Anti‑metastatic effects of arctigenin are regulated by MAPK/AP‑1 signaling in 4T‑1 mouse breast cancer cells

  • Authors:
    • Min‑Gu Lee
    • Kyu‑Shik Lee
    • Kyung‑Soo Nam
  • View Affiliations

  • Published online on: January 13, 2020     https://doi.org/10.3892/mmr.2020.10937
  • Pages: 1374-1382
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Abstract

Arctigenin is a natural lignan that is found in burdock with anti‑viral, ‑oxidative, ‑inflammatory and anti‑tumor activities. In the current study, the effect of arctigenin on metastatic potential was examined in 4T‑1 mouse triple‑negative breast cancer cells. The results indicated that arctigenin inhibited cell motility and invasiveness, which was determined using wound healing and transwell invasion assays. Arctigenin suppressed matrix metalloprotease‑9 (MMP‑9) activity via gelatin zymography, and protein expression of cyclooxygenase‑2 (COX‑2) and MMP‑3. Furthermore, arctigenin attenuated the mRNA expression of metastatic factors, including MMP‑9, MMP‑3 and COX‑2. Based on these results, the effect of arctigenin on the mitogen‑activated protein kinase (MAPK)/activating protein‑1 (AP‑1) signaling pathway was assessed in an attempt to identify the regulatory mechanism responsible for its anti‑metastatic effects. Arctigenin was demonstrated to inhibit the phosphorylation of extracellular signal‑regulated protein kinase (ERK) and c‑Jun N‑terminal kinase (JNK), and the nuclear translocations of the AP‑1 subunits, c‑Jun and c‑Fos. In summary, the present study demonstrated that in 4T‑1 mouse triple‑negative breast cancer cells the anti‑metastatic effect of arctigenin is mediated by the inhibition of MMP‑9 activity and by the inhibition of the metastasis‑enhancing factors MMP‑9, MMP‑3 and COX‑2, due to the suppression of the MAPK/AP‑1 signaling pathway. The results of the current study demonstrated that arctigenin exhibits a potential for preventing cell migration and invasion in triple negative breast cancer.

Introduction

Arctigenin (ATG) is a bioactive natural lignan found in the seeds of Arctium lappa (Burdock) in the family Asteraceae (1). Burdock has long been used as a folk medicine to treat various infectious symptoms such as inflammation and sore throat (2), and several scientific studies have demonstrated that arctigenin has various physiological activities, which include anti-viral, -oxidative, -inflammatory, and anti-tumor effects (212). Furthermore, several recent investigations have reported that ATG exhibits anti-cancer activity in various human cancer cells, including those of breast, pancreatic, hepatic, and colon cancer (1,2,1218).

Breast cancer is the one of the most common causes of female mortality worldwide (19). Breast cancer may be classified as progesterone receptor (PR) and estrogen receptor (ER) positive, HER2 (human epidermal growth factor 2) overexpressing, and triple-negative breast cancer (TNBC). In particular, TNBC cannot treated using a selective target therapy because it lacks HER2, ER, and PR and has a poor prognosis caused by its high metastatic potential (2022).

Metastasis is a complex process that results in secondary tumor formation and is caused by the detachment, migration, invasion, and attachment of cells at secondary sites. This process requires the participations of many proteases to degrade extracellular matrix (ECM) and basement membrane (BM), and the matrix metalloproteinases (MMPs) are known play important roles in development, progression, and in the invasion and migration of breast cancers (23). MMPs are classified into 23 types of proteases [e.g., collagenase, stromelysin, gelatinase, and matrilysin) (24)], and MMP-9 (a gelatinase B type) is known to degrade ECM and BM by breaking down gelatin, and to be an important player during invasion and migration. Notably, MMP-9 has been reported to be an important predictor of cancer invasion, metastasis, prognosis, and angiogenesis in breast cancer (2426). MMP-3 (a stromelysin-1 type) also plays an important role during metastasis and enhances metastasis by activating of MMPs (e.g., MMP-1, MMP-7, and MMP-9) and degrading collagen types II, IV, and IX, proteoglycans, laminin, fibronectin, gelatin, and elastin in ECM and BM (22,23,25,26). In addition, MMP-3 activates MMP-9 via the proteolytic removal of the pro-domain in pro-MMP-9 (27). Flores-Pliego et al showed increased MMP-3 secretion in placental leukocytes was closely linked with MMP-9 secretion and that the activity of MMP-9 was diminished by treating cells with MMP-3 inhibitor (28), which adequately demonstrated MMP-9 and MMP-3 secretions and activities are closely linked.

