Antitumor and Anti-Invasive Effect of Apigenin on Human Breast Carcinoma through Suppression of IL-6 Expression

Interleukin (IL)-6 plays a crucial role in the progression, invasion, and metastasis of breast cancer. Triple-negative breast cancer (TNBC) cell line MDA-MB-231 is known for its aggressive metastasis. Epithelial to mesenchymal transition (EMT) is a critical process in cancer metastasis. The positive correlation between IL-6 and EMT in tumor microenvironment is reported. We found significantly upregulated IL-6 expression in MDA-MB-231 cells. A blockade of IL-6 expression decreased levels of phosphorylated signal transducer and activator of transcription 3 (pSTAT3), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), phosphorylated protein kinase B (pAkt), and cell cycle-related molecules, including cyclin-dependent kinases (CDKs) and cyclins in MDA-MB-231 cells. A short-hairpin RNA (shRNA)-mediated blockade of IL-6 expression inhibited migration and N-cadherin expression and induced E-cadherin expression in MDA-MB-231 cells. Growth rate was slower for the tumors derived from IL-6 shRNA-treated MDA-MB-231 cells than for those derived from control shRNA-treated MDA-MB-231 cells. The expression of pSTAT3, phosphorylated extracellular signal-regulated kinase (pERK), PI3K, pAkt, snail, vimentin, and N-cadherin was significantly lower in tumors from IL-6 shRNA-treated MDA-MB cells. In addition, apigenin treatment significantly inhibited the growth of MDA-MB-231-derived xenograft tumors along with the protein expressions of pSTAT3, pERK, IL-6, PI3K, pAkt, and N-cadherin. Our results demonstrate that the anti-invasive effect of apigenin in MDA-MB-231-derived xenograft tumors is mediated by the inhibition of IL-6-linked downstream signaling pathway.


Introduction
Human breast cancers are classified into three subgroups, namely estrogen or progesterone receptor-positive (ER+ or PR+); Her2/neu-positive (HER2/neu+); and triple-negative breast cancer (TNBC) without ER, PR, and Her2/neu expression [1,2]. TNBC accounts for approximately 15% of all breast cancers and is phenotypically characterized with invasiveness, metastasis, and poor prognosis to chemotherapy as compared to other breast cancer types [3]. The past decade has witnessed tremendous therapeutic progress in the targeting of ER+, PR+, or Her2+ breast cancers, but the therapeutic strategies targeting TNBC human breast cancer are still insufficient [3,4]. The MDA-MB-231 cell line lacks the expression of ER, PR, and HER2 [5] and exhibits relatively high aggressiveness and invasiveness. Hence, the MDA-MB-231 cell line has become one of the major cell lines in the study of human breast cancer [5,6]. Significant difference is shown: * p < 0.05. NEG, negative control; GCSF, granulocyte colonystimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; GRO a/b/g, growthregulated oncogene-a/b/g.

Blockade of IL-6 Expression Decreases the Level of pSTAT3, PI3K, and pAkt Proteins in MDA-MB-231 Cells
To investigate the effects of IL-6 expression blockade on the levels of signaling molecules in MDA-MB-231 cells, we treated cells with anti-IL-6 or IL-6 shRNA. The suppression of IL-6 expression using anti-IL-6 antibody in MDA-MB-231 cells decreased the expression of pSTAT3 protein but had minor effects on the expression levels of PI3K, STAT3, ERK, and pERK ( Figure 2a). The inhibition of IL-6 expression by IL-6 shRNA resulted in a significant reduction in the expression level of pSTAT3, PI3K, and pAkt, all of which are known to be triggered by IL-6 signaling (Figure 2b). Treatment with IL-6 shRNA also resulted in the change in the cellular morphology to a round form (Figure 2c). Significant difference is shown: * p < 0.05. NEG, negative control; GCSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; GRO a/b/g, growth-regulated oncogene-a/b/g.

