Oroxylin A Suppresses the Cell Proliferation, Migration, and EMT via NF-κB Signaling Pathway in Human Breast Cancer Cells

Oroxylin A is a natural extract and has been reported to have a remarkable anticancer function. However, the mechanism of its anticancer activity remains not quite clear. In this study, we examined the inhibiting effects of Oroxylin A on breast cancer cell proliferation, migration, and epithelial-mesenchymal transition (EMT) and its possible molecular mechanism. The cytoactive and inflammatory factors were analyzed via Cell Counting Kit-8 assay and ELISA assay, respectively. Flow cytometry and western blotting were used to assess the cell proliferation. In addition, a wound healing assay and transwell assay were used to detect cell invasion and migration. qRT-PCR and western blot were employed to determine the effect of Oroxylin A on the EMT formation. Moreover, expression level of protein related to NF-κB signaling pathway was determined by western blot. The results revealed that Oroxylin A attenuated the cytoactivity of MDA-MB-231 cells in a dose- and a time-dependent manner. Moreover, cell proliferation, invasion, and migration of breast cancer cells were inhibited by Oroxylin A compared to the control. The mRNA and protein expression levels of E-cadherin were remarkably increased while N-cadherin and Vimentin remarkably decreased. Besides, Oroxylin A suppressed the expression of inflammatory factors and NF-κB activation. Furthermore, we also found that supplement of TNF-α reversed the effects of Oroxylin A on the cell proliferation, invasion, migration, and EMT in breast cancer cells. Taken together, our results suggested that Oroxylin A inhibited the cell proliferation, invasion, migration, and EMT through inactivating NF-κB signaling pathway in human breast cancer cells. These findings strongly suggest that Oroxylin A could be a therapeutic potential candidate for the treatment of breast cancer.


Introduction
Breast cancer is one of the most common cancers and the second leading cause of cancer death in women. Even if advances in inchoate diagnosis and treatment of breast cancer have been made, approximately 30% of patients with breast cancer will develop cureless metastatic disease [1,2] in which the median survival times range from 1 to 4 years [3]. Triple negative (TN) breast cancer is a particular type of breast cancer, which lacks the expression of oestrogen receptor (ER), progesterone receptor (PgR), and ERBB2. The features contribute to biological aggressiveness, worse prognosis, and lack of a therapeutic target compared to other subtypes [4].
Thus, triple negative breast cancer is a huge challenge for modern medicine.
The relationship between cancer and inflammation has been studied for many years. Inflammation in the tumor microenvironment plays an extremely important role in tumor progress [5]. In cancer-related inflammation, major inflammatory cytokines (such as IL-1 , IL-6, and TNF-) and transcription factors (such as NF-B and STAT3) are recognized as key endogenous factors [6]. It has been proved that NF-B participates in initiation and progression of tumor in tissues where cancer-related inflammation easily occurs [7]. NF-B is not only a key coordinator of inflammation and innate immunity, but also regarded as an 2 BioMed Research International important endogenous tumor promoter. In tumor cells, NF-B induces the expression of antiapoptotic genes to accelerate cell growth and activates relative inflammatory cytokines, adhesion molecules, and angiogenic factors to expedite the proliferation of tumor cells [8][9][10][11].
Oroxylin A (OA) is natural compound extracted from Scutellariae radix. It has been proved that the compound exerts broad functions, including anticancer, antiinflammation, neuroprotective, and anticoagulation [12]. The functions have attracted much attention wisely, especially the antitumor activity. OA performs a strong anticancer activity by inducing apoptosis, inhibiting metastasis and invasion, reversing multidrug resistance, suppressing angiogenesis, and so on [13]. In a previous study, OA could suppress the expression of Bcl-2 and procaspase-3 protein in HepG2 cell significantly to exert proapoptotic effect [14]. OA suppressed cell invasion and had antimetastatic effect through downregulating the expression of MMP2 and MMP9 in MDA-MB-435 human breast cancer cells [15]. Moreover, OA prevented inflammation-related tumor through downregulation of inflammatory gene expression by inhibiting NF-B signaling pathway [5]. Further, OA inhibited activated transcription factor NF-B, reducing the expression of LPSinduced iNOS and COX-2 in RAW 264.7 cells [16]. A large number of researches about the anticancer effects of OA have been studied. However, it is unclear whether and how OA can exert the anticancer effects on TN breast cancer by NF-B signaling pathway.
Thus, we explored the inhibiting effects of OA on the proliferation, migration, and EMT on MDA-MB-231 human breast cancer cells in this study and reported the protective effects of OA via suppressing NF-B signaling pathway.

