Matrine inhibits the migratory and invasive properties of nasopharyngeal carcinoma cells

Matrine is a widely used Chinese herbal medicine that has historically been used in the treatment of inflammation and cancer. However, the antimetastatic effects and associated molecular mechanisms of matrine on nasopharyngeal carcinoma (NPC) remain to be elucidated. Therefore, the aims of the present study were to assess the antimetastatic effects of matrine on NPC, and identify the underlying mechanisms. Matrine inhibited the proliferation of NPC cells in vitro and in vivo. Furthermore, matrine inhibited the migration and invasion of NPC tumor cells at doses below the toxic range. Following treatment with matrine for 24 h, there was a decrease in the protein expression levels and activities of matrix metal-loproteinase (MMP)-2 and MMP-9 in NPC-039 cells. In addition, matrine markedly reduced the expression levels of p65 and p50 in the nuclei. Combined treatment of matrine with helenalin, a nuclear factor-κB (NF-κB) inhibitor resulted in a synergistic reduction in MMP-2 and MMP-9 expression levels, and the invasive capabilities of the NPC-039 cells were also reduced. In conclusion, matrine inhibits NPC cell migration and invasion by suppressing the NF-κB pathway. These results suggest that matrine may be a potential therapeutic agent for NPC.


Introduction
Nasopharyngeal carcinoma (NPC) is a common malignancy of the head and neck. There is a high incidence of NPC in the southern regions of China, and it is common among the Inuits of Alaska (1,2). Due to its characteristic epidemiology, pathogenesis and association with the Epstein-Barr virus, NPC markedly differs from other head and neck cancers (3).
In ≤75% of cases of NPC the cancer metastasizes to the neck lymph nodes, which has an adverse effect on prognosis (4). Distant metastasis, to organs including the liver, lungs and bone, is also associated with a high risk of treatment failure (5).
Metastasis is a complex process, which includes reduction of tumor cell adhesion, degradation of the extracellular matrix (ECM), enhancement of cell motility and promotion of neo-vascularization (6). Degradation of the ECM and components of the basement membrane has an important role during the metastatic process (6,7). Therefore, the actions of proteinases, such as matrix metalloproteinases (MMPs), which can degrade the ECM and components of the basement membrane, have an important role in tumor invasion and metastasis. MMP-2 and MMP-9 can degrade the majority of ECM components and are profoundly associated with the process of cancer invasion and metastasis (8,9).
Matrine (C 15 H 24 N 2 O) is derived from the Sophora plant genus and has historically been used in traditional Chinese medicine to treat inflammation (10). Matrine has been shown to produce a wide range of pharmacological effects and has been used to treat various diseases, including viral hepatitis, neuropathic pain and isoproterenol-induced cardiotoxicity (11)(12)(13). No obvious toxicity or side effects of matrine have been reported. Matrine also exhibits an anticancer effect, on malignancies including gastric cancer, rhabdomyosarcoma, acute myeloid leukemia and breast cancer (14)(15)(16)(17). However, to the best of our knowledge, there are currently no published studies regarding the antimetastatic effects of matrine on NPC. The present study aimed to investigate the effects of matrine against NPC cell invasion and metastasis, and identify the underlying mechanisms.

