tetrodotoxin and Prostatic-Neoplasms

<b>Background and Objective:</b> Functional Voltage-Gated Sodium Channels (VGSCs) are expressed in metastatic prostate cancer (PCa) cells. A number of <i>in vitro</i> studies have evaluated the effect of functional VGSC expression on the metastatic cell behavior of PCa cells. This study aimed to evaluate the effect of VGSC inhibition on metastatic cell behavior in PCa cells by meta-analysis. <b>Materials and Methods:</b> Meta-analysis was performed on data taken from 13 publications that examined the effect of VGSC inhibitors on the metastatic cell behavior of metastatic PCa cells expressing functional VGSCs. The measure of effect was calculated according to the random effects model using mean differences and presented with a forest plot graph. Heterogeneity was checked using the Cochran's Q Test (Chi-square statistic) and the I² test statistic. In order to evaluate the objectivity, the funnels-plot graph was used. <b>Results:</b> The g value showing the effect size was calculated as 4.49 (95% CI = 5.35-3.62) in the experiments where Tetrodotoxin (TTX) was used, which has a very high specificity for VGSCs but is not licensed for clinical use. In experiments using licensed inhibitors Lamotrigine, Oxcarbazepine, Phenytoin, Ranolazine, Riluzole and Lidocaine, the g value was 1.37 (95 % CI = 2.02-0.71). Suppression of metastatic cell behavior in both subgroups is statistically significant (p<0.00001). <b>Conclusion:</b> Meta-analysis confirmed that VGSCs are an enhancing factor in the metastasis of PCa cells. The VGSCs appear to be an important target in the diagnosis and development of new treatment options in PCa.

Topics: Humans; Male; Prostatic Neoplasms; Ranolazine; Sodium Channels; Tetrodotoxin; Voltage-Gated Sodium Channel Blockers

Anti-invasive effects of riluzole and ranolazine, a neuro-protectant and an anti-anginal drug, respectively, on Mat-LyLu rat prostate cancer (PCa) cells were tested in vitro (a) at non-toxic doses and (b) under both normoxic and hypoxic conditions, the latter common to growing tumours. Tetrodotoxin (TTX) was used as a positive control. Hypoxia had no effect on cell viability but reduced growth at 48 hours. Riluzole (5 μmol/L) or ranolazine (20 μmol/L) had no effect on cell viability or growth under normoxia or hypoxia over 24 hours. Matrigel invasion was not affected by hypoxia but inhibited by TTX, ranolazine and riluzole under a range of conditions. The expression of Nav1.7 mRNA, the prevailing, pro-invasive voltage-gated sodium channel α-subunit (VGSCα), was up-regulated by hypoxia. Riluzole had no effect on Nav1.7 mRNA expression in normoxia but significantly reduced it in hypoxia. VGSCα protein expression in plasma membrane was reduced in hypoxia; riluzole increased it but only under hypoxia. It was concluded (a) that riluzole and ranolazine have anti-invasive effects on rat PCa cells and (b) that Nav1.7 mRNA and protein expression can be modulated by riluzole under hypoxia. Overall, therefore, riluzole and ranolazine may ultimately be "repurposed" as anti-metastatic drugs against PCa.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Humans; Hypoxia; Male; NAV1.7 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; Prostatic Neoplasms; Rats; Riluzole; Tetrodotoxin

Voltage-gated sodium channels are involved in tumor metastasis, as potentiating or attenuating their activities affects the migration and invasion process of tumor cells. In the present study, we tested the effect of two peptide toxins, JZTX-I and HNTX-III which function as Nav1.7 activator and inhibitor, respectively, on the migration and invasion ability of prostate cancer (PCa) cell line Mat-LyLu. These two peptides showed opposite effects, and subsequently a comparative proteomic analysis characterized 64 differentially expressed membrane proteins from the JZTX-I- and HNTX-III-treated groups. Among these, 15 proteins were down-regulated and 49 proteins were up-regulated in the HNTX-III group. Bioinformatic analysis showed eight proteins are cytoskeleton proteins or related regulators, which might play important roles in the metastasis of Mat-LyLu cells. The altered expressions of four of these proteins, fascin, muskelin, annexin A2, and cofilin-1, were validated by western blot analysis. Further function network analysis of these proteins revealed that the Rho family GTPases RhoA and Rac1 might be of particular importance for the rat PCa cell invasion. Pharmacological data revealed that JZTX-I and HNTX-III could modulate the Rho signaling pathway in a Nav1.7-dependent manner. In summary, this study suggests that the Nav1.7-dependent regulation of Rho GTPase activity plays a vital role in Mat-LyLu cell migration and invasion and provides new insights into the treatment of PCa.

