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Introduction

Casein kinase 2 (CK2) is a ubiquitous serine/threonine protein kinase that is involved in numerous cellular processes, including proliferation and apoptosis, and has been revealed to be overexpressed in a number of human cancer cell lines (1,2). A previous study suggested that CK2 overexpression creates a favorable environment for the development of the tumor phenotype (3). CK2 has been associated with mammary tumorigenesis, as the levels of CK2 protein and activity are increased in breast cancer tissue compared with normal breast tissue (1,4). Furthermore, overexpression of CK2 in the mammary glands of transgenic mice can cause hyperplasia and breast carcinoma, thus demonstrating the oncogenic potential of CK2 (4).

Invasion and metastasis, the fundamental properties of cancer cells, are the primary causes of poor prognosis in patients with breast cancer. The mechanisms of cancer cell invasion and metastasis comprise a multistep biophysical process (5). In the early stages, the proteolytic degradation of extracellular matrix (ECM) components is facilitated by matrix metalloproteinases (MMPs) (6,7). Apigenin is abundant in common fruits and vegetables, and has notable anti-inflammatory, anti-oxidant and anti-carcinogenic properties (8–13). Emodin is an active constituent isolated from Rheum palmatum, a Chinese herb (14) and has been revealed to lead to the inhibition of cell proliferation, cell cycle arrest, inhibition of cell division, and decreased cell motility and invasion (15). TBBz is one of the most efficient inhibitors of CK2 (16), and has been demonstrated to induce apoptosis in cancer cells (17,18). MMP-9 has an important role in the degradation of the ECM during breast cancer cell invasion (19,20). Considering its importance in cancer development and progression, MMP-9 can be suggested to represent an early target in the treatment of breast cancer metastasis (21,22), and the inhibition of MMP-9 expression, activity and/or upstream regulatory activity may represent a potential therapeutic strategy. 12-O-tetradecanoylphorbol-13-acetate (TPA), a selective activator of protein kinase C (PKC) (23), induces breast cancer cell invasion by stimulating MMP-9 synthesis and secretion (21,24) via the activation of transcription factors, including nuclear factor-κB (NF-κB) and activator protein-1 (25–27).

Considering that CK2 has been previously revealed to induce nuclear factor-κB (NF-κB) activation, the present study aimed to investigate whether the inhibition of CK2 affected TPA induced invasion and MMP-9 expression in MCF-7 human breast cancer cells and determine the associated underlying molecular mechanisms.

Materials and methods

Cells and materials

Human breast cancer MCF-7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (cat. no. 16000-044; Gibco; Thermo Fisher Scientific., Inc. Waltham, MA, USA) and 1% Anti-Anti (antibiotic-antimycotic) 100X (cat. no. 15240-062; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a 5% CO2 incubator. CK2 inhibitors [apigenin, 1,3,8-trihydroxy-6-methylanthraquinone (emodin) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (TBBz)], dimethyl sulfoxide used as the solvent of the CK2 inhibitors and TPA (cat. no. P1585), and anti-β-actin (cat. no. A5441) antibodies were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Matrigel was obtained from Corning Incorporated (Corning, NY, USA). Primary antibodies against CK2α (cat. no. 2656), p38 kinase (p38; cat. no. 9212), c-Jun N-terminal kinase (JNK; cat. no. 9252), extracellular signal-regulated kinase (ERK; cat. no. 9102), as well as the phosphorylated forms of p38 (cat. no. 9211), JNK (cat. no. 9261) and ERK (cat. no. 9101), were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies specific to MMP-9 (cat. no. 12759), transcription factor p50 (cat. no. 7178) and transcription factor p65 (cat. no. 372) and proliferating cell nuclear antigen (PCNA; cat. no. 7907), as well as horseradish peroxidase (HRP)-conjugated secondary immunoglobulin G (IgG; cat. no. SC-2004, SC-2005), were all purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

RNA interference

CK2α specific small interfering (si)RNA and negative control siRNA (cat. no. SN-1003) were obtained from Bioneer Corporation (Daejeon, Korea). The siRNA used were as follows: CK2α sense, CAU UUA GUU ACU GGG CAU A(dTdT) and antisense, UAU GCC CAG UAA CUA AAU G(dTdT). Briefly, human breast cancer MCF-7 cells (5×105) were transfected with 100 pmol siRNA using RNAiMAX Transfection Reagent for 24 h at 37°C (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

Determination of cell viability

Cell viability assays were performed using the EZ-Cytox Enhanced Cell Viability Assay kit (DOGEN, Seoul, Korea; www.dogenbio.com/shop/item.php?it_id=1490923054), according to the manufacturer's instructions. Briefly, 3×104 cells/well were seeded into 96-well plates and incubated at 37°C for 24 h to allow the cells to adhere. The cells were then treated with apigenin (20 µM), emodin (20 µM) or TBBz (2 µM) for 24 h at 37°C. Following this, all cells were incubated with EZ-CytoxReagent (10 µl) for 30 min at 37°C. Absorbance at 450 nm was determined using an ELISA plate reader (Sunrise™; Tecan Group, Ltd., Mannedorf, Switzerland).

