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Cancer cell invasion is crucial for metastasis. A major factor in the capacity of cancer cell invasion is the activation of matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix.
Breast cancer is a malignant tumor with the ability to spread beyond the breast tissue. The patients with breast cancer exhibited a high mortality rate (
Invasion and metastasis of cancer cells are characterized by the degradation of the extracellular matrix (ECM) by proteases secreted from cancer cells (
Cytokine and TPA-mediated MMP-9 expression is controlled by the transcription factors, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) in cancer cells (
In the present study, it has been hypothesized that SME may exhibit anticancer properties through the inhibition of cell invasion. Therefore, the molecular mechanism by which SME affects the invasiveness of the breast cancer MCF-7 cell line was investigated. SME reduced TPA-induced cell invasion via the mitogen-activated protein kinase (MAPK)/AP-1 signaling pathways, and decreased MMP-9 expression was associated with the extent of the inhibition of breast cancer cell invasion.
MCF-7 cells were acquired from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 1% antibiotics (10,000 U/ml penicillin, 10,000 µg/ml streptomycin, 25 µg/ml amphotericin B) and 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) at 37°C in an incubator with 5% CO2 saturation. DAPI, TPA, dimethyl sulfoxide and anti-β-actin antibody were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Antibodies against MMP-9 (catalog no. 12759), inhibitory κ B kinase (IKK)α (catalog no. 2682), IKKβ (catalog no. 2678), proliferating cell nuclear antigen (PCNA; catalog no. 7907), IκBα (catalog no. 371), transcription factor p65 (catalog no. 372), and horseradish peroxidase (HRP)-conjugated immunoglobulin G (IgG) (catalog no. 2004, 2005) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies against c-Jun N-terminal kinase (JNK; catalog no. 9252), p38 (catalog no. 9212), extracellular signal-regulated kinase (ERK; catalog no. 9102), phosphorylated (p)-JNK (catalog no. 9252), p-p38 (catalog no. 9211), p-ERK (catalog no. 9101), p-c-Jun (catalog no. 9261), p-IκBα (catalog no. 2859) and p-IKKα/β (catalog no. 2697) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Goat anti-rabbit Alexa Fluor 568 [IgG heavy and light chains (H&L)] (catalog no. A-11036) was obtained from Invitrogen (Thermo Fisher Scientific, Inc.).
Dried roots of S. miltiorrhiza Bunge were purchased from Kwangmyungdang Medicinal Herbs Co., Ltd. (Ulsan, Korea) and authenticated by Professor Guem-San Lee (Department of Herbology, Wonkwang University School of Korean Medicine, Iksan, Korea): A voucher specimen (WKU120302-SM201406A) was deposited at the Department of Herbology, College of Korean Medicine, Wonkwang University (Iksan, Korea).
MCF-7 cells were seeded on 96-well plates (1.5×104 cells/well) and treated with 1, 5, 10, 25 and 50 µg/ml SME for 24 h at 37°C. Then, 100 µl EZ-CyTox assay reagent (Daeil Lab Service Co., Ltd., Seoul, Republic of Korea) was added 100 times diluted to the plate wells. Next, the cells were incubated for 30 min at 37°C prior to measuring the absorbance with a 540-nm filter in an ELISA reader (Molecular Devices, LLC, Sunnyvale, CA, USA).
To analyze the proteolytic activity of MMP-9, zymography was performed as previously described (
Total protein extracts were prepared using an ice-cold M-PER Mammalian Protein Extraction Reagent (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Proteins were quantified using a BioSpec-nano Micro-volume Spectrophotometer (Shimadzu; Columbia, USA). For each lane, 20 µg protein was used. The protein samples were separated using SDS-PAGE (10% gel) and transferred to Hybond™ polyvinylidene fluoride membranes. The membranes were blocked with 2% bovine serum albumin (Sigma-Aldrich; Merck KGaA) or 5% skim milk for 2 h at 4°C. Membranes were then incubated overnight at 4°C with primary antibodies. β-actin (catalog no. A2228) was purchased from Sigma-Aldrich (Merck KGaA). MMP-9 (catalog no. 12759), IKKα (catalog no. 2682), IKKβ (catalog no. 2678), PCNA (catalog no. 7907), IκBα (catalog no. 371) and p65 (catalog no. 372) were obtained from Santa Cruz Biotechnology, Inc. JNK (catalog no. 9252), p38 (catalog no. 9212), ERK (catalog no. 9102), phosphorylated (p)-JNK (catalog no. 9252), p-p38 (catalog no. 9211), p-ERK (catalog no. 9101), p-c-Jun (catalog no. 9261), p-IκBα (catalog no. 2859) and p-IKKα/β (catalog no. 2697) were purchased from Cell Signaling Technology, Inc. All Antibodies used were diluted at 1:2,000. Protein expression levels were measured by Mini HD6 image analyzer using Alliance 1D software (UVItec, Cambridge, UK) with Immobilon™ Western Chemilumi nescent HRP Substrate (enhanced chemiluminescence) kit (EMD Millipore, Billerica, MA, USA).