Blockade of IL-6 Expression Decreases the Level of pSTAT3, PI3K, and pAkt Proteins in MDA-MB-231 Cells
To investigate the effects of IL-6 expression blockade on the levels of signaling molecules in MDA-MB-231 cells, we treated cells with anti-IL-6 or IL-6 shRNA. The suppression of IL-6 expression using anti-IL-6 antibody in MDA-MB-231 cells decreased the expression of pSTAT3 protein but had minor effects on the expression levels of PI3K, STAT3, ERK, and pERK ( Figure 2a). The inhibition of IL-6 expression by IL-6 shRNA resulted in a significant reduction in the expression level of pSTAT3, PI3K, and pAkt, all of which are known to be triggered by IL-6 signaling (Figure 2b). Treatment with IL-6 shRNA also resulted in the change in the cellular morphology to a round form (Figure 2c).

Blockade of IL-6 Expression Decreases the Levels of CDKs and Cyclins and Induces p21 Expression
IL-6-mediated expression of pSTAT3, PI3K, and pAkt is high during the proliferation of triple-negative breast cancer cells [37,38]. Therefore, we examined the expression of cell proliferation-related molecules in response to blockade of IL-6 expression, such as p53, p21, CDK2, CDK4, CDK1, cyclin D1, and cyclin B1, by a western blot analysis. As shown in Figure 3b, the knockdown of IL-6 expression in MDA-MB-231 cells significantly increased the expression levels of p21 proteins and decreased the expression levels of CDKs (CDK2, CDK4, and CDK1) and cyclins (cyclin D1 and cyclin B1) (Figure 3a

Blockade of IL-6 Expression Decreases the Levels of CDKs and Cyclins and Induces p21 Expression
IL-6-mediated expression of pSTAT3, PI3K, and pAkt is high during the proliferation of triplenegative breast cancer cells [37,38]. Therefore, we examined the expression of cell proliferationrelated molecules in response to blockade of IL-6 expression, such as p53, p21, CDK2, CDK4, CDK1, cyclin D1, and cyclin B1, by a western blot analysis. As shown in Figure 3b, the knockdown of IL-6 expression in MDA-MB-231 cells significantly increased the expression levels of p21 proteins and decreased the expression levels of CDKs (CDK2, CDK4, and CDK1) and cyclins (cyclin D1 and cyclin B1) (Figure 3a

Blockade of IL-6 Expression Decreases the Levels of CDKs and Cyclins and Induces p21 Expression
IL-6-mediated expression of pSTAT3, PI3K, and pAkt is high during the proliferation of triplenegative breast cancer cells [37,38]. Therefore, we examined the expression of cell proliferationrelated molecules in response to blockade of IL-6 expression, such as p53, p21, CDK2, CDK4, CDK1, cyclin D1, and cyclin B1, by a western blot analysis. As shown in Figure 3b, the knockdown of IL-6 expression in MDA-MB-231 cells significantly increased the expression levels of p21 proteins and decreased the expression levels of CDKs (CDK2, CDK4, and CDK1) and cyclins (cyclin D1 and cyclin B1) (Figure 3a

Blockade of IL-6 Expression Inhibits Cell Invasion and Metastasis Factors in MDA-MB-231 Cells
To investigate the anti-invasive effect in response to the blockade of IL-6 expression in MDA-MB-231 cells, we evaluated the invasiveness of cells and expression of EMT-related molecules such as E-cadherin and N-cadherin. As shown in Figure 4a, the invasiveness of MDA-MB-231 cells decreased in response to treatment with anti-IL-6 or IL-6 shRNA. Furthermore, the expression of E-cadherin increased and that of N-cadherin significantly decreased in MDA-MB-231 cells transfected with IL-6 shRNA (Figure 4b).
A meta-analysis-based biomarker assessment with Kaplan-Meier Plotter [39] revealed the negative correlation between the blood level of IL-6 and relapse-free survival in patients with TNBC. IL-6 level showed a negative association with relapse-free survival of patients with lymph node metastasis or grade 3 tumors (Figure 4c).