Materials and methods
Ethics statement. Male BALB/c nude mice (age, 4 weeks) were supplied by the Experimental Animal Center of Xi'an Jiaotong University (Xi'an, China). The present study was conducted according to the recommended guidelines for the Care and Use of Laboratory Animals, issued by the Chinese Council on Animal Research. The protocol was approved by the Ethics Committee of Xi'an Jiaotong University. for cell culture. Fetal bovine serum (FBS), penicillin and streptomycin were all purchased from Gibco Life Technologies (Carlsbad, CA, USA). Helenalin was purchased from Sigma-Aldrich. Mouse monoclonal anti-nuclear factor-κB (NF-κB) p50 (sc-271908), mouse monoclonal anti-NF-κB p65 (sc-71676), mouse monoclonal anti-β-actin (sc-376421) and rabbit polyclonal anti-histone H1 (sc-67324) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit polyclonal anti-MMP-2 (#4002) and rabbit polyclonal anti-MMP-9 (#2270) antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA).
Cell culture. The NPC-039 cells were obtained from Academia Sinica (Taipei, China) and were was cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10% FBS. The poorly differentiated human CNE-2Z NPC cell line was obatined from Zhongshan University (Guangzhou, China) and cultured in RPMI-1640 medium (HyClone Laboratories, Inc., Logan, UT, USA) supplemented with 5% FBS and 100 units penicillin/streptomycin. All of the cells were cultured at 37˚C in a humidified incubator containing 5% CO 2 .
Assessment of cell viability. Cell viability was determined using a colorimetric 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT) assay, according to previously described methods (6). Briefly, the cells were plated in 96-well culture plates (2x10 4 /well) and treated with serial concentrations (0, 12.5, 25, 50, 100 and 200 µg/ml) of matrine for 24 h. Following incubation, the cells were washed twice with phosphate-buffered saline (PBS) and incubated with 5 mg/ml MTT (Sigma-Aldrich) for 4 h. The living cells absorbed the reagent and subsequently produced insoluble blue formazan. Following the incubation, the cells were washed again with PBS and solubilized using DMSO. The optical density of the cells was measured at 550 nm using an enzyme-linked immunosorbent assay plate reader (DR-200Bs; Bio-Rad Laboratories, Hercules, CA, USA).
Invasion assays in vitro. Prior to cell seeding, a 24-well cell culture invasion chamber (Corning Inc., Tewksbury, MA, USA) was coated with 8.0 µm Matrigel™ (Becton Dickinson, Bedford, MA, USA), and the CNE-2Z cells were pretreated with 0, 12.5, 25 and 50 µg/ml matrine, or helenalin (5 µM), for 24 h. The cells were then seeded in 200 µl serum-free medium and placed in the upper chambers at 2x10 5 cells, normal growth medium was placed in the bottom chambers. Following a 24 h incubation, the cells on the upper surface of the membrane were removed using cotton swabs, and the cells on the bottom side of the filter were fixed in 90% ethyl alcohol (Xi'an Chemical Reagent Factory, Xi'an, China), stained with Crystal Violet (Sigma-Aldrich) and counted under an Olympus-CX31 microscope (Olympus Corp., Tokyo, Japan). Spontaneous migration in DMSO was designated as the control. The rate of invasion was expressed as a percentage of the control.
Zymography. MMP-2 and MMP-9 activity in the samples was studies using zymography. In brief, the cells were treated with different concentrations (0, 12.5, 25 and 50 µg/ml) of matrine at 37˚C for 24 h. The conditioned media were collected from all of the cells and lysated. The protein concentration was then measured and the final 10 µl sample was subjected to electrophoresis using an 8% SDS-PAGE gel co-polymerized with 0.1% gelatin. Following electrophoresis, the gels were incubated in 2.5% Triton X-100 solution (Sigma-Aldrich) at room temperature for 1 h, and then incubated in reaction buffer (10 mM CaCl 2 , 40 mM Tris-HCl and 0.01% NaN 3 , pH 8.0) overnight at 37˚C. The gels were stained with 0.1% Coomassie brilliant blue R-250 (Sigma-Aldrich) and destained with 30% methanol and 10% acetic acid. Gelatinolytic activities were detected as unstained bands against the background of Coomasie-stained gelatin. The intensities of the bands on the gels were determined using an image analysis system (Quantity One v4.62; Bio-Rad Laboratories).
Animal and tumor xenograft assays. In vivo tumorigenicity was achieved as described by previous methods (18). Briefly, suspensions of NPC-039 tumor cells (5x10 5 viable cells/mouse) were implanted into the right flank region of the BALB/c nude mice. Forty-eight hours after the injection (day 1), the mice were randomly divided into two groups (n=5/group). The animals were pair matched, in order to ensure that the median tumor volume for each group was similar. The treatment group received matrine (60 mg/kg per day) by intragastric administration, and the control group received an equal volume of saline. The tumor volumes were measured twice weekly using calipers, and the volumes (cm 3 ) were calculated according to the following standard formula: (length x width 2 )/2. After three weeks of drug administration, the mice were sacrificed by cervical dislocation, and the tumors were harvested and weighed. The experimental protocols involving mice in the present study were evaluated and approved by the Animal Care and Use Committee of the Medical School of Xi'an Jiaotong University.
Wound healing assays. Wound healing assays were performed on NPC-039 cells. In brief, NPC-039 cells were seeded into a six-well plate and cultured to 60-70% confluency in medium containing 10% FBS. Cell monolayers were wounded using a plastic tip (1 mm) that touched the plate as described previously (6). NPC-039 cells were then incubated in serumcontaining medium (2% serum) with oxymatrine (0, 12.5, 25 and 50 μg/ml) for 24 h. Images were captured at 0 and 24 h following the addition of oxymatrine. The migration distance of the cells was measured under an Olympus-CX31 microscope (Olympus Corp.).
Statistical analysis. The data are expressed as the mean ± standard deviation. Statistical analyses were conducted using SPSS version 16.0 software (SPSS Inc., Chicago, IL, USA), to evaluate statistical differences. Student's t-test was used for comparisons between two groups, and a one-way or two-way analysis of variance was used to analyze statistical differences between the groups under different conditions. P<0.05 was considered to indicate a statistically significant difference. All of the statistical tests were two sided. A correlation analysis by Z test was also performed.