Furthermore, cyclooxygenase-2 (COX-2) is another known metastasis-enhancing factor and catalyzes the synthesis of prostaglandin E2 (PGE2) from arachidonic acid, and thus, enhances metastasis and angiogenesis (23). In one notable study conducted in a COX-2-silenced MDA-MB-231 TNBC xenograft model tumor growth and metastasis to lung were found to be inhibited (29). In another, transfection of siCOX-2 into pancreatic cancer tumors significantly downregulated MMP-9 expression (30). Therefore, it appears the downregulations of MMP-9, MMP-3 and COX-2 are needed to prevent metastatic potential in breast cancer.

The mitogen-activated protein kinases (MAPKs) are typical serine/threonine protein kinases that participate in the regulations of many cellular processes (e.g., growth, proliferation, differentiation, migration, and death) (3133), and several studies have revealed that MAPKs such as extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), and P38 play important roles during tumor development, progression, metastasis, invasion, and angiogenesis (34,35). MMPs and COX-2 contain promoter sites that bind to transcription factors, such as activating protein-1 (AP-1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and signal transducer and activator of transcription 3 (STAT3), and the activities of these transcription factors are regulated by MAPK, Akt, and STAT signaling pathways, respectively (3640). AP-1 is formed by a homodimeric or heterodimeric interactions with Jun, Fos, or ATF subunits and the formed complexes bind to AP-1 binding sites on DNA (3942). Jun/Fos heterodimers are more stable than other AP-1 complexes and have greater DNA binding activity (41,42). Therefore, we evaluated the effect of arctigenin on cancer metastatic potential and investigated whether MAPK/AP-1 signaling is involved in suppression of metastatic potential by arctigenin in 4T-1 mouse TNBC cells.

Materials and methods

Materials

Arctigenin and bovine serum albumin (BSA) were bought from Santa Cruz Biotechnology (Dallas, TX, USA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Pierce™ BCA Protein Assay Kits were purchased from Thermo Scientific (Waltham, MA, USA). Dimethyl sulfate (DMSO) was obtained from Duksan Pure Chemicals (Ansan, Korea) and protease inhibitor cocktail and phosphatase inhibitor cocktail were from GenDEPOT (Barker, TX, USA). Dulbecco's modified Εagles medium (DMEM), antimycotic/antibiotic solution and fetal bovine serum (FBS) were obtained from Welgene (Daegu, Korea), Tris-base and glycine were from BioShop Canada Inc. (Burlington, ON, Canada). Polyvinylidenefluoride (PVDF) membranes were purchased from Pall Life Sciences (Port Washington, NY, USA). Antibodies for ERK1/2 (cat. no. 4695), p-ERK1/2 (cat. no. 4370), JNK1/2 (cat. no. 9258), p-JNK1/2 (cat. no. 4668), P38 MAPK (cat. no. 8690), p-P38 MAPK (cat. no. 4511), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB; cat. no. 8242), p-NF-κB (cat. no. 3033), c-Jun (cat. no. 9265), c-Fos (cat. no. 2250), COX-2 (cat. no. 12282), GAPDH (cat. no. 5174), and histone H3 (cat. no. 14269) were obtained from Cell Signaling Technology (Beverly, MA, USA). β-actin (cat. no. sc-69879) was from Santa Cruz Biotechnology (Dallas, TX, USA); HRP-conjugated anti-rabbit IgG (cat. no. NCI1460KR) and -mouse IgG (cat. no. NCI1430OKR) from Thermo Scientific Fisher Scientific, Inc (Rockford, IL, USA); sodium dodecyl sulfate (SDS) from Amresco (VWR Life Science, Radnor, PA, USA), and 30% polyacrylamide solution from SERVA (Heidelberg, Germany). Collagen type I and matrigel were bought from Corning Life Sciences (Bedford, MA, USA), and hematoxylin and eosin were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

