Human breast cancer cells (MDA-MB-231) from American Type Culture Collection (Manassas, VA, USA) were grown in 10% fetal bovine serum-Dulbecco's modified Eagle's medium (FBS-DMEM) (HyClone Laboratories, Logan, UT, USA).

The dried Zingiber officinale (Z. officinale) was purchased from Gyeong-dong Oriental Medicine Market (Seoul, Republic of Korea), identified by Professor Joa Sub Oh (College of Pharmacy, Dankook University), and deposited at the herbarium of Gyeonggi Biocenter (Suwon, Republic of Korea). One thousand two hundred grams of Z. officinale were extracted three times with 15 liters of ethanol at room temperature for 24 h. The extract was concentrated, suspended in water, and then partitioned three times with 1.5 liters of n-hexane. The n-hexane extract (24 g) was subjected to silica gel column chromatography (Kieselgel 60, 70-230 mesh, 9×25 cm). Among eight fractions eluted from column chromatography, the sixth fraction (0.4 g) was further separated by semi-preparative HPLC (YMC-Pack ODS A column, 250×20 mm I.D.) eluting with acetonitrile-water (acetonitrile gradient from 50 to 100%) at a flow speed of 20 ml/min to yield (S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-tetrade canone (10-gingerol, 22.5 mg). ¹H- and ¹³C-NMR spectra were recorded on a Varian 500 MHz NMR spectrometer (Bruker, Billerica, MA, USA).

¹H-NMR (CDCl₃, 500 MHz) δ 6.83 (1H, d, J=8.0 Hz, H-5′), 6.69 (1H, d, J=2.0 Hz, H-2′), 6.67 (1H, dd, J=8.0, 2.0 Hz, H-6′), 4.03 (1H, m, H-5), 3.88 (3H, s, OCH₃), 2.84 (2H, brd, J=7.5 Hz, H-1), 2.75 (2H, brd, J=7.5 Hz, H-2), 2.58 (1H, dd, J=17.5, 3.0 Hz, H-4b), 2.50 (1H, dd, J=17.0, 9.0 Hz, H-4a), 1.49 (2H, m, H-6), 1.27-1.51 (14H, m, H-7-H-13), 0.89 (3H, t, J=7.0 Hz, H-14); ¹³C-NMR (CDCl3, 125 MHz) δ 211.5 (C-3), 146.4 (C-3′), 144.0 (C-4′), 132.6 (C-1′), 120.7 (C-6′), 114.4 (C-5′), 111.0 (C-2′), 67.7 (C-5), 55.9 (OCH₃), 49.4 (C-4), 45.4 (C-2), 36.5 (C-6), 31.9 (C-1), 29.6 (C-9), 29.5 (C-8), 29.3 (C-10), 29.28 (C-11), 25.5 (C-7), 22.7 (C-13), 14.1 (C-14). The structure of 10-gingerol is presented in Fig. 1A.

The following pharmacological agents and antibodies were purchased from commercial sources: LY294002 (Merck Millipore, Billerica, MA, USA); SB203580 (Cayman Chemical, Ann Arbor, MI, USA); anti-phospho-extracellular signal-regulated kinase (ERK) (T202/Y204), anti-phospho-Akt (S473), anti-phospho-p70^S6K (T421/S424) and anti-phospho-p38 mitogen-activated protein kinase (p38^MAPK) (T180/Y182) (Cell Signaling, Beverly, MA, USA); anti-EGFR, anti-ERK, anti-Akt, anti-p70^S6K, anti-p38^MAPK, anti-Cdk4, anti-Cdk2, anti-cyclin D, anti-cyclin E, anti-actin antibodies, and mouse and rabbit IgG-horseradish peroxidase conjugates (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Subconfluent MDA-MB-231 cells, plated on 6-well plates (5×10⁴ cells/well, BD Biosciences, Bedford, MA, USA), were serum-starved for 24 h in basal DMEM to synchronize cells in G₁/G₀ phase of cell cycle, pretreated with 10-gingerol at different concentrations (0.1-10 μM) in the presence or absence of LY294002 (10 μM) or SB203580 (5 μM) for 30 min, and further incubated with 10% FBS for 24 h. Following culture for 24 h, the number of cells was quantified using trypan blue exclusion method as described previously (23,24). The results from triplicate determinations (mean ± standard deviation) are presented as the fold-increase of untreated controls.