Topics: Actin Depolymerizing Factors; Animals; Annexin A2; Carrier Proteins; Cell Line, Tumor; Cell Movement; Male; Microfilament Proteins; NAV1.7 Voltage-Gated Sodium Channel; Prostatic Neoplasms; Proteome; rac1 GTP-Binding Protein; Rats; rho GTP-Binding Proteins; Signal Transduction; Sodium Channel Blockers; Tetrodotoxin

Tumor fluid accumulation occurs in both human cancer and experimental tumor models. Solid tumors show a tendency to tumor fluid accumulation because of their anatomical and physiological features and this may be influenced by molecular factors. Fluid accumulation in the peri-tumor area also occurs in the Dunning model of rat prostate cancer as the tumor grows. In this study, the effects of tumor fluids that were obtained from Dunning prostate tumor-bearing Copenhagen rats on the strongly metastatic MAT-LyLu cell line were investigatedby examining the cell's migration and tumor fluid's toxicity and the kinetic parameters such as cell proliferation, mitotic index, and labelling index. In this research, tumor fluids were obtained from rats injected with 25105 MAT- LyLu cells and treated with saline solution, and 200 nM tetrodotoxin (TTX), highly specific sodium channel blocker was used. Sterilized tumor fluids were added to medium of MAT-LyLu cells with the proportion of 20% in vitro. Consequently, it was demonstrated that Dunning rat prostate tumor fluid significantly inhibited proliferation (up to 50%), mitotic index, and labeling index of MAT-LyLu cells (up to 75%) (p<0.05) but stimulated the motility of the cells in vitro.

Topics: Adenocarcinoma; Animals; Cell Proliferation; Extracellular Fluid; Humans; Lung Neoplasms; Male; Mitotic Index; Neoplasms, Experimental; Prostate; Prostatic Neoplasms; Rats; Sodium Channel Blockers; Tetrodotoxin; Tumor Cells, Cultured

Resveratrol, a natural plant phenolic found at high concentration in red grapes, has been suggested to have a range of health benefits. Here, we tested its effects on metastatic cell behaviors. The strongly metastatic rat prostate MAT-LyLu cells were used as a model. At 20 μM, resveratrol had no effect on cellular proliferation or viability. However, it suppressed significantly 1) lateral motility by up to 25%; 2) transverse motility by 31%; and invasion by 37%. It also increased the cells' adhesion to substrate by 55%. Electrophysiologically, resveratrol inhibited voltage-gated Na(+) channel (VGSC) activity that has been shown previously to promote metastatic cell behaviors. This effect was dose-dependent with an IC50 of ∼50 μM. Voltage dependencies of current activation and peak were not affected but steady-state inactivation was shifted to more hyperpolarized potentials and recovery from inactivation was slowed. Coapplication of resveratrol with the highly specific VGSC blocker tetrodotoxin did not result in any additive effect on inhibition of both 1) VGSC activity and 2) metastatic cell behaviors. These results suggest 1) that a significant mode of action of resveratrol is VGSC blockage and 2) that resveratrol has promise as a natural antimetastatic agent.

Topics: Animals; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cell Survival; Male; Neoplasm Metastasis; Prostatic Neoplasms; Rats; Resveratrol; Sodium Channel Blockers; Sodium Channels; Stilbenes; Tetrodotoxin

Epigenetic upregulation of voltage-gated sodium channels (VGSCs) has been reported in a number of carcinoma cell lines and tissues. Furthermore, a large body of experimental evidence suggested that functional VGSC expression enhances various in vitro cell behaviours, such as directional motility, that would be involved in the metastatic cascade. However, it is not known if VGSC activity promotes metastasis in vivo. Here, using the Copenhagen rat model of prostate cancer and blocking VGSC activity in primary tumours with tetrodotoxin, we show (1) that the number of lung metastasis is reduced by >40% and (2) that lifespan is significantly improved.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Male; NAV1.7 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; Neoplasm Metastasis; Prostatic Neoplasms; Rats; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

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.