Cell culture and viability assay

4T-1 mouse TNBC cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and routinely cultured in DMEM containing 10% FBS and 1% antibiotic-antimycotic solution at 37°C in a 5% CO2 incubator. The effect of arctigenin on cell viability was evaluated using an MTT assay. Briefly, 2×103 cells/well were seeded into 96-well plates incubated for 24 h at 37°C, treated with 0, 25, 50, 100 or 200 µM arctigenin and cultured for an additional 24, 48 or 72 h. Cell viabilities were determined by measuring absorbances at 570 nm using a Spectramax M2e (Molecular Devices, Sunnyvale, CA, USA).

Wound healing assay

Cells were seeded in 6-well plates coated with collagen type I (Corning Life Sciences, Bedford, MA, USA), grown until confluent, and scratched with a blue tip to create wounds. They were then treated with culture media containing 0, 50, 100, 150 or 200 µM arctigenin. Optical microscopic images were captured from two different areas of each well at 0 and 48 h after wounding.

Transwell invasion assay

The effect of arctigenin on the invasiveness of 4T-1 mouse TNBC cells was determined using transwell chambers (Corning Life Sciences) inserts in 24-well plates. Lower faces of polycarbonate filters (transwell inserts) were coated with matrigel for 1 h at 37°C, and then, 3×104 cells were seeded into matrigel-coated transwell chambers and 750 µl of culture media was added to lower chambers. After 24 h, cells were treated with conditioned media containing 2 or 10% FBS and 0, 50, 100, 150 or 200 µM arctigenin and incubated at 37°C in 5% CO2 atmosphere for 24 h. Cells that migrated across membranes were then fixed and stained using hematoxylin and eosin (H&E) and photographed under an inverted microscope at ×200.

Gelatin zymography

The effect of arctigenin on MMP-9 activity was evaluated by gelatin zymography. Briefly 2×105 cells/well were seeded into 6-well plates and allowed to attach for 24 h. Cells were then serum-starved for 4 h and treated with serum-free media supplemented with various concentrations of arctigenin (0, 50, 100, 150 or 200 µM) for 24 h. Conditioned media were then transferred to new conical tubes and centrifuged to remove cell debris. The supernatants were objected by 8% SDS-polyacrylamide gel electrophoresis (PAGE) containing 0.1% (v/v) gelatin under non-reducing conditions. The gel was then washed with 2.5% Triton X-100 for 1 h at room temperature to remove SDS and gelatinase reactions were performed in reaction buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 0.04% NaN3) at 37°C for 24 h. The gel was then stained with Coomassie staining solution (0.05% Coomassie brilliant blue R, 45% methanol, and 10% acetic acid) and destained at room temperature. Densitometric analysis was performed using ImageJ.

RNA extraction, cDNA synthesis, and RT-qPCR

4T-1 mouse TNBC cells (2×105 cells/well) were seeded into 6-well plates, allowed to attach for 24 h, and cultured in serum-free DMEM containing 0, 25, 50, 100, 150 or 200 µM arctigenin for an additional 24 h. The cells were then collected by trypsinization for RNA extraction, which was performed using the easy-BLUE™ Total RNA extraction kit (iNtRON Biotechnology, Inc., Sungnam, Korean). Extracted total RNA was quantified using a NanoDrop spectrophotometer (Schimazu Scientific Instruments, Kyoto, Japan), and cDNA was synthesized from 1 µg of total RNA in 1X Goscript reaction buffer containing 2 mM MgCl2, 0.5 mM and Goscript™ Reverse Transcriptase (all from Promega, Madison, WI, USA). RT-q PCR was conducted using Q Green SYBR Green Master Mix Kits (Cellsafe, Suwon, Korea) using an Eco™ Real-Time PCR machine (Illumina, San Diego, CA, USA). cDNA amplification reactions were performed as follows: Pre-heating for 5 min at 95°C, 45 cycles at 95°C for 10 sec, 60°C for 15 sec and 72°C for 20 sec. Relative mRNA expressions were calculated automatically using the 2−ΔΔCq method and Eco™ Software v3.1.7 (Illumina, Inc.) (43). The primer sequences used for RT-q PCR were as follows: MMP-9, forward, 5′-TGTCTGGAGATTCGACTTCA-3′ and reverse, 5′-TGAGTTCCAGGGCACACCA-3′; MMP-3, forward, 5′-CTTTGAAGCATTTGGGTTTCTCTAC-3′ and reverse, 5′-AGCTATTGCTCTTCAATATGTGGGT-3′; COX-2, forward, 5′-CCTGCTGCCCGACACCTTCA-3′ and reverse, 5′-AGCAACCCGGCCAGCAATCT-3′; β-actin, forward, 5′-CATCCGTAAAGACCTCTATGCCAAC and reverse, 5′-ATGGAGCCACCGATCCACA-3′.