Blockade of IL-6 Expression Inhibits Cell Invasion and Metastasis Factors in MDA-MB-231 Cells
To investigate the anti-invasive effect in response to the blockade of IL-6 expression in MDA-MB-231 cells, we evaluated the invasiveness of cells and expression of EMT-related molecules such as E-cadherin and N-cadherin. As shown in Figure 4a, the invasiveness of MDA-MB-231 cells decreased in response to treatment with anti-IL-6 or IL-6 shRNA. Furthermore, the expression of Ecadherin increased and that of N-cadherin significantly decreased in MDA-MB-231 cells transfected with IL-6 shRNA ( Figure 4b). A meta-analysis-based biomarker assessment with Kaplan-Meier Plotter [39] revealed the negative correlation between the blood level of IL-6 and relapse-free survival in patients with TNBC. IL-6 level showed a negative association with relapse-free survival of patients with lymph node metastasis or grade 3 tumors (Figure 4c).

Blockade of IL-6 Expression Inhibits the Growth of MDA-MB-231-Derived Tumors
BALB/c nude mice were subcutaneously implanted with 5 × 10 6 MDA-MB-231 cells transfected with control (ctrl) shRNA or IL-6 shRNA. After 14 days, tumor was measured every other day until the volume reached about 1000-1200 mm 3 . Figure 5a,b shows that IL-6 shRNA treatment of MDA-MB-231 cells significantly inhibited the tumor growth as compared with control shRNA treatment. We examined the expression levels of molecules related to IL-6 signaling in the tumor tissues derived from MDA-MB-231 cells. The JAK/STAT3, MAPK, and PI3K-Akt-mTOR pathways are triggered by IL-6 signaling and are associated with tumor cell growth, proliferation, and metastasis [40]. Recent reports have shown that the knockdown of pSTAT3 expression resulted in the reduction in the growth and tumor formation ability of TNBC cells [37]. As seen in Figure 5c,d, the expression of

Blockade of IL-6 Expression Inhibits the Growth of MDA-MB-231-Derived Tumors
BALB/c nude mice were subcutaneously implanted with 5 × 10 6 MDA-MB-231 cells transfected with control (ctrl) shRNA or IL-6 shRNA. After 14 days, tumor was measured every other day until the volume reached about 1000-1200 mm 3 . Figure 5a,b shows that IL-6 shRNA treatment of MDA-MB-231 cells significantly inhibited the tumor growth as compared with control shRNA treatment. We examined the expression levels of molecules related to IL-6 signaling in the tumor tissues derived from MDA-MB-231 cells. The JAK/STAT3, MAPK, and PI3K-Akt-mTOR pathways are triggered by IL-6 signaling and are associated with tumor cell growth, proliferation, and metastasis [40]. Recent reports have shown that the knockdown of pSTAT3 expression resulted in the reduction in the growth and tumor formation ability of TNBC cells [37]. As seen in Figure 5c,d, the expression of pSTAT3, pERK, PI3K, and pAkt proteins was significantly lower in the tumor tissues derived from IL-6 shRNA-treated MDA-MB-231 cells than in those derived from control shRNA-treated MDA-MB-231 cells. Furthermore, a considerable decrease in the expression levels of snail, vimentin, and N-cadherin was observed in the tumor tissues derived from IL-6 shRNA-treated MDA-MB-231 cells. The reduced expression levels of pSTAT3, pAkt, and N-cadherin proteins were also confirmed by immunohistochemistry (IHC) staining of tumor tissues derived from the xenograft mice implanted with IL-6 shRNA-treated MDA-MB-231 cells (Figure 5d). pSTAT3, pERK, PI3K, and pAkt proteins was significantly lower in the tumor tissues derived from IL-6 shRNA-treated MDA-MB-231 cells than in those derived from control shRNA-treated MDA-MB-231 cells. Furthermore, a considerable decrease in the expression levels of snail, vimentin, and Ncadherin was observed in the tumor tissues derived from IL-6 shRNA-treated MDA-MB-231 cells. The reduced expression levels of pSTAT3, pAkt, and N-cadherin proteins were also confirmed by immunohistochemistry (IHC) staining of tumor tissues derived from the xenograft mice implanted with IL-6 shRNA-treated MDA-MB-231 cells (Figure 5d).

