TOV112D, A2780 and SKOV3 ovarian cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). TOV112D cells were cultured in a 1:1 mixture of M199 and MCDB105 media, supplemented with 10% (wt/vol) fetal bovine serum (FBS) (both from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). A2780 and SKOV3 cells were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% (wt/vol) FBS. In addition, A2780 cells were supplemented with 50 µg/ml insulin (Sigma-Aldrich; Merck KGaA). All cells were maintained at 37°C in an incubator containing 5% (vol/vol) CO₂.

Patch-clamp measurements were performed in whole-cell configuration using an Axopatch 200B amplifier controlled by pClamp10 software through an Axon Digidata 1440A system (all from Molecular Devices, LLC, Sunnyvale, CA, USA) (16,17). The extracellular solution contained 140 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl₂, 2.5 mM CaCl₂, 10 mM HEPES and 11 mM glucose, adjusted to pH 7.4 with NaOH. The intracellular solution contained 140 mM CsCl, 2 mM MgCl₂, 0.1 mM CaCl₂, 1.1 mM EGTA and 10 mM HEPES, adjusted to pH 7.2 with CsOH. The sodium currents were activated by depolarization to −10 mV for 50 msec from a holding potential of −110 mV. To test steady-state activation, the sodium currents were measured from a holding potential of −110 mV to test potentials ranging between −110 to +70 mV in 10 mV steps, followed by repolarization to −110 mV. To test steady-state inactivation, currents were elicited to 0 mV following prepulses ranging between −140 and 0 mV in 10 mV steps for 200 msec. EPA was purchased from Sigma-Aldrich; Merck KGaA and dissolved in ethanol to obtain a stock concentration of 10 mM; butylatedhydroxyanisole was added at a ratio of 0.005% w/v to the stock solution, which was then stored at −80°C (18). EPA was added to the extracel-lular solution at the desired final concentrations (1-200 µM), and the sodium currents were recorded before and after the addition of EPA, or after EPA washout. All measurements were performed at 22±2°C. Pipettes had 2-4 MΩ access resistance (19,20). Current densities were calculated according to whole-cell current amplitude and capacitance values obtained from the amplifier following electronic subtraction of the capacitive transients.

Dose-responses of channel inhibition were fitted by the Hill equation, as previously described (21): %_i = %_i,max [B]^h/(IC₅₀^h +[B]^h), where %_i,max is the maximal percentage of channel inhibition by blocker B, h is the Hill coefficient and IC₅₀ is the concentration of an inhibitor required for 50% inhibition. Steady-state activation G-V were fitted by the Boltzmann equation, as described previously (22): G/G_max = 1/(1+exp(V_1/2-V)/K), where G/G_max is the relative conductance normalized by the maximal conductance, V_1/2 is the potential of half activation, V is test pulse, and k is the Boltzmann coefficient. Steady-state inactivation I-V were fitted by the Boltzmann equation: I/I_max = 1/(1+exp(V_1/2-V)/K).

Ovarian cancer cells were rinsed twice with ice-cold PBS, and were then harvested and lysed with lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.02% NaN₃, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate and 1% protease inhibitor cocktail; Sigma-Aldrich; Merck KGaA). Protein concentration was quantified using the bicinchoninic acid assay (Beyotime Institute of Biotechnology, Haimen, China) and proteins (25 µg/lane) were separated by 6% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Bedford, MA, USA). Membranes were blocked at room temperature for 1 h in 5% non-fat milk dissolved in Tris-buffered saline containing Tween-20 (150 mM NaCl, 50 mM Tris, 0.1% Tween-20; pH 7.5), and were incubated with rabbit anti-human Nav1.5 (1:1,000, ASC-013; Alomone Labs, Jerusalem, Israel) and rabbit anti-GAPDH (1:1,000, #5174; Cell Signaling Technology, Inc., Danvers, MA, USA) primary antibodies at 4°C overnight. Subsequently, membranes were incubated with the goat anti-rabbit horse-radish peroxidase-conjugated secondary antibodies (1:10,000, ab6721; Abcam, Cambridge, UK) for 1.5 h at room temperature. Bands were detected by chemiluminescence assay (Beyotime Institute of Biotechnology) and were semi-quantified using ImageJ version 1.4.3 (National Institutes of Health, Bethesda, MD, USA).