Western blot analysis

MCF-7 cells (7×105) pretreated with CK2 inhibitors [20 µM apigenin, 20 µM emodin, or 2 µM TBBz] for 1 h, and cells (3.5×105) transfected with CK2 were subsequently incubated with TPA for 24 h at 37°C. The cells were lysed with ice-cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.), and the protein concentration of the resulting lysates was determined using a BioSpec-nano (Shimadzu Corporation, Kyoto, Japan). Total protein samples were resolved via 10% SDS-PAGE analysis and then transferred to polyvinylidene fluoride membranes (GE Healthcare Life Sciences, Little Chalfont, UK). The membranes were blocked with 5% bovine serum albumin (cat. no. 160069; MP biomedicals Inc.,) or 5% skimmed milk in TBS with 0.5% Tween-20 at 4°C for 2 h, and then incubated overnight at 4°C with primary antibodies. All antibodies used were diluted 1:2,000. HRP-conjugated IgGs were used as secondary antibodies and incubated at 4°C for 1 h. Protein expression levels were visualized using a Mini HD6 image analyzer using Alliance 1D software (UVItec Cambridge; Cleaver Scientific Ltd., Rugby, UK).

Gelatin zymography assay

MMP-9 activity in the conditioned medium (serum-free DMEM) was determined by gelatin zymography. MCF-7 cells (7×105) were pretreated with CK2 inhibitors [apigenin (20 µM), emodin (20 µM) or TBBz (2 µM)] for 1 h, and then incubated with TPA for 24 h at 37°C. The conditioned medium was mixed with non-reducing sample buffer (0.5M Tris-HCl (pH 6.8), Glycerol 2 ml, 10% SDS, 0.1% Bromophenol blue, D.W 1 ml up to 10 ml), and subjected to 10% SDS-PAGE analysis containing 0.1% [weight (w)/volume (v)] gelatin. The gel was then incubated in a renaturing buffer (2.5% Triton™ X-100; Sigma-Aldrich; Merck KGaA) with gentle agitation to remove the SDS at room temperature for 30 min. Following this, gels were incubated in a developing buffer (5 mM CaCl2, 0.02% Brij, pH 7.5 and 50 mM Tris-HCl) overnight at 37°C. The gel was stained for 30 min at room temperature with 0.25% (w/v) Coomassie brilliant blue R-250 at room temperature followed by destaining with washing buffer (10% acetic acid and 10% methanol). Proteolysis was visualized as a white zone in a dark blue field using a digital imaging system (FluorChem R by ProteinSimple; Cell Biosciences, Palo Alto, CA, USA).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Complementary DNA was synthesized from total RNA using a PrimeScript™ RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). mRNA expression levels were determined by qPCR analysis using the StepOnePlus™ Real-Time PCR System and SYBR®-Green (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers used were as follows: MMP-9 sense, 5′-CCTGGAGACCTGAGAACCAATCT-3′ and antisense, 5′-CCACCCGAGTGTAACCATAGC-3′; GAPDH (NM_002046) sense, 5′-ATGGAAATCCCATCACCATCTT-3′ and antisense, 5′-CGCCCCACTTGATTTTGG-3′. qPCR was performed with a preliminary incubation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. MMP-9 mRNA expression levels were quantified relative to GAPDH mRNA expression using the comparative 2−∆∆Cq method (28).

Preparation of nuclear extracts

MCF-7 cells (2×106) were transfected with CK2 siRNA in the presence of 20 nM TPA for 3 h at 37°C and then washed twice with PBS, scraped, resuspended in 1.5 ml ice-cold PBS (pH 7.5), and then centrifuged at 1,500 × g for 4 min at 4°C. Isolation of the nuclear and cytoplasmic extracts was performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (cat. no. 78835; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

Invasion assay

Invasion of MCF-7 cells was assessed using 24-well chambers with 8 µm pore membranes coated with 20 µl Matrigel. The Matrigel coating was rehydrated in 0.5 ml DMEM for 2 h at 37°C immediately prior to experiments. Cell growth medium (0.5 ml) with suspensed cells (3×105) and cells (2×105) transfected with CK2 siNRA were added to the upper chambers, and cell growth medium (0.5 ml) with TPA, alone or with CK2 inhibitors, were added to the bottom well. The upper chambers and bottom well were incubated with DMEM supplemented 10% FBS and 1% antibiotic for 24 h. Following incubation, cells on the upper membrane surface were removed using cotton swabs, and cells that had migrated to the lower membrane surface were fixed by formaldehyde solution (3.6%) at room temperature, stained with crystal violet, and counted in five random fields per chamber using a Leica DM ILLED Inverted Lab microscope (Leica, Wetzlar, Germany) used magnification, ×10.