Materials and Methods
. . Reagents and Antibodies. Oroxylin A was bought from Novartis Pharmaceuticals (Basel, Switzerland) and dissolved in dimethyl sulfoxide (DMSO) as a stock solution. Primary antibodies against CDK2, CyclinE, p27, p-p65, p65, and COX-2 were obtained from Cell Signaling (Beverly, MA, USA). Primary antibodies against E-cad, N-cad, Vimentin, p-I B, I B, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA). Secondary antibodies conjugated with horseradish peroxidase were obtained from Santa Cruz Biotechnology. All other reagents were purchased from Sigma-Aldrich (Louis, MO, USA).
. . Cell Viability Assay. The effect of OA on MDA-MB-231 cell growth was measured by CCK-8 assay. Cells were seeded into 96-well plates and treated with OA at various concentrations (0 M, 10 M, 20 M, and 40 M). Each concentration was repeated five times. After 24 and 48 hours, 10 l CCK-8 (Dojindo, Kumamoto, Japan) reagent was added to each well. Plates were then estimated on a microplate reader after 2 h incubation. Absorbance was measured at 450 nm using microplate spectrophotometry.
. . Flow Cytometric Analysis. Flow cytometry was used to explore the cell proliferation. Treated with OA (20 M) or/and TNF-for 24 h, the cells were harvested and centrifuged at 300 g for 5 min. Then, the cells were stained with a Cell Cycle Detection Kit (KeyGene, Holland) according to the manufacturer's instructions. Finally, the samples were analyzed with the FACSCalibur Flow Cytometer (BD Bioscience, USA) and BD CellQuest software (BD Bioscience, USA).
. . Wound Healing Assay. Cell migration capacity was calculated by a wound healing assay. Cells were seeded in 6-well plates and allowed to growth to 80-90% confluence. The cell monolayers were then wounded with white pipette tips and washed four times with phosphate-buffered saline to remove cell debris. The cells were then incubated in medium with OA (20 M) or/and TNF-for 24 h. Cell migration into the wound surface and number of migrated cells were determined under an inverted microscopy. Five randomly chosen fields were analyzed in each well.

. . Cell Invasion
Assay. An invasion assay was implemented to examine tumor invasion using transwell chamber (6.5 mm in diameter, 8 m pore-size, Corning Costar, Cambridge, MA). Firstly, the transwell chambers were first loaded with 0.1mL of matrigel (Becton Dickinson, Bedford, MA) at 37 ∘ C for 1 h. MDA-MB-231 cells were incubated in DMEM containing 1% FBS and treated with OA (20 M) or/and TNFfor 24h. Then cells were trypsinized and suspended at a final concentration of 5×10 5 cells/mL in DMEM containing 1% FBS. Cell suspensions were then loaded into the upper compartment, and medium with 10% fetal bovine serum was added in the lower compartment. Incubated at 37 ∘ C in 5% CO 2 for 24 h, cells on the upper surface were wiped off with a cotton swab. Then, invaded cells on the lower surface were fixed, stained, and counted under a microscope. Five randomly chosen fields were counted for each group.
. . ELISA Assay. IL-6, IL-8, and TNF-secretion in cell supernatants was measured by ELISA according to the manufacturer's instructions. 6 replicates were established for each group and results were from triplicate experiments.
. . Statistical Analysis. SPSS 20.0 statistical analysis software was used to analyze the experimental data. The results were expressed as mean ± SD. Statistical comparisons were made by two-tailed Student's t-test or one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant. indicated that the protein expression of CDK2 and Cyclin E was extremely decreased compared to the control, while p27 had the opposite results (Figure 2(c)).

. . Effect of OA on the Migration and Invasion of Breast
Cancer Cells. Wound healing assay and transwell assay were applied for investigating the effect of OA on the migration and invasion of MDA-MB-231 cells, respectively. The results showed that cells in the control rapidly spread to the wound area after 24 h incubation, whereas very few cells in OA group spread forward (Figure 3(a)). Additionally, the results from invasion assay revealed that numerous cells invaded through the matrigel in the control, while the number of cells invading to the lower side significantly decreased in the treatment of OA (Figure 3(c)). And the relative cell invasive and migration rates in OA group were both dramatically lower than the control (Figures 3(b) and 3(d)).
. . Effect of OA on EMT Formation. To examine the effect of OA on the formation of EMT, the expression of Ecadherin, N-cadherin, and Vimentin in MDA-MB-231cells was detected by qRT-PCR and western blotting analysis. It was shown that the mRNA and protein expression levels of E-cadherin remarkably elevated, whereas the expression of N-cadherin and Vimentin was considerably decreased when compared to the control (Figure 4). These results suggest that EMT formation could be suppressed by OA.

. . Effect of OA on the Expression of Inflammatory Cytokines.
To explore whether inflammatory cytokines were inhibited in breast cancer in response to OA, the levels of IL-6, IL-8, and TNF-were detected by ELISA. The results showed that OA markedly suppressed the protein expressions of IL-6, IL-8,  . . Inhibitory Effect of OA on NF-B Signaling Pathway. NF-B signaling pathway has been recognized to play vital roles in the regulating of inflammatory pathways in the tumor microenvironment. Thus, the effects of OA on NF-B activity were assessed by western blotting. As shown in Figure 6, OA did not affect the total amounts of p65 and I B but significantly inhibited the phosphorylation of them. Furthermore, the protein level of COX-2 was markedly decreased after treatment with OA compared to the control. These results indicated that OA effectively attenuated activation of the NF-B pathway with the repression of COX-2 in breast cancer cells.