Serum-starved MDA-MB-231 cells, plated on 6-well plates (5×10⁴ cells/well), were pretreated with 10-gingerol (10 μM) for 30 min, followed by 10% FBS for 24 h. Cells were harvested with trypsin-EDTA, rinsed with phosphate buffered saline (PBS), and then fixed with ice-cold 70% ethanol for 3 h. After washing with PBS, cells were stained with Muse™ cell cycle reagent. The profile of cells in the G₁/G₀, S and G₂/M phases of the cell cycle was analyzed with a Muse™ cell analyzer (Merck Millipore) (25).

Serum-starved cells in 100 mm dishes (BD Biosciences) were incubated for 15 min or 24 h in 10% FBS in the presence or absence of 10-gingerol. Cells were rinsed twice with ice-cold PBS and lysed by incubation in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 100 μg/ml 4-(2-amino-ethyl) benzenesulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml pepstatin A, 0.5 μg/ml leupeptin, 80 mM β-glycerophosphate, 25 mM sodium fluoride and 1 mM sodium orthovanadate for 30 min at 4°C. Cell lysates were clarified at 13,000 x g for 20 min at 4°C, and the supernatants were subjected to western blot analysis as described previously (26,27). Bands of interest were integrated and quantified by the use of National Institutes of Health (NIH) ImageJ version 1.34s software.

The upper side of the Transwell insert (Costar, 6.5 mm diameter insert, 8 μm pore size) (Corning Inc., Corning, NY, USA) was coated with 50 μl of 1 mg/ml Matrigel (BD Biosciences) diluted in serum-free DMEM at 37°C. Aliquots (100 μl) of MDA-MB-231 cells (6×10⁵ cells/ml) resuspended in serum-free DMEM were added to the upper compartment of the Matrigel-coated Transwell and 600 μl of serum-free DMEM were added to the lower compartment. After serum starvation for 2 h, MDA-MB-231 cells were pretreated with 10-gingerol (10 μM), LY294002 (10 μM) or SB203580 (5 μM) for 30 min, followed by serum stimulation for 14 h. The inserts were fixed with methanol and using a cotton-tipped swab the non-invasive cells were removed from the top of the membrane. After staining with 0.04% Giemsa solution (Sigma-Aldrich, St. Louis, MO, USA), the number of invasive cells was determined from six different fields using objective magnification, ×200 (28).

Activities of MMPs were measured by zymography (29,30). Aliquots of conditioned medium were diluted in sample buffer, and applied to 10% polyacryl-amide gels containing 1 mg/ml gelatin (Sigma-Aldrich) as a substrate. After electrophoresis, the gels were incubated in 2.5% Triton X-100 for 1 h to remove SDS and allow re-natu-ralization of MMPs, and further incubated in developing buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM CaCl₂, and 150 mM NaCl for 15 h at 37°C. The gels were stained with 0.5% Coomassie Brilliant Blue R-250 in 30% methanol-10% acetic acid for 2 h and followed by destaining with 30% methanol-10% acetic acid. Gelatinolytic activities were detected as unstained bands against the background of the Coomassie Blue-stained gelatin.

Statistical analysis was performed using Student's t-test and was based on at least three different experiments. The results were considered to be statistically significant at P<0.05.