Statistical analysis

Data are expressed as the mean ± standard error. Statistical significance was determined using one-way analysis of variance followed by Scheffe post hoc test in Excel (Microsoft Excel 2013). P<0.005 was considered to indicate a statistically significant difference. All experiments were performed in triplicate.

Results

Inhibition of CK2α inhibits TPA-induced MMP-9 expression in MCF-7 cells

In order to investigate the effects of CK2α inhibition on TPA-induced cell invasion and MMP-9 expression, intracellular CK2α expression was suppressed via transfection with siRNA (Fig. 1A). The siRNA-mediated inhibition of CK2α significantly suppressed the increase in MMP-9 mRNA/protein expression compared with the TPA group (Fig. 1B and C). Furthermore, MCF-7 cells treated with TPA exhibited significantly increased invasion compared with the untreated control cells. However, the inhibition of CK2α significantly suppressed TPA induced MCF-7 cell invasion (Fig. 1D). These results suggest that CK2α may be involved in the underlying mechanism resulting in the TPA-induced increase of cell invasion and MMP-9 expression.

Figure 1. Inhibition of CK2α suppresses TPA-induced MMP-9 expression in MCF-7 cells. (A) CK2α siRNA was transfected into MCF-7 breast cancer cells, and cells transfected with the CK2α inhibitor were revealed to significantly suppress CK2α expression. (B) Transfected cell lysates were analyzed by western blotting. (C) MMP-9 mRNA levels were analyzed by reverse transcription-quantitative polymerase chain reaction, and GAPDH was used as an internal control. (D) Transfected cell lysates were subjected to Matrigel invasion analysis (magnification, ×10). Data represent the mean ± standard error of the mean of three independent experiments. #P<0.005 vs. control; *P<0.005 vs. control + TPA. CK2α, casein kinase 2α; si, small interfering; TPA, 12-O-tetradecanoylphorbol-13-acetate; MMP-9, matrix metalloproteinase-9.

Inhibition of CK2α suppresses TPA-induced mitogen-activated protein kinase (MAPK) and NF-κΒ activation in MCF-7 cells

The role of MAPKs (ERK, p38 and JNK) as upstream modulators of NF-κΒ in the activation of MMP-9 expression has been well established (25,26,29). The effects of CK2α inhibition on the TPA-induced phosphorylation of MAPKs was investigated. Following suppression of CK2α expression via siRNA knockdown, the phosphorylation levels of p38 and JNK, but not of ERK, were significantly suppressed (Fig. 2A). As revealed in Fig. 2B, treatment with TPA significantly enhanced the level of NF-κΒ (p65 and p50 subunits) compared with the control group. However, following suppression of intracellular CK2α expression, the expression levels of NF-κΒ were significantly inhibited (Fig. 2B). These results suggest that CK2α is an upstream regulator of p38 and JNK in the PKC induced MAPK-NF-κΒ signaling pathway responsible for the regulation of MMP-9 expression.

Figure 2. Inhibition of CK2α suppresses TPA-induced activation of mitogen activated protein kinase signaling pathways and NF-κB expression in MCF-7 cells. (A) Western blot analysis of p38, JNK, ERK, and their phosphorylated forms in MCF-7 cells following transfection with CK2α siRNA and treatment with TPA. (B) Nuclear lysates of cells transfected with CK2α siRNA were subjected to western blot analysis using anti-p65 and -p50 antibodies. #P<0.005 vs. control; *P<0.005 vs. control + TPA. CK2α, casein kinase 2α; si, small interfering; TPA, 12-O-tetradecanoylphorbol-13-acetate; MMP-9, matrix metalloproteinase-9; p-, phosphorylated; p38, p38 kinase; JNK, c-Jun N-terminal kinase; NF-κB, nuclear factor-κB; ERK, extracellular signal-regulated kinase; PCNA, proliferating cell nuclear antigen.

