Contributed equally
Arctigenin is a bioactive lignan isolated from the seeds of
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related death in females worldwide, with an estimated 1.7 million cases and 521,900 deaths in 2012 (
Natural products are a very important source providing promising leads for the development of novel cancer therapeutics due to their potentially low toxicity profiles and potential effectiveness (
Arctigenin is the main active constituent that is extracted and isolated from the fruit of
Arctigenin, isolated from the fruit of
The human breast cancer cell line MDA-MB-231 was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS (both from Gibco, Carlsbad, CA, USA), 100 U/ml penicillin G, 2.5 µg/ml amphotericin B and 100 µg/ml streptomycin (complete medium) at 37°C with 5% CO2 in a humidified atmosphere.
The cytotoxic effect of arctigenin on MDA-MB-231 cells was evaluated by the MTT assay. Briefly, MDA-MB-231 cells were placed into 96-well plates at a final concentration of 1×104 cells/well in complete medium. Cells were treated with a range of concentrations of arctigenin. Twenty microliters of MTT (5 mg/ml) were then added at 24, 48 and 72 h after treatment. The cells were incubated for another 4 h at 37°C in the dark. Formed formazan crystals were dissolved in 100 µl DMSO and the absorbance was measured at 570 nm on a microplate reader (Bio-Rad, Hercules, CA, USA).
MDA-MB-231 cells were plated into a 24-well plate at a density of 2×105 cells/well and allowed to form a confluent monolayer for 24 h. Subsequently, the monolayer was scratched with a sterile pipette tip (1,000 µl), washed with medium to remove floating and detached cells and photographed (time 0 h). The cells were successively treated in medium in the presence of different concentrations of arctigenin (20 and 40 µM) along with vehicle DMSO for 24 h. Scratched areas were photographed (magnification, ×40) at 0 h, and then subsequently again 24 h later to assess the degree of wound healing. The percentage of wound closure was estimated by the following equation: Wound closure % = 1 - (wound area at t24/wound area at t0) × 100%, where t24 is the time after wounding and t0 is the time immediately after wounding.
MDA-MB-231 cell migration was also investigated by a modified Boyden chamber assay. The method is based on the passage of cells across porous filters separating the upper and lower wells of the migration chamber. Briefly, cells were incubated in the presence or absence of 40 µM arctigenin for 24 h. After trypsinization, 1×105 cells suspended in 0.1% (v/v) BSA medium were placed in the upper chamber of 8-µm pore size Transwells (24-well; Millipore, Billerica, MA, USA) and incubated for 18 h at 37°C under 5% CO2. For the invasion assay, the upper surface of the Transwell membrane was coated with 1 µg Matrigel. Cells (2×105) (incubated in the presence or absence of 40 µM arctigenin for 24 h) in 0.1% (v/v) BSA medium were placed in the upper part of the Transwell membrane and allowed to migrate for another 24 h. For both the migration and invasion assay, the unmigrated cells were removed from the upper surface of the membrane and the migrated cells on the lower surface of the membrane were fixed in 100% methanol and stained with hematoxylin and eosin. The migration and invasion were determined by counting the number of cells with a microscope at a magnification of ×100. Five visual fields were chosen randomly and the average number of migrating cells in the five fields was obtained for each group.
The enzymatic activities of MMP-2 and MMP-9 were assayed by gelatin zymography in the absence of serum (
MDA-MB-231 cells were exposed to arctigenin for 24 h. The treated cells were collected, washed with phosphate-buffered saline (PBS), and lysed in lysis buffer [25 mM HEPES (pH 7.7), 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 20 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 0.5 mM phenylemethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol, 10 mg/ml aprotinin and 10 mg/ml leupeptin]. The cell lysates were separated by 10% SDS-PAGE and transferred to PVDF membranes using a glycine transfer buffer [192 mM glycine, 25 mM Tris-HCl (pH 8.8) and 20% (v/v) methanol]. After blocking with Block Ace for 4 h at room temperature, the membrane was incubated overnight with primary antibodies, and then for 60 min with secondary antibodies. The primary antibodies were used at a dilution of 1:1,000. The secondary antibodies were used at a dilution of 1:2,000 and visualized with an enhanced chemiluminescence system (Bio-Rad).
The
All data are expressed as the mean ± SD of at least three independent experiments, and the statistical analysis for single comparison was evaluated by performing a Student's t-test. The criterion of statistical significance was p<0.05, p<0.01, p<0.001.