Nuclear fractionation

4T-1 mouse TNBC cells were seeded into 6-well plates at 2×105 cells/well and allowed to attach for 24 h. The cells were then serum-starved for 4 h, treated with conditioned-media containing various concentration of arctigenin (0, 25, 50, 100, 150 or 200 µM) for 24 h, and washed twice with ice-cold phosphate buffered saline. Hypertonic buffer (20 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2] containing protease inhibitor cocktail and phosphatase inhibitor cocktail (GenDEPOT, Barker, TX, USA) was then added to each well. The cells were detached with a rubber policeman (SPL Life Sciences, Pocheon, Korea), transferred to 1.5 ml microtubes, kept on ice for 15 min, treated with 10% NP-40 (final NP-40 concentration 0.125%) with vortex-mixing for 10 sec at the highest setting, and left on ice for 10 min. Cell mixtures were then centrifuged at 3,000 rpm for 10 min at 4°C, supernatants (cytosolic fractions) were removed and pellets were lysed with Cell Extraction Buffer (Invitrogen, Carlsbad, CA, USA) containing phosphatase and protease inhibitor cocktail for 30 min on ice, lysates were centrifuged at 14,000 × g for 30 min at 4°C, and supernatants (nuclear fractions) were collected. Cytosolic and nuclear fractions were stored at −80°C until required.

Western blotting

After treating 4T-1 mouse TNBC cells for 24 h with various concentration of arctigenin (0, 25, 50, 100, 150 or 200 µM), they were lysed with RIPA lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 2 mM ethylenediaminetetraacetic acid] (Biosesang, Seongnam, Korea) containing protease inhibitor cocktail and phosphatase inhibitor cocktail (GenDEPOT, Barker, TX, USA), and centrifuged at 13,000 rpm for 10 min at 4°C. Supernatants (whole cell lysates) were transferred to microtubes and stored at −80°C until required. Total protein in whole cell lysates was quantified using the BCA method, and same amounts of total protein in whole cell lysates were subjected to SDS-PAGE. After transferring proteins to PVDF membranes, membranes were blocked with 1% BSA in Tris-buffered saline (TBS)-Tween (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature, probed with primary antibodies diluted 1:3,000 with 1% bovine serum albumin in TBS-Tween solution overnight at 4°C, washed three times in TBS-Tween, and reacted with secondary antibodies (dilution 1:5,000) for 1 h at room temperature in TBS-Tween. Target proteins were visualized using a homemade chemiluminescent substrate and photographed using a Luminescent Image Analyzer LAS-4000 (Fujifilm, Tokyo).

Statistical analysis

One-way analysis of variance followed by the Tukey's post hoc test was used to determine the significances of differences. The analysis was performed using SPSS Ver. 20.0 software (SPSS, Inc., Chicago, IL, USA), and results are presented as means ± SDs. Statistical significance was accepted for P-values of <0.05.

Results

The effect of arctigenin on the cell viability, migration, and invasion of 4T-1 mouse TNBC cells

We firstly evaluated the effect of arctigenin on the viability of 4T-1 mouse TNBC cells using an MTT assay. As shown in Fig. 1A, arctigenin (100 and 200 µM at 48 and 72 h) slightly reduced cell viability. Furthermore, arctigenin inhibited cell migration and invasiveness as determined by the matrigel invasion and wound healing assays in a concentration-dependent manner, respectively. Lignans should enters into cells with simple diffusion or a low affinity transporter (44). Therefore, these results suggest arctigenin should inhibit invasion and migration by 4T-1 mouse TNBC cells and we postulated the effect was mediated via the regulation of signaling molecules by arctigenin entered into the cells with simple diffusion or a low affinity transporter.