Treatment of MDA-MB-231 Cells with Apigenin Decreases the Expression Level of Snail and Ncadherin via IL-6 Inhibition
Apigenin is a flavonoid compound with anticancer effects on breast cancer cells that are mediated via apoptotic induction and cell cycle regulation [41,42]. However, studies reporting the antimetastatic effect of apigenin are few, and the molecular mechanism underlying the inhibitory effect of apigenin on cancer cell metastasis is incompletely understood. We investigated whether apigenin (Figure 6a) inhibits the production IL-6 and exerts anti-invasive effects via the suppression of IL-6 expression in MDA-MB-231 cells. Figure 6a,b shows that IL-6 production from MDA-MB-231 cells was significantly inhibited following apigenin treatment in a dose-dependent manner ( Figure  6b  Apigenin is a flavonoid compound with anticancer effects on breast cancer cells that are mediated via apoptotic induction and cell cycle regulation [41,42]. However, studies reporting the antimetastatic effect of apigenin are few, and the molecular mechanism underlying the inhibitory effect of apigenin on cancer cell metastasis is incompletely understood. We investigated whether apigenin (Figure 6a) inhibits the production IL-6 and exerts anti-invasive effects via the suppression of IL-6 expression in MDA-MB-231 cells. Figure 6a,b shows that IL-6 production from MDA-MB-231 cells was significantly inhibited following apigenin treatment in a dose-dependent manner (Figure 6b 3 . The growth rate of tumor was significantly inhibited in mice treated with both the doses of apigenin (Figure 7a,b). Apigenin treatment significantly decreased the expression levels of pSTAT3, pERK, PI3K, and pAkt proteins in the tumor tissues derived from MDA-MB-231-implanted xenograft models (Figure 7c,d). We confirmed the decrease in the expression levels of pSTAT3, pAkt, and N-cadherin by IHC staining of the tumor tissues derived from MDA-MB-231-implanted xenograft mice that were orally administrated with apigenin ( Figure 7e).  significantly inhibited in mice treated with both the doses of apigenin (Figure 7a,b). Apigenin treatment significantly decreased the expression levels of pSTAT3, pERK, PI3K, and pAkt proteins in the tumor tissues derived from MDA-MB-231-implanted xenograft models (Figure 7c,d). We confirmed the decrease in the expression levels of pSTAT3, pAkt, and N-cadherin by IHC staining of the tumor tissues derived from MDA-MB-231-implanted xenograft mice that were orally administrated with apigenin ( Figure 7e).