CK2 inhibitors suppress TPA-induced cell invasion and MMP-9 expression in MCF-7 cells

To further elucidate the therapeutic potential of CK2 inhibition for the prevention of PKC induced breast cancer cell invasion, three CK2 inhibitors were investigated. The cytotoxicity of these three CK2 inhibitors was investigated using a EZ-cytox assay. As revealed in Fig. 3A, treatment with 20 µM apigenin, 20 µM emodin and 2 µM TBBz did not cause any significant changes in cell viability. Furthermore, the CK2 inhibitors were revealed to suppress TPA-induced increase in MMP-9 mRNA/protein expression and cell invasion (Fig. 3B-D). These results suggest that CK2 inhibitors have the ability to inhibit cell invasion via regulating MMP-9 expression.

Figure 3. Inhibition of CK2 suppresses TPA-induced invasion and MMP-9 expression in MCF-7 cells. (A) To investigate the cytotoxic effects of the CK2 inhibitors, cell viability following incubation with CK2 inhibitors was determined. The optical density of the control was regarded as 100%. (B) The activity of MMP-9 following treatment with CK2 inhibitors and TPA was investigated using a zymography assay. (C) The mRNA expression of MMP-9 was analyzed via reverse transcription-quantitative polymerase chain reaction. (D) The invasive ability of cells following treatment with CK2 inhibitors and TPA was determined using a Matrigel invasion assay (magnification, ×10). Data are presented as the mean ± standard error of the mean of three independent experiments. #P<0.005 vs. control; *P<0.005 vs. TPA only. CK2α, casein kinase 2α; si, small interfering; TPA, 12-O-tetradecanoylphorbol-13-acetate; MMP-9, matrix metalloproteinase-9; TBBz, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; zymo, zymography; A, apigenin; E, emodin; T, TBBz.

Discussion

The stages of breast cancer are classified as 0–4. Number staging systems usually use the TNM system to divide cancers into stages. The majority of cancer types are classified into 4 stages, numbered from 1 to 4, which are important for diagnosis and treatment (30). From stage 2 onwards, metastases develop and the 5 year survival rate begins to decline (31). Mortality from metastatic breast cancer are on the rise, and thus suppression of metastasis has emerged as a major challenge in breast cancer treatment (32,33). In the early stages of tumor metastasis, individual tumor cells or clusters invade the ECM surrounding the primary tumor (34,35). The ECM consists of type IV collagen and other matrix proteins (19). Type IV collagen is a major component of the basement membrane and type IV collagenase MMP-9 has previously been revealed to be an important molecule in tumor progression and invasion in mammary tumors (36).

Elevated MMP-9 levels are functionally associated with breast cancer metastasis, and MMP-9 serves a role in TPA induced invasion of MCF-7 cells (21,24,37). Therefore, the suppression of MMP-9 expression may represent a novel therapeutic strategy for the prevention of tumor metastasis. TPA upregulates MMP-9 expression in MCF-7 cells; however, the underlying molecular mechanisms have not been fully established. Therefore, the present study aimed to investigate the role of CK2 in the regulation of MMP-9 expression in MCF-7 cells. The results of the present study revealed that the inhibition of CK2α suppressed TPA-induced MMP-9 expression and invasion in MCF-7 cells. Furthermore, the results of the present study revealed that CK2α is a regulator of PKC-induced invasion in MCF-7 cells.

In addition, the inhibition of CK2 was demonstrated to suppress the activation of NF-κB in MCF-7 cells. The MAPK/NF-κB signaling cascade serves a role in PKC-mediated MMP-9 expression in MCF-7 cells (23,24,38,39). Furthermore, the MAPK signaling pathway has been previously demonstrated to be important for the activation of NF-κB (40,41). CK2 has been reported to induce NF-κB activation (27,42). In the present study, it was revealed that CK2α inhibition markedly suppresses TPA-induced activation of p38, JNK and NF-κB. In addition, it was revealed that the treatment of MCF-7 cells with CK2 inhibitors suppressed TPA-induced invasion and MMP-9 expression in MCF-7 cells.

In conclusion, the results of the present study revealed that suppression of CK2 exerts anti-invasive effects in PKC-induction condition via the regulation of MMP-9 expression levels. Therefore, CK2 may represent a novel anti-invasive target for cancer therapy.

Acknowledgements

Not applicable.

Funding

The present study was supported by a National Research Foundation of Korea grant funded by the Korea Government (grant nos. 2011-0030130 and 2013R1A1A2007181).

Availability of data and materials

The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

JMK and EMN performed most of the experiments. HKS, YOY and KBK analyzed the data and provided comments. SJ and JSK helped with the experiments. HJY designed the project. YRL designed the analysis. KBK, YRL and HJY wrote the manuscript. All authors reviewed the manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

Authors declare that they have no competing interests.

References