Arctigenin inhibited MMP-9 activity and its gene expression in 4T-1 mouse TNBC cells

Due to its important role on metastasis in breast cancer and the associations between MMP-9 activity and cell migration and invasiveness, we evaluated the effects of arctigenin on MMP-9 activity and its mRNA level. Arctigenin was found to reduce both the protein and mRNA levels of MMP-9 dose-dependently (Fig. 2A and B), which suggested that the suppression of MMP-9 expression by arctigenin was responsible for its inhibition of cell migration and invasion.

Arctigenin inhibited MMP-3 mRNA expression in 4T-1 TNBC cells

Active MMP-9 is produced by cleavage of the prodomain in pro-MMP-9 mediated by MMP-3 and MMP-9 and as mentioned above, its activity is also positively associated with MMP-3 activity. We found arctigenin at 150 or 200 µM suppressed MMP-3 transcription (Fig. 2C). Mehner et al showed metastatic potential and MMP-3 expression are associated in breast carcinoma (45), and Chu et al found breast cancer tumorigenesis and metastasis were prevented by MMP-3 knockdown by mir-519d (46). These reports indicate MMP-3 activity is closely linked with its gene expression. Therefore, our observation suggest arctigenin might inhibit MMP-9 activity by downregulating MMP-3 expression in 4T-1 mouse TNBC cells.

Arctigenin inhibited COX-2 protein and mRNA levels in 4T-1 mouse TNBC cells

COX-2 is another important factor of metastasis in breast cancer and MMP-9 activity is positively associated with COX-2 expression. We found arctigenin dose-dependently downregulated COX-2 protein and mRNA levels (Fig. 3), which suggests that arctigenin might also reduce cancer cell growth and metastatic potential by inhibiting COX-2 expression in 4T-1 mouse TNBC cells.

Arctigenin inhibited nuclear c-Jun and c-Fos levels via the ERK1/2 and JNK1/2 signaling pathways

Because arctigenin appeared to inhibit 4T-1 TNBC cell migration and invasion by suppressing MMP-9, MMP-3, and COX-2. Therefore, we investigated the effect of arctigenin on ERK1/2 and JNK1/2 signaling pathways, which are key regulators of the expressions of MMP-9, MMP-3, and COX-2. The results obtained showed that arctigenin inhibited the phosphorylations of ERK1/2 and JNK1/2 but not p38 MAPK (Fig. 4). Arctigenin did not change the whole cell expressions of c-Jun and c-Fos (AP-1 subunits), but attenuated their nuclear expressions and reduced AP-1 transcriptional activity (Fig. 5). Consequently, our results suggest that anti-metastatic activity of arctigenin is governed by reduction in nuclear c-Jun and c-Fos levels and associated inhibitions of the phosphorylations of ERK1/2 and JNK1/2.

Discussion

Although various studies have reported arctigenin has anti-cancer effects on some types of cancer cells, comparatively little is known of its effects on metastasis (2,13). In this study, we evaluated the effect of arctigenin on metastatic potential in 4T-1 mouse TNBC cells. We found arctigenin suppressed the migration and invasiveness of these cells (Fig. 1B and C) but did not significantly decrease cell viability in 24 h (Fig. 1A). These results suggest arctigenin has therapeutic potential for preventing the invasion and migration that are important roles in metastasis in triple-negative breast cancer.

