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 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.     

Comparative studies of intracellular Ca2+ in strongly and weakly metastatic rat prostate cancer cell lines

The metastatic ability of prostate cancer cells involves differential expression of ionic mechanisms. In the present study, using electrophysiological recordings and intracellular Ca2+ measurements, we investigated Ca2+ related signalling in two rat prostate cancer (MAT-LyLu and AT-2) cell lines of markedly different metastatic potential. Whole-cell voltage clamp experiments indicated the absence of an inward current carried through voltage-dependent Ca2+ channels in either cell line. A Ca2+-dependent component was also absent in the voltage-activated outward K+ currents. Indo-1 microfluorimetry confirmed these results and also revealed marked differences in the resting level of intracellular Ca2+ and the ability of the two cell lines to regulate intracellular Ca2+. The weakly metastatic AT-2 cells displayed a significantly higher resting intracellular Ca2+ than the related but strongly metastatic MAT-LyLu cell line. Increasing extracellular K+ decreased intracellular Ca2+ in the AT-2 but had no effect on intracellular Ca2+ levels in the MAT-LyLu cells. Furthermore, increasing extracellular Ca2+ increased intracellular Ca2+ in AT-2 but, again, had no effect on MAT-LyLu cells. These results suggested the presence of a tonic, voltage-independent Ca2+ permeation mechanism operating specifically in the AT-2 cells. The influx of Ca2+ into the AT-2 cells was suppressed by both CdCl2 (100-300 microM) and SKF-96365 (10-30 microM). It is concluded that the strongly metastatic MAT-LyLu cell line lacks a voltage-independent basal Ca2+ influx mechanism that is present in the weakly metastatic AT-2 cells.     

Patterning of endocytic vesicles and its control by voltage-gated Na<Superscript>+</Superscript> channel activity in rat prostate cancer cells: fractal analyses

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 cells endocytic activity was determined by horseradish peroxidase uptake. Digital images of the patterns of vesicular staining were evaluated by multifractal analyses: generalized fractal dimension (D_q) and its Legendre transform f(), 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.     

Small-cell Lung Cancer (Human): Potentiation of Endocytic Membrane Activity by Voltage-gated Na<Superscript>+</Superscript> Channel Expression in Vitro

The possible functional role of voltage-gated Na⁺ channel (VGSC) expression in controlling endocytic membrane activity in human small-cell lung cancer (SCLC) cell lines (H69, H209, H510) was studied using uptake of horseradish peroxidase (HRP). The normal human airway epithelial (16HBE14o) cell line was used in a comparative approach. Uptake of HRP was vesicular, strongly temperature-sensitive and suppressed by cytoskeletal poisons (cytochalasin D and colchicine), consistent with endocytosis. Compared with the normal cells, HRP uptake into SCLC cells was kinetically more efficient, resulting in more than four-fold higher uptake under optimized conditions. Importantly, HRP uptake into SCLC cells was inhibited significantly by the specific VGSC blocker tetrodotoxin, as well as lidocaine and phenytoin. These effects were dose-dependent. None of these drugs had any effect on the uptake into the 16HBE14o cells. Uptake of HRP into SCLC cells was reduced by ∼66% in Na⁺-free medium and was partially (∼30%) dependent on extracellular Ca²⁺. The possibility that the endocytic activity in the H510 SCLC cells involved an endogenous cholinergic system was investigated by testing the effects of carbachol (a cholinergic receptor agonist) and eserine (an inhibitor of acetylcholinesterase). Both drugs inhibited HRP uptake, thereby suggesting that basal cholinergic activity occurred. It is concluded that VGSC upregulation could enhance metastatic cell behavior in SCLC by enhancing endocytic membrane activity.     

Contact stimulation of prostate cancer cell migration: the role of gap junctional coupling and migration stimulated by heterotypic cell-to-cell contacts in determination of the metastatic phenotype of Dunning rat prostate cancer cells

BACKGROUND INFORMATION: Motile activity of tumour cells is regarded as a critical factor determining their metastatic potential. We have shown previously that contrary to the majority of normal cells, homotypic contacts between some tumour cells, among them low metastatic (AT-2) and highly metastatic (MAT-LyLu) rat prostate cancer cells, increase the speed of their movements. The aim of the present study was to determine the effect of heterotypic cell-to-cell contacts on the migration of rat prostate cancer cells differing in metastatic potential, and to correlate it with the intensity of homo- and heterologous gap junctional coupling. RESULTS: MAT-LyLu and AT-2 cells moving on the surface of fibroblasts displayed significantly greater cell displacement than those moving on plastic substrata. This effect correlated with the polarization (contact guidance) and increased speed of cell movements. However, in contrast with the migration on plastic substrata, where MAT-LyLu cells displayed considerably higher motility than AT-2 cells, no differences between both cell lines were observed on the surface of fibroblasts. On the other hand, in contrast with AT-2, Mat-LyLu cells displayed extensive homologous coupling mediated by connexin43 and were able to couple with normal fibroblasts. CONCLUSION: Heterotypic contacts between migrating prostatic cancer cells and normal fibroblasts can strongly stimulate their migration during invasion; however, this effect does not correlate with the gap junctional coupling between cancer cells and normal fibroblasts.     