Discussion
The present study demonstrates the inhibitory effects of apigenin through the suppression of IL-6 expression on tumor progression and invasiveness of MDA-MB-231 cells in vitro and in vivo.
The overexpression of IL-6 in MDA-MB-231 cells has been reported in many studies [20,43,44], but the implications of IL-6 expression in breast cancer are yet unclear. MCF-7 is a noninvasive breast cancer cell line, whereas MDA-MB-231 is known as a highly invasive breast cancer cell line [5]. We confirmed the higher expression of IL-6 in MDA-MB-231 cells than in MCF-7 cells, suggestive of the positive correlation between the expression of IL-6 and invasiveness of MDA-MB-231 cells (Figure 1a,b). Many studies have reported that chronic inflammation is closely related to tumor progression, migration, and metastasis in various cancers [45][46][47]. Cancer inflammatory environment may be established by several factors, including pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 [7]. Of these, IL-6 released from MDA-MB-231 cells was reported to be associated with chronic inflammation [8,48] and is known to selectively induce the activation of the JAK/STAT3 and PI3K/Akt pathways [49]. IL-6-linked JAK/STAT3 and PI3K/Akt signaling pathways have been implicated in cancer progression [50][51][52]. We investigated the effect of knockdown of IL-6 expression using anti-IL-6 or IL-6 shRNA on the expression of inflammation-linked signaling molecules in MDA-MB-231 cells. As a result, we found that the neutralization of IL-6 expression or IL-6 shRNA treatment significantly inhibited the expression level of pSTAT3 (Figure 2a). However, the expression of PI3K and pAkt was downregulated only after treatment with IL-6 shRNA (Figure 2b). Neutralization of IL-6 using anti-IL-6 was probably insufficient to completely block IL-6 secretion, as low levels of IL-6 may be continuously released from MDA-MB-231 cells.
The interaction between IL-6 and IL-6R was shown to induce antiapoptotic effects on human esophageal carcinoma and multiple myeloma through the phosphorylation of gp130 and STAT3 [53,54]. The JAK/STAT3, MAPK, and PI3K/Akt/mTOR pathways are activated by IL-6 signaling and are closely related to cell growth and metastasis in TNBC [55]. In particular, STAT3, a transcription factor stimulated by IL-6, was shown to be consistently expressed in most breast cancer cells. The expression of the phosphorylated form of STAT3 is high in TNBC and thought to be related to the aggressiveness and invasiveness of tumors [15,56]. Blocking the expression of STAT3 by shRNA treatment in mouse breast cancer cells was shown to inhibit tumor growth, progression, and metastasis [37]. In addition, the activation of the PI3K-pAkt-mTOR pathway triggered by IL-6 was shown to be related to cell proliferation and metastasis in a variety of cancers [57,58]. In our study, we investigated the effect of IL-6 expression blockade by shRNA treatment on the expression of the molecules related to cell cycle progression in MDA-MB-231 cells. As shown in Figure 3a,b, the downregulation of IL-6 expression significantly increased the expression level of p21 (Figure 3a,b). Steiner demonstrated that the overexpression of IL-6 in prostate cancer resulted in a significant increase in cell proliferation through the upregulated expression of CDK2 and increased expression of pERK [18]. The results of our study suggest similar phenotypic characteristics in TNBC with respect to IL-6-mediated expression of pSTAT3, pERK, and cell cycle-associated molecules (Figures 2 and 3).
Studies have highlighted the crucial role of EMT in cancer metastasis [19]. EMT is known to correlate with increased cell migration and invasiveness [59]. Cancer cells undergoing the EMT process exhibit upregulated expression of N-cadherin, vimentin, fibronectin, and snail and downregulated expression of E-cadherin [60]. The circulating high levels of IL-6 in patients with breast cancer were shown to be positively associated with cancer metastasis and poor survival rate [10,11], consistent with the results of meta-analysis from a Kaplan-Meier Plotter (Figure 4c). We examined whether blocking IL-6 expression may inhibit the growth and metastasis of breast cancer in vivo using a xenograft mouse model. Blockade of IL-6 expression significantly delayed the growth of MDA-MB-231-derived tumor (Figure 5a,b). The tumor tissues derived from IL-6 shRNA-treated MDA-MB-231 cells showed decreased expression of pSTAT3, pERK, PI3K, and pAkt proteins (Figure 5c). The expression levels of snail, vimentin, and N-cadherin proteins, representative biomarkers of EMT process, were also significantly inhibited in the tumors derived from IL-6 shRNA-treated MDA-MB-231 cells (Figure 5c), indicating that IL-6 expression is directly associated with tumor metastasis through the regulation of snail, vimentin, and N-cadherin expression.
Apigenin is known to have many biological activities, including anti-inflammatory, antioxidant, and anticancer effects [21,22,61]. The anticancer effects of apigenin have been reported in several cancer types, including breast cancer [41,61,62]. Anticancer activities are generally mediated via antiproliferative, antiapoptotic, and/or antimetastatic effects on cancer cells. Very few studies have reported the antimetastatic activity of apigenin [63,64]. Furthermore, the molecular mechanism underlying the antimetastatic effects of apigenin is still under investigation. The expression of TNF-α, IL-1α, and IL-6 from MDA-MB-231 cells was shown to be inhibited by apigenin [35], and the inhibition of TNF-α-induced CCL2 release by apigenin was thought to suppress tumor migration and metastasis through the regulation of tumor microenvironment [35]. We found that apigenin significantly inhibited the production of IL-6 from MDA-MB-231 cells in a dose-dependent manner (Figure 6b,c). We also observed the inhibitory effect of apigenin on the migration and invasion of MDA-MB-231 cells (Figure 6e,f) and reported, for the first time, the significant delay in the growth of tumors in MDA-MB-231 xenograft mice orally treated with apigenin (Figure 7a,b). This effect was directed through the inhibition of IL-6-mediated pSTAT3, pERK, PI3K, and pAkt expression as well as EMT signal-linked N-cadherin expression (Figure 7c-e). The inhibitory effect of apigenin on hepatocyte growth factor-promoted metastasis of MDA-MB-231 cells was shown to be involved in the suppression of the PI3K/Akt pathway; this observation is partly in agreement with the results of the present study [36].
In summary, we demonstrate that the blockade of IL-6-associated inflammation positively correlates with the inhibition of tumor growth and EMT process in human breast cell line MDA-MB-231 and that oral administration of apigenin results in the suppression of IL-6-related downstream signaling pathways via anticancer and anti-invasive effects.

