The present study used the highly metastatic Mat-LyLu cell line and the weakly metastatic AT-2 cell line, which were derived from the same Dunning rat prostate tumor (25). These cell lines were obtained from Imperial College, London (UK), and were cultured in RPMI-1640 medium supplemented with 1% heat inactivated fetal bovine serum, 1% l-glutamine and 0.5% dexamethasone. The cells were maintained under cell culture conditions of 37°C and 5% CO₂ in a humidified chamber. All chemicals for the cell culture were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

H. helix L. (family, Araliaceae) was collected in winter from rural areas of Mersin, located in the Mediterranean region of Turkey. The plant samples were identified by Professor Tuna Ekim from Istanbul University (Istanbul, Turkey). Voucher specimens of the plant were stored in the Herbarium of the Faculty of Science at the University of Istanbul (ISTF Herbarium number, 40074). Following identification, shiny, light green H. helix leaves and unripened fruits were washed and dried in a chamber at 40°C for 24 h. The dry samples (500 g) were powdered mechanically and extracted using ethanol (>98%; Honeywell Riedel-de Haën AG, Seelze, Germany; 1:10 w/v) in an orbital shaker at room temperature for at least 24 h. These extracts were filtrated using Whatman filter paper no. 4. Following filtration, the supernatant was lyophilized at −40°C under a vacuum (Edwards, Crawley, UK). Following lyophilization, the weight of crude extracts was 11 and 7 g for H. helix leaves and fruits, respectively.

Stock solutions (10 mg/ml) of the extracts were prepared using RPMI medium with the aforementioned supplements. Stock solutions were filtered using 0.45- or 0.20-µm-diameter disposable filters. The final concentrations of the HLE were 25, 50 and 75 µg/ml and the final concentrations of the HFE were 18, 20 and 22 µg/ml. The extracts were diluted in conditioned RPMI medium. The concentrations were selected as result of preliminary toxicity assays (described below). TTX was prepared according to the manufacturer's protocol (Alomone Labs, Jerusalem, Israel). Briefly, 1 mg TTX was dissolved in 1 ml sterile distilled water. The stock solution (3,132 µM) of TTX was frozen in aliquots and stored at −20°C until use. The test concentration of TTX (200 nM) was diluted in conditioned RPMI medium.

Trypan blue exclusion assays, which are based on living cells excluding trypan blue dye (4), were performed to determine whether the ivy extracts and TTX had toxic effects on the cell lines. After treatment of the cell lines with the ivy extracts or TTX for 48 h, trypan blue dye (4%; Sigma-Aldrich, St. Louis, MO, USA) was added into the cell culture dishes (Greiner BioOne GmbH, Frickenhausen, Germany). The live/dead cell number was counted by using an inverted microscope (Olympus, Tokyo, Japan) (40X objective).

The effects of the HLE and HFE on the proliferation of Dunning model rat prostate cancer cell lines were assayed spectrophotometrically using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reagent (Sigma-Aldrich), as described previously (5). A total of 3×10⁴ cells per dish were plated into 35-mm-diameter cell culture dishes. The cells were left to incubate for 24 h undisturbed, and following this, the medium was replaced daily for 2 days while incubation continued. In the control dishes, fresh supplemented RPMI medium was used. In the test dishes, fresh test medium of various concentrations was used. To determine the cell proliferation, spectrophotometric measurements were performed using the MTT assay 48 h subsequent to the addition of the test medium. Absorbance of MTT was measured at 570 nm. The cell counts were normalized using the absorbance values of the test groups compared with the control group. Additionally, the cell inhibition rates were calculated proportionally on the basis of the absorbance value of the control group.

The mitotic index (MI) was determined by observing the effects of the extracts on the mitotic activity of prostate cancer cell lines. The cells were plated on coverslips and treated with control or test media for 48 h. The cells were then fixed using Clarke's fluid (ethanol:acetic acid, 3:1) and stained using the Feulgen method (26). The number of cells in the mitotic phases (including the late prophase, metaphase, anaphase and telophase; n) per total cells (4,000–6,000; C) was determined by the same person. The MI (%) was calculated using the following formula: MI=(n/C)×100.

The effect of H. helix extracts on cell motility was evaluated using wound healing assays (5). Cells were plated in 35-mm-diameter cell culture dishes at an initial concentration of 15×10⁴ cells per dish. Following 24 h of incubation at 37°C, three parallel scratches per dish were made using a sterile pipette tip. The initial widths (mm) of these wounds (W₀) were measured using a graticule on an inverted microscope (Olympus) (10X objective). A total of 24–48 h after the initial measurement, the widths of the wounds were measured again (W_t). The control and test media were replaced at 24 and 48 h following plating. Motility index (MoI) was calculated using the formula: MoI=1-(W_t/W₀).

Data are presented as the mean ± standard error. Student's t-test, the comparison test of two ratios and the Pearson correlation test were used to compare the data. P<0.05 was considered to indicate a statistically significant difference. Statistical tests were performed using Microsoft Office Excel 2007 (Microsoft Corporation, Redmond, WA, USA) and SPSS version 11.0 (SPSS, Inc., Chicago, IL, USA).