SKOV3 ovarian cancer cells were transfected with siRNA directed against sodium voltage-gated channel α subunit 5 (SCN5A) mRNA (Nav1.5-targeting siRNA; forward, 5′-GAACGGCACCUCUGAUGU GTT-3′ and reverse, 5′-CACAUCAGAGGUGCCGUUCTT-3′) or scramble siRNA-A (forward, 5′ GCAACAAUGUGCGUC CUGGTT-3′ and reverse, 5′-CCAGGACGCACAUUGUUG CTT-3′), which was used as a control (SiCtl). siRNAs were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Cells (80% confluence) were transfected with 20 or 40 nM siRNA using Lipofectamine^® RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Subsequently, the migration and cell proliferation assays, and the patch clamp technique, were performed 24 h post-transfection.

The pRc/CMV plasmid containing the sequence of the human Nav1.5 channel was kindly provided by Dr Alfred L. George Jr (Northwestern University, Chicago, IL, USA). The pRc/CMV-hNav1.5 plasmid contains a 6.1 kb cDNA sequence encoding the human voltage-gated sodium channel Nav1.5 (SCN5A gene product) inserted into the NotI (5′) and XbaI (3′) sites of the mammalian expression vector, pRc/CMV (5.4 kb). Plasmids containing cDNA encoding human Nav1.5 channels (pRc/CMV-hNav1.5) and green fluorescent protein (GFP; pRc/CMV-GFP) were co-transfected into 293T cells (cat. no. CRL-3216; American Type Culture Collection), which do not possess endogenous Nav1.5 channels. Cells (80% confluence) were transfected with 1 µg/µl plasmids using Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The cells were then cultured for 24 h in Dulbecco's modified Eagle's medium supplemented with 10% FBS (both from Gibco; Thermo Fisher Scientific, Inc.). Patch clamp experiments were conducted 24-48 h after 293T cells were reseeded on coverslips in 6-well plates containing RPMI-1640 medium. A coverslip with 293T cells was placed in a recording chamber containing bath solution on the stage of a fluorescence micro-scope (Nikon Corporation, Tokyo, Japan), and the transfected cells were identified by the fluorescent signal emitted from GFP (data not shown).

SKOV3 cells were seeded onto 24-well plates (10,000 cells/well). For the wound-healing assay, scratch lesions were created on confluent SKOV3 cells using a 10 µl pipette tip. Wounded cultures were incubated at room temperature for 36 h, and three randomly selected fields at the lesion border were subsequently examined by inverted phase contrast microscopy (IX73; Olympus Corporation, Tokyo, Japan) in order to assess cell migration.

Cell Counting kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to study SKOV3 cell proliferation, according to the manufacturer's protocol. SKOV3 cells were seeded at a density of 5×10³ cells/well in 96-well plates, after which 10 µl CCK-8 solution was added to each well. Absorbance was measured at 450 nm using a multiplate reader (Lambda Bio-20; Beckman Coulter, Inc., Brea, CA, USA). Cell viability was expressed as a percentage of that of the control (untreated) cells.

All data are presented as the means ± standard error of the mean. The n value denotes the number of independent experiments conducted, unless otherwise stated. Significance between means was determined using either the two-tailed Student's paired t-test or one-way analysis of variance with Dunnett's multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

The voltage-gated sodium channel Nav1.5 has been implicated in ovarian cancer (12), the present study detected Nav1.5 expression in the epithelial ovarian cancer cell lines TOV112D, A2780 and SKOV3. The results of a western blot analysis indicated that Nav1.5 channel protein was expressed in the three ovarian cancer cells (Fig. 1A and B). Although the voltage-gated sodium channels have been associated with ovarian cancer development, to the best of our knowledge, the functional sodium currents have not been examined in ovarian cancer cells (11,12). Using whole cell patch-clamp configuration, the present study observed fast inactivation currents in ovarian cancer cells (Fig. 1C). The currents elicited by depolarization to −10 mV were further measured prior to and after the addition of the specific voltage-gated sodium channel blocker tetrodotoxin (TTX); Sigma-Aldrich; Merck KGaA) to the bath solution; currents were partially inhibited by 10 µM TTX (Fig. 1D). TTX had IC₅₀ values of 5.8, 9.2 and 3.1 µM in SKOV3, A2780, and TOV112D ovarian cancer cells, respectively (Fig. 1E and Table I). Since the IC₅₀ values were in the micromolar range, it was suggested that these cell lines were of the TTX-resistant subtype (23).

To further analyze sodium currents in ovarian cancer cells, the RNA interference approach was used to knockdown Nav1.5 expression in SKOV3 cancer cells. Transfection with Nav1.5 siRNA resulted in a significant reduction in the protein expression levels of Nav1.5, whereas SiCtl did not alter Nav1.5 expression (Fig. 2A and B). The reduction in Nav1.5 protein expression was associated with >90% reduction of the maximal currents recorded when the cells were depolarized to −10 mV (Fig. 2C and D). These results indicated that the sodium currents were predominantly carried by the Nav1.5 channel, and that Nav1.5 channels are functionally expressed in ovarian cancer cells.