Cell migration and invasiveness are closely associated with the activity of MMP-9 in breast cancer and MMP-9 is activated by proteolytic cleavage of the prodomain in pro-MMP-9 by MMP-3. Furthermore, MMP-9 gene expression is known to be closely associated with that of MMP-3 (26,28). It has also been well-established that the activity and expression of MMP-9 importantly contribute to breast cancer metastasis. Several authors have demonstrated that reductions in MMP-9 activity and expression in breast cancer cells are associated with reduced metastatic potential (22,47,48). In the present study, arctigenin decreased MMP-9 activity and suppressed its gene expression (Fig. 2A and B) and also downregulated MMP-3 mRNA expression (Fig. 2C). Furthermore, arctigenin also inhibited COX-2 at the protein and mRNA levels (Fig. 3), and as mentioned above, COX-2 also affects metastatic potential and MMP-9 activity and expression in cancer cells (29,30). Therefore, our results indicate arctigenin inhibits the migration and invasion of 4T-1 mouse TNBC cells by suppressing the activity and mRNA levels of MMP-9, transcription of MMP-3, and the protein and mRNA expression of COX-2.

The MAPK/AP-1 signaling pathway plays an important role in the regulations of various metastasis-associated gene expressions. AP-1 is a transcription factor regulated by MAPKs, such as ERK1/2, JNK1/2, and p38 MAPK, and the transcriptional activity of AP-1 is determined by the nuclear levels of AP-1 subunits, which are in turn, governed by the regulation of MAPK phosphorylation. In the present study, we found that arctigenin suppressed the gene expressions of MMP-9, MMP-3 and COX-2 and the phosphorylations of ERK1/2 and JNK1/2, which were associated with reductions in the nuclear levels of c-Jun and c-Fos (AP-1 subunits) (Figs. 2B and C and 35). Furthermore, down-regulations of the gene expressions of MMP-9, MMP-3, and COX-2 corresponded to diminished phosphorylations of ERK1/2 and JNK1/2 and decreased nuclear c-Jun and c-Fos. However, p38 MAPK phosphorylation did not affected by arctigenin (Fig. 4). The promotor sites on MMP-9, MMP-3 and COX-2 genes contain AP-1 binding site, and thus, their gene expressions are closely linked with the nuclear levels of c-Jun and c-Fos (36,37,41,42,49,50). Consequently, our results suggest that the inhibitions of MMP-9, MMP-3, and COX-2 by arctigenin are mediated via partial suppression of MAPK/AP-1 signaling pathway. Also, these investigations implies that the inhibitory effects of arctigenin are not associated with the direct inhibition of protein kinase C.

Our findings suggest arctigenin reduces the metastatic potential of 4T-1 mouse TNBC cells by reducing cell motility, invasiveness and MMP-9 activity and that these effects are linked with suppression of the gene expressions of MMP-9, MMP-3, and COX-2 via the partial suppression of MAPK/AP-1 signaling pathway. Taken together, our observations suggest arctigenin be considered a potential means of preventing metastatic potential in triple negative breast cancer.

Acknowledgements

Not applicable.

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Korean Ministry of Education (grant no. 2015R1D1A1A01058841).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

MGL, KSL and KSN designed the experiments. MGL performed experiments. MGL, KSL and KSN analyzed the data. MGL and KSL wrote the manuscript. KSN reviewed the manuscript. All authors confirmed and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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March-2020
Volume 21 Issue 3

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Spandidos Publications style
Lee MG, Lee KS and Nam KS: Anti‑metastatic effects of arctigenin are regulated by MAPK/AP‑1 signaling in 4T‑1 mouse breast cancer cells. Mol Med Rep 21: 1374-1382, 2020
APA
Lee, M., Lee , K., & Nam, K. (2020). Anti‑metastatic effects of arctigenin are regulated by MAPK/AP‑1 signaling in 4T‑1 mouse breast cancer cells. Molecular Medicine Reports, 21, 1374-1382. https://doi.org/10.3892/mmr.2020.10937
MLA
Lee, M., Lee , K., Nam, K."Anti‑metastatic effects of arctigenin are regulated by MAPK/AP‑1 signaling in 4T‑1 mouse breast cancer cells". Molecular Medicine Reports 21.3 (2020): 1374-1382.
Chicago
Lee, M., Lee , K., Nam, K."Anti‑metastatic effects of arctigenin are regulated by MAPK/AP‑1 signaling in 4T‑1 mouse breast cancer cells". Molecular Medicine Reports 21, no. 3 (2020): 1374-1382. https://doi.org/10.3892/mmr.2020.10937