In the present study, an initial evaluation was performed to determine the non-toxic concentrations of HLE and HFE on the two cell lines (Mat-LyLu and AT-2) by using a trypan blue exclusion assay. HLE (25, 50 and 75 µg/ml) and HFE (18, 20 and 22 µg/ml) were not toxic against the cells following 48 h of incubation. Thus, these non-toxic concentrations were used in the subsequent experiments. In addition, 200 nM TTX did not demonstrate toxicity against the two cell lines. Metastatic properties of Mat-LyLu and AT-2 cells are closely associated with VGSCs, as has been demonstrated in studies on TTX, which specifically blocks VGSCs (4,7,8,11). In the present study, 200 nM TTX was used as the positive control, as it has been previously reported that TTX does not produce changes in the proliferation of either cell line even at higher doses (600 nM and 6 µM) than 200 nM (5,7,27). TTX reduced the movement distance of Mat-LyLu cells in a lateral direction, and this effect was found to be statistically significant. The MoI was 0.81±0.02 and 0.76±0.02 for the control (non-treated) and test (TTX-treated) groups, respectively (P<0.01). The MoI for AT-2 cells was 0.85±0.01 in the control and 0.87±0.03 in the test groups. No significant difference was observed between the control and TTX-treated groups in AT-2 cells (P>0.05; Fig. 1). The aforementioned data revealed that the cell lines maintained their metastatic properties (Mat-LyLu strongly metastatic and AT-2 weakly metastatic) associated with VGSCs.

HLE treatment of Mat-LyLu and AT-2 cells for 48 h resulted in significant inhibition of cell proliferation at 50 and 75 µg/ml (P<0.01). Normalized cell counts (NCCs) of Mat-LyLu cells were 0.87±0.03, 0.81±0.02 and 0.53±0.02 following treatment with 25, 50 and 75 µg/ml of HLE for 48 h, respectively. Following 48 h of incubation, the NCCs of AT-2 cells were 0.78±0.03, 0.70±0.02 and 0.51±0.02 for 25, 50 and 75 µg/ml HLE, respectively (Fig. 2A). The inhibition rates (%) of Mat-LyLu cell proliferation were 19.3±1.20 for 50 µg/ml and 46.9±2.34 for 75 µg/ml HLE. HLE treatment for 48 h produced increased rates of inhibition on AT-2 cell proliferation compared with Mat-LyLu cells. The inhibition rates (%) for AT-2 cells were 30.3±1.51 and 48.9±2.44 for 50 and 75 µg/ml HLE, respectively. The half-maximal (50%) inhibitory concentration (IC₅₀) of HLE on cell growth at 48 h was 88.9±4.44 µg/ml for Mat-LyLu cells and 77.3±3.90 µg/ml for AT-2 cells (Fig. 2B). Following HLE treatment, mitotic activity decreased in both cell lines. The MI (%) was 4.25±0.24 in the Mat-LyLu cell control group. The Mat-LyLu cell MI (%) decreased to 3.24±0.41, 2.70±0.16 and 2.70±0.32 as result of treatment with 25, 50 and 75 µg/ml HLE for 48 h, respectively. In a similar manner, the MI (%) of AT-2 cells decreased from 4.88±0.35 (control group) to 2.07±0.35 (75 µg/ml HLE treatment; Fig. 2C).

HFE did not produce a significant change in proliferation (Fig. 3A) or mitotic activity (Fig. 3B) in the Mat-LyLu cell line. However, HFE significantly suppressed proliferation and mitotic activity of AT-2 cells at 18 and 22 µg/ml concentrations, with inhibition rates (%) of 13.6±0.68 and 22.8±1.14, respectively (Fig. 3A-C). The MI (%) of AT-2 cells was 3.41±0.19 following treatment with 22 µg/ml HFE for 48 h, while the MI (%) was 4.88±0.35 in the control group (Fig. 3B).

In the present study, the effect of HLE and HFE on lateral motility of both cell lines was investigated using wound healing assays. HLE reduced the distance of movement in a lateral direction in both cell lines (Table I; Fig. 4A). MoI was 0.89±0.01 in the Mat-LyLu cell control group. Following 75 µg/ml HLE treatment for 48 h, the MoI of Mat-LyLu cells reduced to 0.81±0.02 (P<0.01). The MoI of the AT-2 cells was 0.84±0.01 and 0.71±0.02 in the control and 75 µg/ml HLE test groups, respectively. It was determined that the difference between the control and test groups was statistically significant (P<0.01). The MoI decreased by 8.83 and 15.7% in the Mat-LyLu and AT-2 cell lines, respectively. Considering the changing MoI and rate of inhibition of cell proliferation, it was suggested that the MoI may decrease due to the inhibition of proliferation in the investigated cell lines (Fig. 4B and C). A strong positive correlation was noted between the decrease of MoI and proliferation (r²=0.97; P<0.01). In addition, HFE treatment reduced the movement distance of Mat-LyLu cells, but not AT-2 cells (Table II; Fig. 4D). The MoI for Mat-LyLu cells was 0.89±0.01 and 0.87±0.02 in the control and 22 µg/ml test groups, respectively (P<0.05). However, no difference in MoI was observed between the control and test groups for the AT-2 cells. Notably, HFE treatment did not affect the kinetic parameters (proliferation and mitotic activity) of Mat-LyLu cells, while suppressing the growth of AT-2 cells. Additionally, no significant correlation was observed between the decrease of MoI and proliferation of Mat-LyLu cells (r²=0.05; P>0.05). Due to this, the present study concluded that HFE treatment reduced the motility of Mat-LyLu cells without affecting their proliferation (Fig. 4E and F).