After examining Nav1.5 channel currents in ovarian cancer cells, the present study aimed to determine whether EPA exerted effects on them (Fig. 3). EPA was previously reported to block the Nav1.5 channel in cardiomyocytes (14), and to inhibit ovarian cancer cell growth and reduce cancer metastasis (15). The present study hypothesized that EPA may affect the Nav1.5 channel in ovarian cancer cells; the results indicated that treatment with 15 µM EPA blocked ~60% sodium currents in SKOV3 cancer cells (Fig. 3A, B and E), and this inhibition could be completely reversed following EPA washout (Fig. 3C and D). Furthermore, 15 µM EPA blocked sodium currents to similar extents in A2780 and TOV112D cancer cells (Fig. 3F and G). Various concentrations of EPA were then used to generate a dose-response curve; the IC₅₀ values for EPA in SKOV3, A2780 and TOV112D cells were 11.8, 19.6 and 18.1 µM, respectively (Fig. 3H and Table I). These results indicated that EPA may effectively block Nav1.5 sodium currents in ovarian cancer cells. Subsequently, the present study aimed to explore the potential mechanism by which EPA inhibits Nav1.5 currents.

Previous studies demonstrated that EPA induces Nav1.5 channels to enter an inactive state and stabilizes the channel, thus resulting in a reduction of sodium currents (24,25). The present study investigated the effects of EPA on SKOV3 cancer cells (Fig. 4); the results indicated that 15 µM EPA shifted the steady-state inactivation of sodium currents by ~20 mV to the hyperpolarizing direction (Fig. 4A and C and Table II), whereas it had almost no effect on channel activation (Fig. 4B and C and Table III). The window currents were obtained by plotting the area where steady-state inactivation and activation overlap; EPA significantly reduced the Nav1.5 sodium window currents (Fig. 4D). In addition, the shifts of inactivation induced by EPA, along with the rise of EPA concentration, were gradually enhanced to the hyperpolarizing direction, indicating that the shift of steady-state inactivation is EPA dose-dependent (Fig. 5 and Table II). In the other two ovarian cancer cells (A2780 and TOV112D cells), 15 µM EPA also altered steady-state inactivation curves to the hyperpolarizing direction, and did not significantly alter steady-state activation curves (Fig. 6 and Tables II and III). Subsequently, the present study transfected 293T cells with Nav1.5 cDNA; the results revealed that EPA hyperpolarized the slow inactivation curve of Nav1.5 channels and reduced Nav1.5 channel availability, which are consistent with the results in ovarian cancer cells (Fig. 7). In conclusion, these data suggested that EPA may enhance sodium channel inactivation and stabilize the inactivation gate of the channel, thus indicating that EPA-induced reductions in sodium currents may be due to more channels remaining in the inactivated state post-activation.

The present study also aimed to examine the function of Nav1.5 during the process of ovarian cancer development. The Nav1.5 channel has been reported to promote cancer cell invasion (26); therefore, inhibition of this channel was expected to reduce cell migration. The present study used a wound-healing assay to investigate the effects of either Nav1.5 knockdown or Nav1.5 functional inhibition by EPA on SKOV3 ovarian cancer cell migration. The cells were divided into five groups: Group 1, control; group 2, cells transfected with Nav1.5 siRNA; group 3, cells transfected with SiCtl; group 4, cells treated with 200 µM EPA; group 5, cells transfected with Nav1.5 siRNA and treated with 200 µM EPA (Fig. 8). The results demonstrated that EPA was able to inhibit SKOV3 cell migration in a dose-dependent manner (Fig. 8C), and migration was significantly reduced in Nav1.5 siRNA-transfected cells compared with in SiCtl cells (Fig. 8A and B). Notably, the migration of Nav1.5 siRNA-transfected cells was not further reduced by 200 µM EPA, thus indicating that the impaired invasion of these cells was due to the absence of Nav1.5 activities (Fig. 8B). These results suggested that the Nav1.5 channel may have a critical role in ovarian cancer migration, and the effects of EPA on cell migration were due to the blockade of Nav1.5 sodium channels.

Using the CCK-8 assay, it was revealed that EPA inhibited the proliferation of SKOV3 cells in a dose-dependent manner (Fig. 9A). Knockdown of Nav1.5 in SKOV3 cells resulted in a marked decrease in the inhibitory effect of 15 µM EPA on SKOV3 cell proliferation. In addition, 15 µM EPA alone still inhibited SKOV3 cell proliferation in SiCtl-transfected cells (Fig. 9B). Collectively, these data indicated that inhibition of Nav1.5 by EPA may reduce ovarian cell proliferation.