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.     

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

We have developed a simple yet effective apparatus, based upon negative pressure directed to the tip of a micro-pipette, to measure the adhesiveness of single cells. The “single cell adhesion measuring apparatus” (SCAMA) could differentiate between the adhesion of strongly versus weakly metastatic cancer cells as well as normal cells. Adhesion was quantified as “detachment negative pressure” (DNP) or “DNP relative to cell size” (DNPR) where a noticeable difference in cell size was apparent. Thus, for rat and human prostate and human breast cancer cell lines, adhesiveness (DNPR values) decreased in line with increased metastatic potential. Using the SCAMA, we investigated the effect of tetrodotoxin (TTX), a specific blocker of voltage-gated Na⁺ channels (VGSCs), on the adhesion of rat and human prostate cancer cell lines of markedly different metastatic potential. Following pretreatment with TTX (48 h with 1 μM), 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 (∼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.     

A Study of Membrane Activity in Rat Prostate Cancer Cells: An Evaluation of the FM1-43 Dye Technique

A study was initiated to test whether the FM1-43 dye technique could beapplied to the study of endocytic membrane activity in two rodent prostatecancer (MAT-LyLu and AT-2) cell lines of markedly different metastaticability. The lipophilic dye FM1-43, which has frequently been used tomonitor endo/exocytic activity in excitable cells was employed. We found,as in excitable tissues, that both strongly metastatic (MAT-LyLu) andweakly metastatic (AT-2) cells in culture take up FM1-43 to give vesicularstaining of a variable pattern, which appeared to differ between the twocell lines. However, unlike excitable tissues, neither cell linesubsequently released the dye. Indeed, both cell lines retained the dyethrough several rounds of cell division suggesting that dye incorporatedby cells does not enter the endo/exocytotic cycle. Uptake of dye wasindependent of temperature, Na⁺/K+ gradients, pH or metabolism. Wesuggest that passive accumulation of FM1-43 can occur in cancer cells andshould not, automatically, be interpreted as evidence of endocytosis.     

Sodium-dependent calcium efflux from adrenal chromaffin cells following exocytosis. Possible role of secretory vesicle membranes.

Although cytosolic Ca2+ transients are known to influence the magnitude and duration of hormone and neurotransmitter release, the processes regulating the decay of such transients after cell stimulation are not well understood. Na(+)-dependent Ca2+ efflux across the secretory vesicle membrane, following its incorporation into the plasma membrane, may play a significant role in Ca2+ efflux after stimulation of secretion. We have measured an enhanced 45Ca2+ efflux from cultured bovine adrenal chromaffin cells following cell stimulation with depolarizing medium (75 mM K+) or nicotine (10 microM). Such stimulation also causes Ca2+ uptake via voltage-gated Ca2+ channels and secretion of catecholamines. Na+ replacement with any of several substitutes (N-methyl-glucamine, Li+, choline, or sucrose) during cell stimulation inhibited the enhanced 45Ca2+ efflux, indicating and Na(+)-dependent Ca2+ efflux process. Na+ deprivation did not inhibit 45Ca2+ uptake or catecholamine secretion evoked by elevated K+. Suppression of exocytotic incorporation of secretory vesicle membranes into the plasma membrane with hypertonic medium (620 mOsm) or by lowering temperature to 12 degrees C inhibited K(+)-stimulated 45Ca2+ efflux in Na(+)-containing medium but did not inhibit the stimulated 45Ca2+ uptake. Enhancement of exocytotic secretion with pertussis toxin resulted in an enhanced 45Ca2+ efflux without affecting calcium uptake. The combined results suggest that Na(+)-dependent Ca2+ efflux across secretory vesicle membranes, following their incorporation into the plasma membrane during exocytosis, plays a significant role in regulating calcium efflux and the decay of cytosolic Ca2+ in adrenal chromaffin cells and possibly in related secretory cells.     

Tetrodotoxin suppresses morphological enhancement of the metastatic MAT-LyLu rat prostate cancer cell line

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 μM 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 μM 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. Received: 9 March 1998 / Accepted: 5 October 1998     

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.     

