate transporters, down-regulating the expression of the Na+-dependent component whilst enhancing the K+-dependent citrate transporter.

Carrier Proteins↗

PubMed2004

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.

Animals↗

PubMed2004

Epidermal growth factor, neurotrophins and the metastatic cascade in prostate cancer.

Although cancer of the prostate (CaP) is the most commonly occurring cancer in males, there are major limitations in its diagnosis and long-term cure. Consequently, understanding the molecular mechanisms involved in the progression of CaP is of particular importance for production of pharmacological and biological agents to manage the disease. The development of the normal prostate is regulated by stromal-epithelial interactions via endocrine and paracrine factors, such as androgens and growth factors, which act as precise homeostatic regulators of cellular proliferation. Importantly, after a period of hormonal therapy, CaP shifts from an androgen-dependent to an androgen-independent state with a concomitant switch from paracrine to autocrine growth factor stimulation and subsequent upregulation of growth factor expression. Thus, growth factors and their receptors have a pivotal role in CaP. This is emphasized by current evidence obtained from clinical specimens as well as several in vitro and in vivo models strongly suggesting that epidermal growth factor and the neurotrophins (nerve growth factor, brain derived neurotrophin factor, neurotrophin-3 and neurotrophin-4/5) together with their tyrosine kinase receptors could play a very significant role in CaP progression.

Androgens↗

PubMed2004

Citrate transport in the human prostate epithelial PNT2-C2 cell line: electrophysiological analyses.

Although prostate synthesizes and releases large amounts of citrate, the mechanism of the release is not well understood. Most known citrate transporters mediate uptake of citrate from extracellular space and, consequently, are driven by the transmembrane Na+ gradient, which would not be appropriate for prostatic function. In the present study, we investigated citrate transport in a normal human prostate cell line, PNT2-C2, using mainly electrophysiological methods. Intracellular application of citrate through the patch pipette in the whole-cell recording mode induced an outward current whilst in response to extracellular citrate an inward current was recorded. Membrane currents induced by citrate were bigger than those elicited by other (equimolar) Krebs cycle intermediates. Both inward and outward citrate-induced currents had the same ionic dependence, inhibitor profile and reversal potential. In particular, the currents were strongly dependent on the transmembrane K+ gradient. Uptake and release of citrate and their K+ dependence were confirmed by spectrophotometric enzyme analyses. Citrate-induced membrane currents were also sensitive to pH, consistent with the transporter preferring the trivalent form. Application of intracellular Zn2+ generated an outward current which had the same quantitative K+ dependence as the citrate-induced currents. Extracellular application of a membrane-permeant Zn2+ chelator generated an inward current. These experiments suggested that m-aconitase was tonically active in PNT2-C2 cells. Determination of 'forward' and 'reverse' K+ stoichiometry both suggested a citrate: K+ ratio of 1: 4. We conclude that normal prostatic epithelial cells possess an electrogenic citrate transporter which mediates the cotransfer of 1 trivalent citrate anion alongside 4 K+ out of cells and thus generates a net outward current.

Carrier Proteins↗

PubMed2004

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 alpha-subunits of multiple voltage-gated sodium channel subtypes were present but indicated that Na(v)1.5 was the predominant subtype, consistent with the TTX-resistant nature of the recorded currents. Importantly, 10 microM 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.

Humans↗

PubMed2004

Cellular mechanisms of direct-current electric field effects: galvanotaxis and metastatic disease.

Endogenous direct-current electric fields (dcEFs) occur in vivo in the form of epithelial transcellular potentials or neuronal field potentials, and a variety of cells respond to dcEFs in vitro by directional movement. This is termed galvanotaxis. The passive influx of Ca(2+) on the anodal side should increase the local intracellular Ca(2+) concentration, whereas passive efflux and/or intracellular redistribution decrease the local intracellular Ca(2+) concentration on the cathodal side. These changes could give rise to 'push-pull' effects, causing net movement of cells towards the cathode. However, such effects would be complicated in cells that possess voltage-gated Ca(2+) channels and/or intracellular Ca(2+) stores. Moreover, voltage-gated Na(+) channels, protein kinases, growth factors, surface charge and electrophoresis of proteins have been found to be involved in galvanotaxis. Galvanotactic mechanisms might operate in both the short term (seconds to minutes) and the long term (minutes to hours), and recent work has shown that they might be involved in metastatic disease. The galvanotactic responses of strongly metastatic prostate and breast cancer cells are much more prominent, and the cells move in the opposite direction compared with corresponding weakly metastatic cells. This could have important implications for the metastatic process and has clinical implications. Galvanotaxis could thus play a significant role in both cellular physiology and pathophysiology.

Animals↗