Matrine inhibits the proliferation of NPC cells in vitro.
Matrine reduced the viability of the two human NPC cell lines, in a dose-dependent manner, following treatment with 0-200 µg/ml matrine for 24, 48 and 72 h (NPC-039, Fig. 1A; CNE-2Z, Fig. 1B). At concentrations <50 µg/ml (24 h), the antiproliferative effects of matrine were not obvious; therefore, a concentration range lower than this was chosen for use in all subsequent experiments regarding the anti-metastatic effects of matrine.

Matrine inhibits the migration and invasion of NPC-039 cells.
As shown in Fig. 2A, the movement of NPC-039 cells was significantly reduced in response to treatment with matrine; the rate of inhibition was ~15.79, 42.86 and 56.68.1% at 24 h with 12.5, 25 and 50 µg/ml matrine, respectively (Fig. 2B). The effects of matrine on the invasiveness of NPC-039 cells treated with 0, 12.5, 25 and 50 µg/ml matrine for 24 h are shown in Fig. 2C. Matrine significantly reduced the invasiveness of the NPC-039 cells. Similar antimetastatic effects of matrine were observed in the CNE-2Z cells (data not shown). Quantification indicated that the invasiveness of the NPC-039 cells was reduced by 4.71, 56.75 and 68.77% in response to treatment with 12.5, 25 and 50 µg/ml of matrine (Fig. 2D), respectively.

Matrine inhibits the protein expression and activity of MMP-2 and MMP-9 in NPC-039 cells.
MMP-2 and MMP-9 have important roles in the invasion of cancer cells (6). The present study analyzed the effects of matrine on the protein expression levels and the activity of MMP-2 and MMP-9. Matrine significantly reduced the protein expression levels ( Fig. 3A and B) and the relative activity of MMP-2 and MMP-9 ( Fig. 3C and D).