RNA isolation from cells was performed using the FastPure™ RNA kit (Takara Bio, Inc., Otsu, Japan). Complementary DNA was synthesized using a PrimeScript™ RT Reagent kit (Takara Bio, Inc.). qPCR analysis was performed using the StepOnePlus™ Real-Time PCR System and SYBR Green (both Applied Biosystems; Thermo Fisher Scientific, Inc.) to determine mRNA levels. The thermocycling conditions were as follows: 50°C for 2 min and 95°C for 10 min, followed by 50 cycles of 95°C for 15 sec and 60°C for 1 min. The primers used were as follows: MMP-9 (NM 004994) sense, 5′-CCTGGAGACCTGAGAACCAATCT-3′ and antisense, 5′-CCACCCGAGTGTAACCATAGC-3′; and GAPDH (NM 002046) sense, 5′-ATGGAAATCCCATCACCATCTT-3′ and antisense, 5′-CGCCCCACTTGATTTTGG-3′. The mRNA levels were normalized to the GAPDH housekeeping gene expression levels. The method of quantification was 2−ΔΔCq method (
Cells were fixed with 4% paraformaldehyde at room temperature for 30 min, and then washed three times with cold PBS. Next, the cells were incubated for 45 min at room temperature with blocking buffer to prevent nonspecific antibody binding. Then, anti-p-c-Jun (red) antibodies (dilution, 1:1,000) were added and the cells were incubated for 24 h at 4°C. Subsequently, the cells were washed three times and then incubated for 1 h at room temperature with DAPI (blue) and 1:1,000 diluted goat anti-rabbit Alexa Fluor 568 (IgG H&L) in 0.1% triton X-100 for nuclear and p-c-Jun staining. Images were captured by an ArrayScan™ VTI system using Cellomics VHCS: View Software, version 1.6.30 (Cellomics, Inc., Pittsburgh, PA, USA).
MCF-7 cells (2×106) were treated with SME and/or TPA for 4 h at 37°C. Following this, the cells were centrifuged at 1,500 × g for 5 min at 4°C. Nuclear and cytoplasmic extracts were separated by using NE-PER Nuclear and Cytoplasmic Extraction Reagent (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.
The invasion (
The data were analyzed by one-way analysis of variance and Duncan's multiple range tests using SAS software, version 9.1; (SAS Institute Inc., Cary, NC, USA). P<0.05 was considered to indicate a statistically significant difference.
The treatment of cells with SME did not affect cell viability until concentrations reached 50 µg/ml (
To confirm whether SME is involved in MAPK activation, western blot analysis was performed. Exposure to TPA for 15 or 30 min markedly elevated the phosphorylation levels of p38/JNK/ERK, and pre-treatment with SME markedly decreased them (
To confirm this TPA-induced activation of NF-κB (p65/p50) and AP-1 (c-Jun/c-Fos), immunofluorescence and western blotting were used. TPA induced substantial p-c-Jun expression, whereas pre-treatment with SME blocked it (
To demonstrate whether SME inhibits the invasion and migration abilities of MCF-7 cells,
In the present study, it was revealed that SME blocked TPA-induced cell invasion and MMP-9 expression through inhibition of the MAPK/AP-1 signaling pathway. Concurrently, SME did not affect NF-κB signaling. These results suggest that SME blocks cell invasion by suppressing MMP-9 expression, mediated by the activity of the MAPK/AP-1 signaling pathway, in MCF-7 breast cancer cells.
The ECM provides biochemical and physical barriers to the proliferation and spread of cancer cells, and cancer cell invasion requires its degradation (
It has been indicated in several previous studies that the active component of
MAPK families serve an important role in the activation of AP-1. ERK enhances AP-1 activation through c-Fos, whereas JNK leads to the phosphorylation of c-Jun (
The present study was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Nuclear Research & Development Program of the National Research Foundation (grant no. NRF-2012M2A2A6011335) and by the Korean government (the Ministry of Education, Science Technology); grant no. 2011-0030130), Republic of Korea.
SME inhibits TPA-induced MMP-9 expression in MCF-7 cells. (A) SME was treated at various concentrations (0, 1, 5, 10, 25 and 50 µg/ml) for 24 h. An MTT assay was used to measure cell viability. (B) MCF-7 cells in a monolayer were treated with the indicated SME concentrations in the presence of TPA for 24 h. Cell lysates were analyzed by western blotting with an anti-MMP-9 antibody. The blot was re-probed with an anti-β-actin antibody to confirm equal loading amounts. Conditioned medium was prepared and used for gelatin zymography. (C) MMP-9 mRNA levels were analyzed by quantitative polymerase chain reaction using GAPDH mRNA as an internal control. Each value represents the mean ± standard error of the mean of three independent experiments. *P<0.05 vs. TPA only. SME,
SME inhibits TPA-induced mitogen-activated protein kinase activation in MCF-7 cells. Cells were pretreated with SME for 1 h and then stimulated with TPA for 15 and 30 min. Western blotting for p-p38, p-ERK and p-JNK was performed as described in the Materials and methods section. SME,
SME suppresses TPA-induced transcriptional activation of MMP-9 by inhibiting AP-1. (A) Cells were pretreated with SME in the presence of TPA. Following 4 h of incubation, the expression of p-c-Jun in the nucleus was assessed by immunofluorescence analysis (magnification, ×10). (B) Cells were pretreated with SME for 1 h and then exposed to TPA for 4 h. Western blotting was performed to determine the nuclear levels of p65 and activator protein 1 (p-c-Jun) subunits. PCNA was used as loading control. (C) Cells were pretreated with SME for 1 h and then stimulated with TPA for 4 h. Western blotting was performed to determine the cytoplasmic levels of IκBα, p-IκBα, IKKα, IKKβ and p-IKKαβ. SME,
SME inhibits TPA-induced invasion and migration in MCF-7 cells. Change in (A) invasion and (B) migration in MCF-7 cells (magnification, ×10). Each value represents the mean ± standard error of the mean of three independent experiments. #P<0.05 vs. control, *P<0.05 vs. TPA only. SME,