The MTT assay was used as an indirect measure to determine the viability of the MDA-MB-231 cells exposed to arctigenin. The MDA-MB-231 cells were treated with arctigenin for 24 h at various concentrations (0–40 µM). As shown in
To determine whether arctigenin could suppress the migration of MDA-MB-231 cancer cells, mechanical wounds were introduced into confluent monolayers, and wound closure was measured by microscopy. As shown in
To further confirm the activity of arctigenin on cell invasion, a Boyden chamber invasion assay was performed to evaluate the anti-invasion effect of arctigenin on MDA-MB-231 cells. As shown in
To determine whether the inhibitory effect of arctigenin on the invasion of MDA-MB-231 cells was related to the activity of MMPs, a gelatin zymography assay was performed to examine the activity of MMP-2 and MMP-9. As shown in
Degradation of extracellular matrix (ECM) proteins is an essential step in the invasion and metastasis of cancer cells and is mainly mediated by MMPs such as MMP-2 and MMP-9 (
The CAM assay is the method commonly used for screening anti-angiogenic drugs and studying angiogenic mechanisms
Metastasis, the spread of malignant cells from a primary tumor to distant sites, poses the biggest problem to cancer treatment and is the main cause of death of cancer patients (
Matrix metalloproteinases (MMPs), a family of structurally and functionally related zinc-dependent enzymes, significantly contribute to the promotion of metastasis and tumor growth and the mechanisms include proteolytic degradation of ECM components and possibly regulation of tumor cell growth itself (
Heparanase, an endo-β-D-glucuronidase, has the ability to cleave heparin sulfate (HS) chains of heparan sulfate proteoglycans (HSPGs) in the extracellular matrix (ECM) and basement membrane (BM) and plays a critical role in tumor cell invasion, migration and angiogenesis (
As it is widely known, tumor cell invasion alone is not sufficient to produce distant metastasis; it requires the transport of malignant cells through the blood and/or lymph vessels. Angiogenesis is a crucial factor in tumor growth and metastases and constitutes an important point in the control of cancer progression (
In conclusion, the authors demonstrated that arctigenin suppressed the metastasis of MDA-MB-231 cancer cells by downregulation of MMP-2, MMP-9 and heparanase. However, more studies are still required to determine the exact mechanisms involved and to explore signal transduction pathways to better understand the biological mechanisms.
The present study was kindly supported by the Zhejiang Chinese Medical University Research Fund Project (2015ZR06), the Zhejiang Provincial Natural Science Fund (LZ15H310001), and the Open Project of First-Class Key Discipline for Science of Chinese Materia Medica, Zhejiang Chinese Medical University (Yao2016017).
Effects of arctigenin on the cell viability in the MDA-MB-231 cells. The MDA-MB-231 cells in exponential growth were placed at a final concentration of 1×104 cells/well in a 96-well plate and incubated for 3 h. After incubation, the cells were treated with various concentrations of arctigenin or with the vehicle (vehicle control, 0.5% DMSO) for 24, 48 and 72 h. The cell viability was then determined by the MTT assay and is expressed as the mean ± SD of three separate experiments.
Effects of arctigenin on the cellular migration of the MDA-MB-231 cells. The effect of arctigenin on cell migration was measured by wound healing assay. MDA-MB-231 cell monolayers were scraped with a sterile micropipette tip, and the cells were treated for 24 h with various concentrations of arctigenin (20 and 40 µM) or with the vehicle. Scratched areas were photographed (magnification, ×40) at 0 h, and then subsequently again 24 h later to assess the degree of wound healing. The data are expressed as mean ± SD from three independent experiments; ***p<0.001 compared with the control.
Effects of arctigenin on cell migration and invasion by Boyden chamber migration and invasion assays. (A) For the migration assay, cells were pretreated with arctigenin (40 µM) for 24 h. Then, an aliquot of cells (1×105) was transferred to each upper well. Cells were allowed to migrate for 18 h. (B) For the invasion assay, cells (2×105) pretreated with arctigenin (40 µM) for 24 h were transferred to each of the upper wells and allowed to migrate for 24 h. For both of the migration and invasion assays, after incubation, migratory and invasive cells on the bottom of the insert membrane were fixed in 100% methanol and stained with hematoxylin and eosin. Migration and invasion were determined by counting the cells with a microscope at a magnification of ×100. Five visual fields were chosen randomly and the average number of invasive cells in the five fields was obtained for each group. Each experiment was performed in triplicate. The data are presented as the mean ± SD of three replicate experiments; ***p<0.001 compared with the untreated control.
Effects of arctigenin on MMP-2 and MMP-9 activity by gelatin zymography assay. Subconfluent monolayers of MDA-MB-231 cells pretreated for 24 h with arctigenin (20 and 40 µM) were cultured for another 24 h in serum-free DMEM. Culture supernatants from compound-treated cultures were subjected to electrophoresis in a gelatin-embedded SDS-polyacrylamide gel. After electrophoresis, strips of gel were incubated with an incubation buffer. After 42 h of incubation, the gel strips were stained with Coomassie Brilliant Blue, and the locations of the gelatinolytic enzymes were visualized as clear bands on the blue background. An image of each gel was scanned. The intensity of the gelatin zymography bands was quantified by densitometry analysis using ImageJ software v1.47. The data are representative results of three independent experiments; **p<0.01, ***p<0.001 compared with the control.
Effects of arctigenin on MMP-2, MMP-9 and heparanase protein expression in MDA-MB-231 cells. The cells were pretreated with arctigenin (20 and 40 µM) for 24 h. The cell lysates were collected and subjected to western blot analysis to detect MMP-2/−9 and heparanase expression. The data are representative results of three independent experiments.
Effects of arctigenin on angiogenesis in the chick embryo chorioallantoic membrane (CAM) assay. Representative images illustrate the inhibitory effects of arctigenin on the CAM. The images show the inhibition of angiogenesis in the presence of arctigenin (50 µM, 10 µl/egg).