Matrine inhibits the nuclear translocation of p50 and p65 in NPC-039 cells, and the effects of helenalin and matrine on cell invasion and MMP-2 and MMP-9 expression in NPC-039 cells.
NF-κB has an important role in controlling tumor cell migration; therefore, the present study hypothesized that the reduction in NPC cell invasion may be a result of downregulation of NF-κB. The protein expression levels of p65 and p50 in the nuclei of NPC-039 cells were markedly downregulated following treatment with matrine ( Fig. 4A and B). NF-κB has previously been reported as a downstream target of matrine in numerous types of cells, which results in the upregulation of MMP-2 and MMP-9 expression (19)(20)(21). The results of the present study indicate that combined treatment of an NF-κB inhibitor, henenalin, and matrine, results in a synergistic reduction in cell invation (Fig. 4C and D) as well as MMP-2 and MMP-9 expression (Fig. 4E and F).

In vivo inhibition of NPC tumor growth by matrine.
To evaluate the in vivo effects of matrine on tumor growth, NPC-039 cells were xenografted into nude mice, as described by previous methods (18). The time course of NPC-039 xenograft growth,   with and without matrine treatment, is shown in Fig. 5A. A significant inhibition of tumor growth was observed in response to treatment with matrine. In the matrine-treated (60 mg/kg/day) mice, 21 days after cell implantation the NPC-039 xenograft volumes were significantly inhibited. At the end of the experiment, the xenograft tumors were harvested and weighed. Matrine was found to significantly decrease the solid tumor mass, as compared with the control group (Fig. 5B).

Discussion
Metastasis is currently considered to be the main obstacle in the clinical management of NPC. Prevention, prediction and inhibition of NPC metastasis is critical to further improve the survival rate. The present study demonstrated that matrine was a strong metastatic inhibitor for NPC. Matrine has previously been proposed as a potential drug for certain types of tumor (22,23). However, the antimetastatic effects and related mechanisms in NPC cells remained unclear. In the present study, matrine suppressed the migratory and invasive ability of NPC cells, at doses below its toxic range. This is the first scientific report, to the best of our knowledge, regarding the antimetastatic effects of matrine on NPC.
To determine the effects of matrine on the motility, migration and invasiveness of NPC cells, a wound healing migration assay and a Boyden chamber invasion assay were employed, respectively. Matrine significantly inhibited the migration and invasion of the NPC cells. Metastasis is a complex process, during which degradation of components of the ECM is a key step (6); since the loss of ECM integrity allows the cancer cells to invade the blood or lymphatic system, and spread to other tissues and organs. MMP-2 and MMP-9 have important roles in the degradation of ECM (24)(25)(26). Previous studies have shown that matrine can reduce the expression of MMP-2 and MMP-9 in numerous types of cells (19)(20)(21)27). Therefore, the present study investigated the effects of matrine on the expression levels of MMP-2 and MMP-9 in NPC-039 cells, and demonstrated that matrine could significantly reduce the expression and activity of MMP-2 and MMP-9. These results indicate that matrine may inhibit the invasion of NPC-039 cells by regulating the expression and activity of MMP-2 and MMP-9.
NF-κB is able to upregulate MMP-2 and MMP-9 expression (19)(20)(21), and has previously been reported as a downstream target of matrine in numerous cell types (28)(29)(30)(31). In the present study, matrine was shown to significantly increase the expression levels of p65 and p50 within the nuclei of NPC-039 cells.
The results of the present study also indicated that cellular motility inhibited by matrine may be enhanced by blocking the NF-κB pathway, thus providing further evidence that NF-κB is a key signaling pathway by which matrine regulates the migratory and invasive abilities of NPC cells.
In vivo analyses of the present study demonstrated that matrine decreased the growth of NPC-039 cell tumor xenografts in nude mice by inhibiting cell proliferation. In conclusion, the present study demonstrated the inhibitory effects of matrine on the migration and invasion of NPC cells. Furthermore, the decreased expression levels of MMP-2 and MMP-9, induced by matrine, may be attributed to inhibition of the NF-κB pathway. These results reveal a novel potential therapeutic application of matrine in antimetastatic therapy for NPC.