Topics: Animals; Biophysics; Cell Adhesion; Cell Line, Tumor; Equipment Design; Humans; Male; Neoplasm Metastasis; Neoplasms; Prostatic Neoplasms; Rats; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

The main aim of this investigation was to determine whether a functional relationship existed between epidermal growth factor (EGF) and voltage-gated sodium channel (VGSC) upregulation, both associated with strongly metastatic prostate cancer cells. Incubation with EGF for 24 h more than doubled VGSC current density. Similar treatment with EGF significantly and dose-dependently enhanced the cells' migration through Transwell filters. Both the patch clamp recordings and the migration assay suggested that endogenous EGF played a similar role. Importantly, co-application of EGF and tetrodotoxin, a highly selective VGSC blocker, abolished 65% of the potentiating effect of EGF. It is suggested that a significant portion of the EGF-induced enhancement of migration occurred via VGSC activity.

Topics: Animals; Cell Movement; Epidermal Growth Factor; Ion Channel Gating; Male; Prostatic Neoplasms; Quinazolines; Rats; Sodium Channels; Tetrodotoxin; Tyrphostins; Up-Regulation

Although a high level of functional voltage-gated sodium channel (VGSC) expression has been found in strongly metastatic human and rat prostate cancer (PCa) cells, the mechanism(s) responsible for the upregulation is unknown. The concentration of epidermal growth factor (EGF), a modulator of ion channels, in the body is highest in prostatic fluid. Thus, EGF could be involved in the VGSC upregulation in PCa. The effects of EGF on VGSC expression in the highly metastatic human PCa PC-3M cell line, which was shown previously to express both functional VGSCs and EGF receptors, were investigated. A quantitative approach, from gene level to cell behaviour, was used. mRNA levels were determined by real-time PCR. Protein expression was studied by Western blots and immunocytochemistry and digital image analysis. Functional assays involved measurements of transverse migration, endocytic membrane activity and Matrigel invasion.. Exogenous EGF enhanced the cells' in vitro metastatic behaviours (migration, endocytosis and invasion). Endogenous EGF had a similar involvement. EGF increased VGSC Nav1.7 (predominant isoform in PCa) mRNA and protein expressions. Co-application of the highly specific VGSC blocker tetrodotoxin (TTX) suppressed the effect of EGF on all three metastatic cell behaviours studied.. 1) EGF has a major involvement in the upregulation of functional VGSC expression in human PCa PC-3M cells. (2) VGSC activity has a significant intermediary role in potentiating effect of EGF in human PCa.

Topics: Cell Line, Tumor; Cell Movement; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Male; Microscopy, Confocal; NAV1.7 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; Neoplasm Metastasis; Prostatic Neoplasms; RNA, Messenger; Sodium Channels; Tetrodotoxin

This study tested the possible functional relationship of two signalling mechanisms shown previously to be involved in human prostate cancer (PCa), Notch and voltage-gated sodium channel. Notch1 and Notch2 were differentially expressed in PCa cell lines of varying metastatic potential (LNCaP, PC-3, PC-3M) in comparison to a normal prostate cell line (PNT2), whereas Notch3 and Notch4 were not expressed. The Notch ligand Jagged1, but not Jagged2, was increased in all cell lines, whereas the Notch downstream target Deltex was not expressed. In comparison to the LNCaP cell line, Hes1, another downstream target, showed elevated expression in the metastatic PC-3 and PC-3M cells and promoted lateral motility. In contrast, the Notch ligand Delta-like1 (Dll1) levels were higher in LNCaP compared with PC-3 and PC-3M cells. Importantly, decreasing Dll1 expression increased the lateral motility of PC-3 cells, whereas blocking voltage-gated Na(+) channel activity with tetrodotoxin decreased motility. However, the effect of Dll1 was independent of Notch signalling through Hes1 and voltage-gated Na(+) channel expression/activity.