. . TNF-Reverses the Functions of OA on TN Breast
Cancer Cells. TNF stimulates the growth of normal mammary epithelial cells and the mammary cancer cells [18]; thus we studied the effects of TNF-on the proliferation, migration, and EMT of breast cancer cells with or without OA. Flow cytometry demonstrated that OA suppressed and TNF-promoted cell proliferation compared to the control, while the OA +TNF group showed that OA-inhibited cell proliferation was promoted by TNF- (Figures 7(a) and  7(b)). In addition, the results from wound healing assay and transwell assay showed that OA suppressed and TNFpromoted cell migration and invasion, while treatment of OA +TNF promoted OA-inhibited cell migration and invasion (Figures 7(c) and 7(d)) than the OA group. Furthermore, the results from a western blot revealed that OA inhibited and TNF-enhance EMT phenotype formation of MDA-MB-231 cells while the OA +TNF group reversed the EMT formation inhibited by OA (Figure 8). Taken together, these findings suggest that the effects of OA on the suppression of proliferation, migration, and EMT formation on breast cancer cells could be reversed by TNF-.

Discussion
OA, one of the main bioactive flavonoids of Scutellariae radix, exerts obvious antitumor activities [19]. It has been proved that OA inhibited breast cancer progression by proapoptosis, antiproliferation, antiangiogenesis, and so on [15,[20][21][22]. However, the mechanism of OA defenses against breast cancer remains not to be fully elucidated.
In the present study, we found that OA significantly reduced the cytoactivity of MDA-MB-231 cells in a dosedependent manner. Moreover, compared with the control, OA inhibited proliferation, migration, and EMT formation and the expression of inflammatory cytokines of breast cancer cells obviously. The experiments also showed that OA inhibited NF-B activation by suppressing the phosphorylation of p65 and I B, as well as the expression of COX-2. Furthermore, it was found that TNF-reversed the effects of OA on breast cancer cells.
The inflammatory antitumor response plays a critical role in inhibiting tumor growth in human cancers [23]. NF-B is one of the most important regulators of proinflammatory gene expression [24], contributing to the progress of tumor. Recent studies have revealed that NF-B is involved in regulation of expression of COX-2 [25]. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme, regulates the formation of carcinogens, apoptosis, and angiogenesis in tumor progression [26]. Previous study showed that Oroxylin A modulated NF-B signaling to hold back inflammation related tumor [5]. Moreover, it is reported that Oroxylin A suppressed LPS-induced expression of iNOS and COX-2 related to inflammatory processes by inhibition of NF-B activation in macrophages [27]. In the present study, we found that OA significantly suppressed the activation of NF-B signaling in MDA-MB-231cells by suppressing phosphorylation of I B and p-65, which are important steps in NF-B activation. What is more, the protein expression of COX-2 was found to be decreased, which may be associated with inhibition of NF-B signaling.    Chronic inflammation is a main activator in the metastatic cascade [28]. Epithelial-mesenchymal transition (EMT) is a complex process, losing cell-cell adhesion, contributing to cancer invasion and metastasis. The results of the present study indicated that OA inhibited the EMT formation in breast cancer cells by upregulation of Ecadherin levels and downregulation of N-cadherin and Vimentin levels. EMT programs stimulate the production of proinflammatory factors in cancer environment while inflammation is a potent inducement for EMT in tumors [9]. What is more, NF-B activity promotes the collection of inflammatory cells and secretion of proinflammatory cytokines like IL-1, IL-6, IL-8, and TNF- [29]. The present results demonstrated that OA markedly suppressed the secretion of IL-6, IL-8, and TNF-protein in MDA-MB-231cells. These findings indicate OA has a repressive influence on EMT and inflammation progress in breast cancer cells.
TNF-has been proved to have pleiotropic effects on cells death, growth, or differentiation under different concentrations and context [30,31]. TNF has anticancer and anti-infection effects in high concentrations because of its cytotoxic effects on the tumor vessel [32]. However, it is shown that TNF can promote the proliferation and migration of normal epithelial cell and breast cancer cells both at low doses [33,34]. One study demonstrated that TNFsecreted by tumor cells stimulated the growth of malignant mammary epithelial cells, which contributed to mammary tumorigenesis in neu/erbB2 transgenic mice [18]. Another study showed that TNF-can promote transendothelial migration of breast cancer via upregulation of LOX [35]. In the present study, we found OA-suppressed cell proliferation, migration, invasion, and EMT phenotype formation were promoted by TNF-, leading to aggravation of breast cancer process, which is consistent with previous reports. On these bases, we hypothesized that the blockade of TNF-may enhance the antitumor effects of OA for breast cancer patients and this speculation warrants further investigation.

Conclusion
In summary, the present study showed that OA could suppress cell proliferation, migration, invasion, and EMT formation and downregulate the expressions of inflammatory cytokines. Furthermore, OA exerted protective effect on breast cancer by inhibiting NF-B signaling pathway. The results provided a theoretical foundation for Oroxylin A as a potential drug for the treatment of breast cancer.

Data Availability
The data used to support the findings of this study are included within the article.