Specimen Preparation
Apigenin (≥95% pure) was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA), followed by dilution in a medium or drinking water for in vitro or in vivo experiments, respectively.

Cell Lines and Treatments
Human breast cancer cell lines MDA-MB-231 and MCF-7 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). MDA-MB-231 and MCF-7 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Corning Inc, New York, NY, USA) and Eagle's minimum essential medium (EMEM, ATCC, Manassas, VA, USA), respectively, supplemented with 10% heat-inactivated fetal bovine serum (FBS, Young-In Frontier, Seoul, South Korea) and 100 U/mL of penicillin and streptomycin (Gibco, Grand Island, NY, USA) at 37 • C in a humidified atmosphere of 5% CO 2 . Cells were cultured at a density of 5 × 10 5 cells in 6-well plate overnight and treated with three different concentrations of apigenin (0 µM, 50 µM, and 100 µM) for 6 h.

Human Cytokine Array
Cytokines released from cells were analyzed using Human Cytokine Array C1 (RayBiotech, Norcross, GA, USA). All experiments were performed according to manufacturer's instructions. Briefly, membranes were treated with a blocking buffer for 30 min and incubated overnight in the supernatants harvested from cells at 4 • C on a rocker. The membranes were washed with a wash buffer and incubated in biotinylated antibody cocktail diluted in blocking buffer for 2 h at room temperature. Following incubation, the membranes were washed and treated with horseradish peroxidase (HRP)-streptavidin for 2 h at room temperature. The membranes were washed and incubated in a detection buffer for 2 min at room temperature. The spots were analyzed using ChemiDoc (Protein Simple, San Jose, CA, USA).

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
The level of mRNA was analyzed by RT-qPCR using SYBR Green (BioLine, London, UK). Total cellular RNA was extracted with TaKaRa MiniBEST Universal RNA Extraction Kit (#9767, TaKaRa, Kusatsu, Japan), and cDNA synthesis was conducted using PrimeScript TM 1st Strand cDNA Synthesis Kit (#6110A, TaKaRa, Shiga, Japan) in accordance with the manufacturer's instructions. Briefly, cells were treated as indicated and harvested for total RNA extraction. The extracted RNA was quantitated with Nanodrop (MicroDigital Co., Seoul, South Korea), and 1 µg of RNA was used for 50-ng/µL cDNA synthesis. RT-qPCR was performed under the following conditions: 20 s at 94 • C for denaturation, 20 s at 60 • C for annealing, and 1 min at 72 • C for extension for 40 cycles. PCR primer sequences used were as follows: human IL-6F (5 -GCC CAG CTA TGA ACT CCT TCT-3 ) and IL-6R (5 -GAA GGC AGC AGG CAA CAC-3 ).