Topics: Amyloid Precursor Protein Secretases; Basic Helix-Loop-Helix Transcription Factors; Calcium-Binding Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dimethyl Sulfoxide; Enzyme Inhibitors; Epithelial Cells; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Ion Channel Gating; Jagged-1 Protein; Male; Membrane Proteins; Poisons; Prostatic Neoplasms; Proto-Oncogene Proteins; Receptor, Notch1; Receptor, Notch2; Receptor, Notch3; Receptor, Notch4; Receptors, Notch; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serrate-Jagged Proteins; Signal Transduction; Sodium Channels; Solvents; Tetrodotoxin; Transcription Factor HES-1; Tumor Cells, Cultured

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.

Topics: Carrier Proteins; Cell Line, Tumor; Citric Acid; Gene Expression; Humans; Hydrogen-Ion Concentration; Ion Channel Gating; Male; Membrane Potentials; Neoplasm Metastasis; Prostatic Neoplasms; RNA, Messenger; Sodium; Tetrodotoxin; Zinc

Prostate cancer is the second leading cause of cancer deaths in American males, resulting in an estimated 37,000 deaths annually, typically the result of metastatic disease. A consequence of the unsuccessful androgen ablation therapy used initially to treat metastatic disease is the emergence of androgen-insensitive prostate cancer, for which there is currently no prescribed therapy. Here, three related human prostate cancer cell lines that serve as a model for this dominant form of prostate cancer metastasis were studied to determine the correlation between voltage-gated sodium channel expression/function and prostate cancer metastatic (invasive) potential: the non-metastatic, androgen-dependent LNCaP LC cell line and two increasingly tumorogenic, androgen-independent daughter cell lines, C4 and C4-2. Fluorometric in vitro invasion assays indicated that C4 and C4-2 cells are more invasive than LC cells. Immunoblot analysis showed that voltage-gated sodium channel expression increases with the invasive potential of the cell line, and this increased invasive potential can be blocked by treatment with the specific voltage-gated sodium channel inhibitor, tetrodotoxin (TTX). These data indicate that increased voltage-gated sodium channel expression and function are necessary for the increased invasive potential of these human prostate cancer cells. When the human adult skeletal muscle sodium channel Na(v1.4) was expressed transiently in each cell line, there was a highly significant increase in the numbers of invading LC, C4, and C4-2 cells. This increased invasive potential was reduced to control levels by treatment with TTX. These data are the first to indicate that the expression of voltage-gated sodium channels alone is sufficient to increase the invasive potential of non-metastatic (LC cells) as well as more aggressive cells (i.e., C4 and C4-2 cells). Together, the data suggest that increased voltage-gated sodium channel expression alone is necessary and sufficient to increase the invasive potential of a set of human prostate cancer cell lines that serve as a model for prostate cancer metastasis.

Topics: Cell Line, Tumor; Humans; Male; Muscle Proteins; NAV1.4 Voltage-Gated Sodium Channel; Neoplasm Metastasis; Prostatic Neoplasms; Sodium Channels; Tetrodotoxin

Fractal methods were used to analyze quantitative differences in secretory membrane activities of two rat prostate cancer cell lines (Mat-LyLu and AT-2) of strong and weak metastatic potential, respectively. Each cell's endocytic activity was determined by horseradish peroxidase uptake. Digital images of the patterns of vesicular staining were evaluated by multifractal analyses: generalized fractal dimension (Dq) and its Legendre transform f(alpha), as well as partitioned iterated function system -- semifractal (PIFS-SF) analysis. These approaches revealed consistently that, under control conditions, all multifractal parameters and PIFS-SF codes determined had values greater for Mat-LyLu compared with AT-2 cells. This would agree generally with the endocytic/vesicular activity of the strongly metastatic Mat-LyLu cells being more developed than the corresponding weakly metastatic AT-2 cells. All the parameters studied were sensitive to tetrodotoxin (TTX) pre-treatment of the cells, which blocked voltage-gated Na+ channels (VGSCs). Some of the parameters had a "simple" dependence on VGSC activity, whereby pre-treatment with TTX reduced the values for the MAT-LyLu cells and eliminated the differences between the two cell lines. For other parameters, however, there was a "complex" dependence on VGSC activity. The possible physical/physiological meaning of the mathematical parameters studied and the nature of involvement of VGSC activity in control of endocytosis/secretion are discussed.