We first investigated the effect of 10-gingerol on cell proliferation in ER-negative MDA-MB-231 breast cancer cells. 10-gingerol treatment inhibited mitogen-stimulated cell proliferation in a dose-dependent manner (Fig. 1B), and did not alter cell viability at the highest concentration used in this study (data not shown), indicating that 10-gingerol-mediated inhibition of cell proliferation is not mediated by the induction of apoptosis or cytotoxicity. In addition, our initial experiments indicate that 10-gingerol markedly inhibited ER-positive MCF-7 breast cancer cell proliferation to levels similar to that observed in MDA-MB-231 cells (data not shown). These findings demonstrate the anti-proliferative activity of 10-gingerol in breast cancer cells, independently of ER expression status. We next examined the effect of 10-gingerol on the cell cycle by DNA content analysis (Fig. 2A). Mitogenic stimulation for 24 h increased the percentage of cells in S phase (6.3 vs. 14.9%) and G₂/M phase (13.3 vs. 29.8%), and simultaneously decreased the percentage of cells in G₁ phase (80.4 vs. 55.3%), compared with untreated controls. However, 10-gingerol prevented the increase in S phase (14.9 vs. 13.9%) and G₂/M phase (29.8 vs. 22.3%), and the decrease in G₁ phase (55.3 vs. 63.8%) associated with mitogenic stimulation. These observations suggest that 10-gingerol inhibits the transition from G₁ phase of the cell cycle to S phase, resulting in G₁ arrest, which is well correlated with inhibition of cell proliferation (Fig. 1B). Based on these findings, we analyzed the changes of cell cycle regulatory proteins in 10-gingerol-treated MDA-MB-231 cells. 10-Gingerol treatment markedly suppressed mitogen-induced expression of cyclin-dependent kinases (Cdks) and cyclins to levels observed in untreated controls (Fig. 2B). Collectively, these findings indicate that 10-gingerol downregulates the expression of cell cycle regulatory proteins, resulting in inhibition of cell cycle progression and proliferation.

We next examined the effect of 10-gingerol on cell invasion which plays pivotal roles in cancer progression. 10-Gingerol treatment markedly inhibited mitogen-induced invasion of MDA-MB-231 cells (Fig. 3A). Expression and activation of MMPs have been reported to enhance cell migration and invasion by degrading the components of extracellular matrix and cell surfaces (6-9). Based on 10-gingerol-mediated inhibition of cell invasion, we examined the activity of MMPs in MDA-MB-231 cells. 10-gingerol treatment marginally inhibited the activity of MMP-2, but not MMP-9, suggesting that inhibition of cell invasion by 10-gingerol is mediated, at least in part, through the suppression of MMP-2 activity (Fig. 3B).

To investigate the molecular mechanism by which 10-gingerol modulates cell proliferation and invasion, we examined the changes in activation of intracellular signaling pathways such as Akt, p70^S6K, ERK and p38^MAPK in 10-gingerol-treated MDA-MB-231 cells. Mitogenic stimulation increased the phosphorylation/activation of Akt, ERK and p38^MAPK, but not that of p70^S6K, as compared with unstimulated controls. 10-Gingerol treatment markedly inhibited the phosphorylation of Akt and p38^MAPK in MDA-MB-231 cells (Fig. 4). Finally, pretreatment of cells with LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K)/Akt pathway, or SB203580, an inhibitor of p38^MAPK pathway, mimicked the suppressive effects of 10-gingerol on cell proliferation and invasion in MDA-MB-231 cells (Fig. 5A and B). Co-treatment with 10-gingerol did not significantly enhance the anti-proliferative activity of these pharmacological inhibitors, indicating that 10-gingerol and these inhibitors may share similar roles and mechanisms of action in regulating cellular processes of MDA-MB-231 cells.

The EGFR is a receptor tyrosine kinase which is highly expressed or activated in various types of human cancers including breast cancer (4,31). Therefore, EGFR and its down-stream signaling pathways have been known as key therapeutic targets for cancer treatment. 10-Gingerol treatment markedly suppressed mitogen-induced expression of EGFR in MDA-MB-231 cells to levels observed in unstimulated controls (Fig. 5C). Taken together, these observations suggest that anti-proliferative and anti-invasive activities of 10-gingerol in MDA-MB-231 breast cancer cells might be correlated with suppression of EGFR expression.