Functional expression of choline transporter-like protein 1 (CTL1) in human neuroblastoma cells and its link to acetylcholine synthesis

We examined the molecular and functional characterization of choline uptake into human neuroblastoma cell lines (SH-SY5Y: non-cholinergic and LA-N-2: cholinergic neuroblastoma), and the association between choline transport and acetylcholine (ACh) synthesis in these cells. Choline uptake was saturable and mediated by a single transport system. Removal of Na(+) from the uptake buffer strongly enhanced choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium. The increase in choline uptake under Na(+)-free conditions was inhibited by a Na(+)/H(+) exchanger (NHE) inhibitor. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), NHE1 and NHE5 mRNA are mainly expressed. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. ChAT mRNA was expressed at a much higher level in LA-N-2 cells than in SH-SY5Y cells. The conversion of choline to ACh was confirmed in both cells, and was enhanced in Na(+)-free conditions. These findings suggest that CTL1 is functionally expressed in both SH-SY5Y and LA-N-2 cells and is responsible for choline uptake that relies on a directed H(+) gradient as a driving force, and this transport functions in co-operation with NHE1 and NHE5. Furthermore, choline uptake through CTL1 is associated with ACh synthesis in cholinergic neuroblastoma cells.     

Iodoacetate action on endocytic uptake of different fluid-phase markers by OK renal epithelial cells

When grown in monolayer culture, OK cells display endocytic uptake of soluble fluid-phase markers such as lucifer yellow (LY) and horseradish peroxidase (HRP). The response of this process to metabolic inhibitors was characterized in the present study. Inhibition of cell metabolism by cyanide produced a decrease in cell ATP content which was accompanied by a decrease in uptake of both LY and HRP, confirming the energy-dependence of fluid-phase endocytosis in OK cells. Use of iodoacetate also decreased cell ATP content but its action on endocytosis was unexpected. Cell uptake of HRP was decreased by iodoacetate, similar to the effect of cyanide, but there was a marked increase in LY uptake. Additional studies showed that cyanide did not change intracellular Na+ or intracellular K+ and did not interfere with the Na(+)-dependency of Pi uptake. In contrast, iodoacetate produced a marked increase in Na+, a decrease in K+, and abolished the Na(+)-dependency of Pi transport. The latter was due primarily to a 10-fold increase in Na(+)-independent uptake of Pi. These findings suggest, indirectly, that plasma membrane permeability to Na+, K+, Pi, and small molecules such as LY, may be increased by iodoacetate, possibly through its action as an alkylating agent. This mechanism may allow increased cell uptake of LY through a non-endocytic pathway, and may mask the inhibitory action of iodoacetate on endocytic uptake of LY. These additional effects complicate the use of iodoacetate to interrupt endocytosis.     

On the biophysics of cathodal galvanotaxis in rat prostate cancer cells: Poisson-Nernst-Planck equation approach

Rat prostate cancer cells have been previously investigated using two cell lines: a highly metastatic one (Mat-Ly-Lu) and a nonmetastatic one (AT-2). It turns out that the highly metastatic Mat-Ly-Lu cells exhibit a phenomenon of cathodal galvanotaxis in an electric field which can be blocked by interrupting the voltage-gated sodium channel (VGSC) activity. The VGSC activity is postulated to be characteristic for metastatic cells and seems to be a reasonable driving force for motile behavior. However, the classical theory of cellular motion depends on calcium ions rather than sodium ions. The current research provides a theoretical connection between cellular sodium inflow and cathodal galvanotaxis of Mat-Ly-Lu cells. Electrical repulsion of intracellular calcium ions by entering sodium ions is proposed after depolarization starting from the cathodal side. The disturbance in the calcium distribution may then drive actin polymerization and myosin contraction. The presented modeling is done within a continuous one-dimensional Poisson-Nernst-Planck equation framework.     

Fractal analysis and ionic dependence of endocytotic membrane activity of human breast cancer cells

The endocytic membrane activities of two human breast cancer cell lines (MDA-MB-231 and MCF-7) of strong and weak metastatic potential, respectively, were studied in a comparative approach. Uptake of horseradish peroxidase was used to follow endocytosis. Dependence on ionic conditions and voltage-gated sodium channel (VGSC) activity were characterized. Fractal methods were used to analyze quantitative differences in vesicular patterning. Digital quantification showed that MDA-MB-231 cells took up more tracer (i.e., were more endocytic) than MCF-7 cells. For the former, uptake was totally dependent on extracellular Na⁺ and partially dependent on extracellular and intracellular Ca²⁺ and protein kinase activity. Analyzing the generalized fractal dimension (D _q ) and its Legendre transform f(α) revealed that under control conditions, all multifractal parameters determined had values greater for MDA-MB-231 compared with MCF-7 cells, consistent with endocytic/vesicular activity being more developed in the strongly metastatic cells. All fractal parameters studied were sensitive to the VGSC blocker tetrodotoxin (TTX). Some of the parameters had a “simple” dependence on VGSC activity, if present, whereby pretreatment with TTX reduced the values for the MDA-MB-231 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.     