Topics: Animals; Cell Line, Tumor; Computer Simulation; Fractals; Image Interpretation, Computer-Assisted; Male; Models, Biological; Prostatic Neoplasms; Rats; Sodium Channels; Tetrodotoxin; Transport Vesicles

Voltage-gated Na+ channel (VGSC) expression has previously been shown to be upregulated in strongly metastatic prostate cancer cells (rat and human) and its activity shown to potentiate a variety of cellular behaviours integral to the metastatic cascade. However, the mechanism(s) responsible for the Na+ channel upregulation is not known. As a step towards evaluating the role of the extracellular biochemical environment in this regard, we have determined the effects of serum concentration on characteristics of Na+ channel expressed in the strongly metastatic Mat-LyLu rat prostate cancer cell line. Whole-cell patch-clamp recording techniques were used to study the effects of serum concentrations, above and below the normal 1%. Both the amplitude and the kinetics of the currents were analysed. The following results were obtained: (1) Adding 1% foetal calf serum to cells starved of serum for 24h increased Na+ current density; however, increasing serum concentration further (to 5%) caused a reduction. (2) Serum-free medium produced Na+ currents with slower kinetics of activation (time to peak) and inactivation (exponential decay). (3) Increased serum concentration (a) shifted steady-state inactivation to more positive potentials without affecting conductance and (b) increased tetrodotoxin sensitivity. It is concluded that serum concentration is an important determinant of the Na+ channel characteristics leading to possible transcriptional and post-translational modifications of channel expression and/or activity. Experiments are now needed to determine which constituents (protein hormones, growth factors, etc.) are responsible for these effects.

Topics: Animals; Cattle; Cell Line, Tumor; Electric Conductivity; Fetal Blood; Male; Patch-Clamp Techniques; Prostatic Neoplasms; Rats; Sodium Channels; Tetrodotoxin

The secretory membrane activities of two rat prostate cancer cell lines of markedly different metastatic potential, and corresponding electrophysiological characteristics, were studied in a comparative approach. In particular, voltage-gated Na(+) channels (VGSCs) were expressed in the strongly metastatic MAT-LyLu but not in the closely related, but weakly metastatic, AT-2 cells. Uptake and release of the non-cytotoxic marker horseradish peroxidase (HRP) were used as indices of general endocytotic and exocytotic membrane activity, respectively. The amount of tracer present in a given experimental condition was quantified by light microscopic digital imaging. The uptake of HRP was an active process, abolished completely by incubating the cells at low temperature (5 degrees C) and suppressed by disrupting the cytoskeleton. Interestingly, the extent of HRP uptake into the strongly metastatic MAT-LyLu cells was almost twice that into the weakly metastatic AT-2 cells. Vesicular uptake of HRP occurred in a fast followed by a slow phase; these appeared to correspond to cytoplasmic and perinuclear pools, respectively. Importantly, the overall quantitative difference in the uptake disappeared in the presence of 1 microM tetrodotoxin which significantly reduced the uptake of HRP into the MAT-LyLu cells. There was no effect on the AT-2 cells, consistent with functional VGSC expression occurring selectively in the former. A similar effect was observed in Na(+)-free medium. The uptake was partially dependent upon extracellular Ca(2+) but was not affected by raising the extracellular K(+) concentration. We suggest that functional VGSC expression could potentiate prostate cancer cells' metastatic ability by enhancing their secretory membrane activity.

Topics: Animals; Calcium; Cell Membrane; Cytoskeleton; Endocytosis; Exocytosis; Horseradish Peroxidase; Male; Membrane Potentials; Models, Biological; Neoplasm Metastasis; Prostatic Neoplasms; Rats; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Tumor Cells, Cultured

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 microM) and phenytoin (at 50 microM) both decreased the motility index of the MAT-LyLu cell line by 47 and 11%, respectively. Veratridine (at 20 microM) and brevetoxin (at 10 nM) had no effect on the motility of either cell line, whilst aconitine (at 100 microM) 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.

