The human A2780 ovarian cancer cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and cells were routinely cultured in RPMI-1640 containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin (Macgene Biotechnology Ltd., Beijing, China) in 5% CO₂ at 37°C. Monoclonal rabbit anti-human ERK1/2 (cat. no. 4695; 1:1,000 dilution), monoclonal rabbit anti-human phospho-ERK1/2 (cat. no. 4370; 1:1,000 dilution), monoclonal rabbit anti-human p38 (cat. no. 8690; 1:1000 dilution), monoclonal rabbit anti-human phospho-p38 (cat. no. 4631; 1:1,000 dilution), monoclonal rabbit anti-human JNK (cat. no. 9252; 1:1,000 dilution), monoclonal rabbit anti-human phospho-JNK (cat. no. 4668; 1:1,000 dilution), monoclonal rabbit anti-human MMP-2 (cat. no. 13132; 1:1,000 dilution) and monoclonal rabbit anti-human MMP-9 (cat. no. 13667; 1:1,000 dilution) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). NC was purchased from Shanghai Tauto Biotech Co., Ltd. (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO). U0126, a selective inhibitor of ERK, was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell viability and proliferation were assessed using a 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Beyotime Institute of Biotechnology, Haimen, China) assay. A2780 cells (5,000 cells/well) in 100 µl medium were seeded into 96-well plates. Following stimulation with NC of various concentrations (0, 1, 2.5, 5 and 10 µM) for various times (0, 6, 12, 24 and 48 h), 20 µl MTT (5 mg/ml) was added into each well. Following incubation for 4 h at 37°C, 100 µl of DMSO was added to each well for another 15 min. Finally, the absorbance values were determined using a microplate luminometer (iMark; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 490 nm. A2780 cells stimulated with 0 µM NC at various time points (0, 6, 12, 24 and 48 h) were used as the control group.

Total RNA was extracted from A2780 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and then reverse transcribed to cDNA using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. qPCR analysis was performed using a LightCycler (Bio-Rad Laboratories Inc.). The primer sequences used for qPCR were as follows: MMP-2, forward: 5′-TTG ATG GCA TCG CTC AGA TC-3′ and reverse: 5′-TTG TCA CGT GGC GTC ACA GT-3′; MMP-9, forward: 5′-GAC GCA GAC ATC GTC ATC CA-3′ and reverse: 5′-CAC AAC TCG TCA TCG TCG AAA-3′. The cycling conditions were as follows: 95°C for 10 sec, followed by 40 cycles of 95°C for 5 sec, 60°C for 30 sec and a final extension at 72°C for 3 min. SYBR Green I was purchased from Invitrogen (Thermo Fisher Scientific, Inc.) Melting curves were assessed to confirm the specificity of the products generated for each set of primers. Following amplification, the ΔΔCq comparative method was then used to normalize the relative levels of gene expression to GAPDH (21). Experiments were performed in triplicate.

Following treatment, the cells were harvested and then the protein was extracted using protein lysis buffer (Beyotime Institute of Biotechnology). Centrifugation was performed at 10,000 × g for 15 min at 4°C. Total cell protein concentrations were determined using the bicinchoninic acid protein assay kit (Pierce Biotechnology Inc., Rockford, IL, USA). Equal protein from cell lysates was loaded onto 12% SDS-PAGE gels (Bio-Rad Laboratories, Inc.). Following electrophoresis, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and then blocked with 5% fat-free milk at room temperature for 1 h, and incubated with the indicated primary antibodies overnight at 4°C. Subsequently, the membranes were washed with Tris-buffered saline and Tween 20 and incubated with polyclonal goat anti-rabbit horseradish peroxidase-conjugated IgG (cat. no. ZDR-5306; 1:10,000 dilution; ZSGB-BIO, Beijing, China) secondary antibody for 1 h at room temperature. Immune complexes were detected with enhanced chemiluminescence reagents and the blots were quantified by densitometric analysis using the Alpha Imager 2200 (Alpha Innotech Corp., Santa Clara, CA, USA). Reactions were performed once per PVDF membrane.

A scratch wound healing assay was used to assess the migration ability of the A2780 cells. In brief, the A2780 cells (1×10⁶/well) were seeded in 6-well plates and cultured with RPMI-1640 supplemented with 10% FBS. When reaching confluency, each well was scratched with a 200 µl pipette tip. To assess the effects of NC on the migration of A2780 cells, 5 µM NC was added to the plates. After 24 h of incubation, images of the wound healing areas were captured and then the distance between two cell edges was analyzed by ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA).

To evaluate the effect of NC on the invasive ability of A2780 cells, the transwell system was used. A2780 cells were cultured in Boyden chambers, with 8-µm pore filter inserts, in 24-well plates (Corning Life Sciences, Corning, NY, USA). The pore inserts were pre-coated with Matrigel (BD Biosciences, San Jose, CA, USA) overnight. A2780 cells (1×10⁵ cells/well) were suspended in 100 µl RPMI-1640 supplemented with 1% FBS and added to the upper chamber. RPMI-1640 with 10% FBS and 5 µM NC were added to the lower chamber. The cells on the top of the membrane were gently scraped using a swab. After 24 h of incubation at 37°C, the cells attaching to the lower surface were fixed with methanol and stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) for 15 min at room temperature. A total of five random high-power fields (magnification, ×200; Nikon E100; Nikon Corp, Tokyo, Japan) of each sample were selected and counted to evaluate the average number of invasive cells.