Characterisation of inorganic phosphate transport in bovine articular chondrocytes.

In mineralising tissues such as growth plate cartilage extracellular organelles derived from the chondrocyte membrane are present. These matrix vesicles (MV) possess membrane transporters that accumulate Ca(2+) and inorganic phosphate (P(i)), and initiate the formation of hydroxyapatite crystals. MV are also present in articular cartilage, and hydroxyapatite crystals are believed to promote cartilage degradation in osteoarthritic joints. In the present study, P(i) transport pathways in isolated bovine articular chondrocytes have been characterised. P(i) uptake was temperature-sensitive and could be resolved into Na(+)-dependent and Na(+)-independent components. The Na(+)-dependent component saturated at high concentrations of extracellular P(i), with a K(m) for P(i) of 0.17 mM. In solutions lacking Na(+), uptake did not fully saturate, implying that under these conditions carrier-mediated uptake is supplemented by a diffusive pathway. Both Na(+)-dependent and Na(+)-independent components were sensitive to the P(i) transport inhibitors phosphonoacetate and arsenate, although a fraction of Na(+)-independent P(i) uptake was resistant to these anions. Total P(i) uptake was optimal at pH 7.4, and reduced as pH was made more acidic or more alkaline, an effect that represented reduced Na(+)-dependent influx. RT-PCR analysis confirmed that two members of the NaPi III family, Pit-1 and Pit-2, are expressed, but that NaPi II transporters are not.     

Ca(2+) signaling by TRPC3 involves Na(+) entry and local coupling to the Na(+)/Ca(2+) exchanger

TRPC3 has been suggested as a key component of phospholipase C-dependent Ca(2+) signaling. Here we investigated the role of TRPC3-mediated Na(+) entry as a determinant of plasmalemmal Na(+)/Ca(2+) exchange. Ca(2+) signals generated by TRPC3 overexpression in HEK293 cells were found to be dependent on extracellular Na(+), in that carbachol-stimulated Ca(2+) entry into TRPC3 expressing cells was significantly suppressed when extracellular Na(+) was reduced to 5 mm. Moreover, KB-R9743 (5 microm) an inhibitor of the Na(+)/Ca(2+) exchanger (NCX) strongly suppressed TRPC3-mediated Ca(2+) entry but not TRPC3-mediated Na(+) currents. NCX1 immunoreactivity was detectable in HEK293 as well as in TRPC3-overexpressing HEK293 cells, and reduction of extracellular Na(+) after Na(+) loading with monensin resulted in significant rises in intracellular free Ca(2+) (Ca(2+)(i)) of HEK293 cells. Similar rises in Ca(2+)(i) were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na(+) subsequent to stimulation with carbachol. These increases in Ca(2+)(i) were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 (5 microm), chelation of extracellular Ca(2+), or dominant negative suppression of TRPC3 channel function. This suggests that Ca(2+) entry into TRPC3-expressing cells involves reversed mode Na(+)/Ca(2+) exchange. Cell fractionation experiments demonstrated co-localization of TRPC3 and NCX1 in low density membrane fractions, and co-immunoprecipitation experiments provided evidence for association of TRPC3 and NCX1. Glutathione S-transferase pull-down experiments revealed that NCX1 interacts with the cytosolic C terminus of TRPC3. We suggest functional and physical interaction of nonselective TRPC cation channels with NCX proteins as a novel principle of TRPC-mediated Ca(2+) signaling.     

Genistein inhibits the contact-stimulated migration of prostate cancer cells

The results of several epidemiological studies have suggested that a soybean-based diet is associated with a lower risk of prostate cancer. We investigated the effect of the soy isoflavone genistein on the proliferation and contact-stimulated migration of rat prostatic carcinoma MAT-LyLu and AT-2 cell lines. Genistein almost completely inhibited the growth of both MAT-LyLu and AT-2 cells in the concentration range from 25 to 100 μM, but the addition of 1 μM genistein to the medium significantly stimulated the proliferation of both cell lines. Additionally, at concentrations above 25 μM, genistein showed a potent cytotoxic effect. However, the central finding of this study is that at physiologically relevant concentrations (1 μM and 10 μM), genistein inhibits the motility of prostate cancer cells stimulated by homo-and heterotypic contacts. These results show that at physiological concentrations, genistein exerts an inhibitory effect on the migration of prostate cancer cells and suggest that it may be one of the factors responsible for the anti-metastatic activity of plant isoflavonoids