Topics: Aconitine; Animals; Carcinoma; Cell Division; Cell Movement; Cnidarian Venoms; Cytoskeletal Proteins; Cytoskeleton; Gene Expression Regulation, Neoplastic; Ion Transport; Male; Marine Toxins; Neoplasm Metastasis; Oxocins; Patch-Clamp Techniques; Phenytoin; Prostatic Neoplasms; Rats; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Tumor Cells, Cultured; Veratridine

A two-part hypothesis has been tested, which proposes that (1) prostate cancer cells are galvanotactic (i.e. respond to an electric field by moving directionally) and (2) voltage-gated Na+ channel activity, which was shown previously to be expressed specifically by strongly metastatic cells, controls galvanotaxis. Two well-defined rat ('Dunning') cell lines, originally derived from the same prostate tumour but differing markedly in their metastatic ability, were used. Cells were exposed to exogenous direct-current electric fields of physiological strength (0.1-4.0 V cm(-1)), their reactions were recorded by light microscopy and analysed by a quantitative tracking method. Voltage-gated Na+ channel activity was modulated pharmacologically using a range of concentrations of a specific channel blocker (tetrodotoxin) or an opener (veratridine). The results showed that the highly metastatic MAT-LyLu cells responded to the application of the electric field strongly by migrating towards the cathode. By contrast, the weakly metastatic AT-2 cells gave no such response. Tetrodotoxin suppressed the galvanotactic response of the MAT-LyLu cells whereas veratridine enhanced it. Both compounds had little effect on the AT-2 cells. These results are consistent with functional voltage-gated Na+ channel expression occurring specifically in highly metastatic cells. This is also the first demonstration of control of galvanotaxis, in any cell type, by voltage-gated Na+ channel activity. The possible underlying mechanisms and the in vivo relevance of these findings are discussed.

Topics: Anesthetics, Local; Animals; Calcium; Cell Movement; Electromagnetic Fields; Ion Channel Gating; Male; Membrane Potentials; Prostatic Neoplasms; Rats; Sodium Channels; Tetrodotoxin; Tumor Cells, Cultured; Veratridine

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.. Several voltage-gated ion channel modulators were tested for their effects on proliferation over 54 hr, using an in vitro assay. The modes of action of the chemicals were monitored by electrophysiological (patch-clamp) recording.. The voltage-gated K(+) channel blockers 4-aminopyridine (4-AP; 2 mM), margatoxin (5 nM), charybdotoxin (4.5 nM), and verapamil (50 microM) inhibited the K(+) channels of both cell lines by between 38-65% and reduced the proliferation of the AT-2 cell line, in a dose-dependent manner, by 8-51%. However, only 4-AP reduced proliferation of the MAT-LyLu cell line. Tetrodotoxin (6 microM) blocked completely the voltage-gated Na(+) channel expressed selectively in the MAT-LyLu cell line, but had no effect on the proliferation of either cell line. On the other hand, the presumed Na(+) channel "opener" veratridine (10-50 microM) reduced significantly, in a dose-dependent manner, the proliferation of both cell lines by up to approximately 30%.. We conclude that the mechanism(s) controlling the proliferation of the weakly metastatic AT-2 cells involves voltage-gated K(+) channels. In contrast, the proliferation of strongly metastatic MAT-LyLu cells is much less dependent upon voltage-gated K(+) channel activity.

Topics: 4-Aminopyridine; Animals; Calcium Channel Blockers; Cell Division; Charybdotoxin; Dose-Response Relationship, Drug; Formazans; Ion Channel Gating; Male; Neurotoxins; Patch-Clamp Techniques; Potassium Channels; Prostatic Neoplasms; Rats; Scorpion Venoms; Sodium Channels; Tetrazolium Salts; Tetrodotoxin; Tumor Cells, Cultured; Verapamil; Veratridine

Voltage-gated Na+ channels are expressed by highly metastatic MAT-LyLu cells, but not by poorly metastatic AT-2 cells, derived from the rodent Dunning model of prostatic cancer. We have investigated the possible involvement of these channels in the morphological development of the cells. Incubation of both the MAT-LyLu and the AT-2 cell line for 24 h with the Na+ channel blocker tetrodotoxin (TTX) at 6 microM altered the morphology only of the MAT-LyLu cell line. TTX produced significant decreases in: (a) cell process length and (b) field diameter, and increases in (c) cell body diameter and (d) process thickness. Importantly, 6 microM TTX had no significant effects on proliferation rates or cellular toxicity. The results suggest that Na+ channel activity plays a significant role in determining the morphological development of MAT-LyLu cells in such a way as to enhance their metastatic potential.