For the migration assay, cells were added to upper chambers without coated Matrigel. The assay was performed as described above for the invasion assay. The cells on the lower surface were also counted in five randomly selected fields and then the cell number was analyzed statistically.

The data are expressed as the mean ± standard deviation. All experiments were repeated at least three times. Comparisons among values for all groups were performed by one-way analysis of variance. Holm's t-test was used for analysis of differences between different groups. P<0.05 was considered to indicate a statistically significant difference.

The structure of NC is shown in Fig. 1A. To determine the specific role of NC on the cell viability of A2780 cells, an MTT assay was used as described in Materials and methods. As shown in Fig. 1B, various concentrations (0, 1, 2.5, 5 and 10 µM) of NC were added to the cultured A2780 cells. At a dose of 10 µM, NC significantly inhibited the viability of A2780 cells after 24 h incubation. However, at concentrations <10 µM (1, 2.5 and 5 µM), the inhibitory effect was not significant. Furthermore, 5 and 10 µM NC were selected to stimulate the cells at different time points (0, 6, 12, 24 and 48 h). As shown in Fig. 1C, after 24 h stimulation, NC significantly inhibited the cell viability at a dose of 10 µM, while 5 µM NC did not significantly inhibit cell viability. However, after 48 h stimulation, 5 and 10 µM NC significantly inhibited cell viability. As a result, 5 µM NC for 24 h was selected in the subsequent migration and invasion experiments in order to exclude the effect of cell viability.

The wound healing assay was used to investigate the effect of NC on the migration of ovarian cancer cells. A2780 ovarian cancer cells were treated with NC at a concentration of 5 µM for 24 h. As shown in Fig. 2A, migration of A2780 cells was inhibited by NC (5 µM). The results of the wound healing assay demonstrated that healing over the scratch was significantly deceased following treatment with NC. In order to further examine the effect of NC on cell migration and invasion, a Transwell assay was performed. A2780 cells were treated with NC at a concentration of 5 µM for 24 h. As shown in Fig. 2B, the migration and invasion of A2780 cells were significantly inhibited by NC (5 µM). The results of the Transwell assay indicated that NC could inhibit the migratory and invasive ability of ovarian cancer cells.

Numerous evidence has supported that MMPs, particularly MMP-2 and MMP-9, are important in the process of cancer cell migration and invasion (22,23). The present study thus determined the effect of NC on MMP-2 and MMP-9 expression in A2780 ovarian cancer cells. Various concentrations of NC (0, 2.5, 5 and 10 µM) were added to the A2780 cells, respectively, and the cells were then cultured for another 24 h. As shown in Fig. 3A, NC treatment significantly decreased the expression of MMP-2 and MMP-9 in A2780 cells at a concentration of 5 and 10 µM. The A2780 cells were then stimulated with 5 µM NC for different time periods (0, 6, 12 and 24 h). As shown in Fig. 3B, following stimulation with 5 µM NC for 6 h, the expression of MMP-2 and MMP-9 was not altered. However, when the stimulating time was extended to 12 and 24 h, the expression of MMP-2 and MMP-9 was significantly decreased compared with the control group. In addition, identical treatments were performed and qPCR was applied to detect the mRNA levels of MMP-2 and MMP-9. As shown in Fig. 3C, the relative mRNA expression of MMP-2 and MMP-9 was significantly decreased when treated with 5 and 10 µM NC. Similar to the protein level, it also decreased gradually following stimulation with 5 µM for 6, 12 and 24 h (Fig. 3D). The results above suggest that NC inhibited the production of MMP-2 and MMP-9 in a dose- and time-dependent manner at the mRNA and protein levels. The results above also indicate that the inhibitory effect of NC on A2780 cell invasion and migration may be associated with MMP-2 and MMP-9 production.

The MAPK signaling pathway is important in the development of various types of cancer. Inhibition of MAPK activation has been demonstrated to suppress tumor cell migration and invasion (24). To determine the association between NC and the production of MMP-2 and MMP-9, the relative expression of three members of the MAPK family, including phospho-JNK, phospho-p38 and phospho-ERK1/2 were investigated following treatment of A2780 cells with 5 µM NC for 0, 5, 15, 30 and 60 min. As shown in Fig. 4A and B, the relative expression of phospho-ERK1/2 was significantly downregulated compared with the control group. The activity of phospho-ERK1/2 rapidly decreased after 5–30 min stimulation and gradually increased after 1 h stimulation. However, the expression of phospho-JNK and phospho-p38 was not altered. The results above suggested that the ERK pathway may be involved in MMP-2 and MMP-9 production of ovarian cancer cells.

To further investigate whether the effect of NC is ERK-dependent, U0126 (ERK inhibitor) was applied to inhibit ERK activation. As shown in Fig. 4C and D, inhibition of ERK activity with U0126 could further downregulate the expression of MMP-2 and MMP-9 compared with NC treatment. These results further demonstrated that NC-inhibited MMP-2 and MMP-9 production may be ERK dependent.

To further investigate whether the anti-metastatic effect of NC was attributed to ERK signaling suppression, the effect of U0126 on A2780 cell migration in the presence or absence of NC was examined. As shown in Fig. 5A, the results of the wound healing assay demonstrated that the NC-induced inhibition of wound healing was significantly enhanced by using an ERK inhibitor. Similar results were observed using the Transwell assay (Fig. 5B), which also demonstrated that inhibition of ERK activation could significantly enhance the NC-induced inhibition of ovarian cancer cell invasion. These results above support the theory that suppression of metastasis by NC is regulated by inhibition of MMP-2/9 production though the ERK signaling pathway in ovarian cancer cells.