Short-Hairpin RNA (shRNA) Lentivirus Plasmid Transfection Using Lipofectamine 2000
Human IL-6 shRNA lentivirus plasmid with pGFP vector kit (#TL312162, Origene, Rockville, MD, USA) was used for blocking IL-6 expression in MDA-MB-231 cells. Briefly, cells were seeded at a density of 5 × 10 5 cells/well in a six-well plate and incubated overnight. After incubation, the supernatant was aspirated, and the cells were treated with the mixture of shRNA plasmid-Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) for 5 h at 37 • C. After incubation, the medium was replaced with fresh medium supplemented with 10% FBS and 100 U/mL of penicillin-streptomycin, and the cells were cultured at 37 • C. For selection, cells were incubated in a medium containing puromycin at appropriate concentration.

Invasion Assay
Invasive capability of MDA-MB-231 cells was evaluated with a transwell system (Corning Inc, New York, NY, USA). Briefly, a transwell insert was coated with 2 mg/mL of Matrigel (Corning Inc, New York, NY, USA). Cells were seeded into the upper chamber of the transwell in a 200-µL volume of medium with serum at a density of 2-5 × 10 5 cells/mL. The lower chamber was filled with medium containing 10% serum. The cells were incubated for 12 or 24 h at 37 • C. After incubation, the medium was aspirated and the cells were washed twice with Dulbecco's phosphate-buffered saline (DPBS). The cells were fixed with 100% methanol for 30 min at room temperature, followed by washing and staining with 0.2% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) diluted in DPBS for 30 min at room temperature in the dark. The invasive cells were counted under a light microscope (×100). Apigenin was added into the lower chamber of the transwell.

Scratch Motility Assay
Briefly, cells were plated at a density of 2 × 10 5 cells/well in a 24-well plate and incubated overnight. A wound was created by making a scratch (1 mm width). The cells were washed twice with DPBS and incubated with fresh medium. Cells were treated with several concentrations (0, 10, 20, and 40 µM) of apigenin for different time points (0, 6, 12, and 24 h). Images were captured under a light microscope (×100).

Western Blot Analysis
Cells were seeded at a density of 5 × cells into the dorsum next to the right hind leg. After 14 days, the tumors of mice were measured every other day using standard caliper; tumor size was calculated using the formula (tumor length (mm) × width (mm) 2 )/2, as previously described [65,66]. To evaluate the antitumor effects of apigenin, another set of mouse group were prepared and they were subcutaneously implanted with 5 × 10 6 MDA-MB-231 cells. After 14 days, mice were orally administrated with drinking water or two doses of apigenin (25 or 50 mg/kg) for another 2 weeks.

Immunohistochemistry (IHC) Staining of Tumor Tissues
Tumor tissues excised from mice were frozen with Frozen Section Compound (Leica, Wetzlar, Germany) and stored at −80 • C. Tumor tissues were sliced at 0.5-mm widths and placed on slides. The tissue sections were hydrated in 70% ethanol for 5 min and permeabilized in 3% hydrogen peroxide (H 2 O 2 ) diluted in methanol for 10 min. Tissues were washed in tap water for 10 min, followed by three washes in 1× PBS for 5 min and incubation in a blocking buffer (10% bovine serum albumin (BSA) + 0.05% Tween-20 in PBS) at room temperature. After 1 h, the tissues were blotted with the first antibody in PBST overnight at 4 • C, followed by the treatment with the second and third antibodies (;ABC kit, VECTASTAIN, Burlingame, CA, USA) for 2 h at room temperature. The blotted tissues were washed thrice in 1× Tris-buffered saline (TBS) for 5 min. Tissues were then stained with DAB (Vector Laboratories, Burlingame, CA, USA) and visualized under a microscope (×200). The first antibodies, pAkt (#4060), pSTAT3 (#9134), and N-cadherin (#13116), were purchased from Cell signaling technology (CST, Danvers, MA, USA), while the second antibody used was mouse anti-rabbit IgG-HRP (#sc-2537, Santa Cruz, Dallas, TX, USA).

Statistical Analysis
Data were processed using Microsoft Excel, and the results are presented as mean ± standard deviation (SD). Comparisons of several means were performed with one-way analysis of variance followed by Fisher's Least Significant Difference as a post hoc test. Differences among groups were considered significant at a value of p < 0.05.

Conflicts of Interest:
The authors have no conflicts of interest.