Topics: Animals; Cell Division; Male; Neoplasm Metastasis; Prostatic Neoplasms; Rats; Sodium Channel Blockers; Tetrodotoxin; Tumor Cells, Cultured

Voltage-gated Na+ channels, classically associated with impulse conduction in excitable tissues, are also found in a variety of epithelial cell types where their possible functions are not known so well. We have previously reported expression of a voltage-gated Na+ channel specifically in the highly metastatic Mat-LyLu rat prostate cancer cell line; blockage of the current with tetrodotoxin (TTX) significantly reduced the invasiveness of the cells in vitro, suggesting that the channel may have a functional role in metastasis. The aim of the present study was to characterize this current using the whole-cell patch clamp recording technique, and compare it to Na+ currents found in various other tissues. The inward current of the Mat-LyLu cells was abolished completely, but reversibly, in Na+-free solution, confirming that Na+ was indeed the permeant ion. Activation occurred at -40 mV and currents reached a maximal amplitude at around 6 mV. Boltzmann fits to current activation and steady-state inactivation revealed that the currents were half activated at about -15 mV and half inactivated at -80 mV. Both current inactivation and recovery from inactivation followed a double-exponential time course with fast and slow components. The Na+ currents were highly sensitive to block by TTX (IC50 approximately 18 nM), whilst 1 microM mu-conotoxin GIIIA mostly had no effect. 100 microM Cd2+ also had no effect on the current, whilst 2.5 mM Cd2+, Mn2+, and Co2+ each caused a depolarizing shift in activation and a reduction in peak conductance of around 20%. In conclusion, the Na+ channel expressed in the highly metastatic Mat-LyLu cell line appeared to have electrophysiological and pharmacological properties of TTX-sensitive channels. Further work is needed, however, to elucidate the exact nature of the channel protein and the mechanism(s) of its involvement in cellular invasiveness.

Topics: Animals; Cations, Divalent; Conotoxins; Electric Stimulation; Ion Channel Gating; Male; Patch-Clamp Techniques; Peptides, Cyclic; Prostatic Neoplasms; Rats; Sodium; Sodium Channels; Tetrodotoxin; Tumor Cells, Cultured

Ion channels are important for many cellular functions and disease states including cystic fibrosis and multidrug resistance. Previous work in the Dunning rat model of prostate cancer has suggested a relationship between voltage-activated Na+ channels (VASCs) and the invasive phenotype in vitro. The objectives of this study were to 1) evaluate the expression of VASCs in the LNCaP and PC-3 human prostate cancer cell lines by Western blotting, flow cytometry, and whole-cell patch clamping, 2) determine their role in invasion in vitro using modified Boyden chambers with and without a specific blocker of VASCs (tetrodotoxin). A 260-kd protein representing VASCs was found only in the PC-3 cell line, and these were shown to be membrane expressed on flow cytometry. Patch clamping studies indicated that functional VASCs were present in 10% of PC-3 cells and blocking these by tetrodotoxin (600 nmol/L) reduced their invasiveness by 31% (P = 0.02) without affecting the invasiveness of the LNCaP cells. These results indicate that the reduction of invasion is a direct result of VASC blockade and not a nonspecific action of the drug. This is the first report of VASCs in a human prostatic cell line. VASCs are present in PC-3 but not LNCaP cells as determined by both protein and functional studies. Tetrodotoxin reduced the invasiveness of PC-3 but not LNCaP cells, and these data suggest that ion channels may play an important functional role in tumor invasion.

Topics: Animals; Blotting, Western; Brain; Carcinoma; Diffusion Chambers, Culture; Electrophysiology; Flow Cytometry; Humans; Male; Neoplasm Invasiveness; Patch-Clamp Techniques; Prostatic Neoplasms; Rats; Sodium Channels; Tetrodotoxin; Tumor Cells, Cultured

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.

Topics: Animals; Epithelial Cells; Ion Channel Gating; Male; Neoplasm Invasiveness; Prostatic Neoplasms; Rats; Sodium Channels; Tetrodotoxin; Tumor Cells, Cultured