A Benzochalcone Derivative , ( E )-1-( 2-hydroxy-6-methoxyphenyl )-3-( naphthalen-2-yl ) prop-2-en-1-one ( DK-512 ) , Inhibits Tumor Invasion through Inhibition of the TNF α-Induced NF-κ B / MMP-9 Axis in MDA-MB-231 Breast Cancer Cells

1Department of Biological Sciences, College of Biological Science and Biotechnology, KonkukUniversity, Seoul 05029, Republic of Korea 2Cancer and Metabolism Institute, Konkuk University, Seoul 05029, Republic of Korea 3Division of Bioscience and Biotechnology, BMIC, Konkuk University, Seoul 05029, Republic of Korea 4Department of Applied Chemistry, Dongduk Women’s University, Seoul 02748, Republic of Korea


Introduction
Breast cancer is the most common malignancy in women and ranks second after lung cancer as a cause of cancer death in Western and Korean women [1,2].Metastatic breast cancer spreads to other parts of the body, such as the bones, liver, lungs, and brain [3].Such distant metastases are the main cause of death [4].
The extracellular matrix (ECM) is a highly dynamic structure, composed of a variety of fibrous proteins and proteoglycans that are present throughout interstitial tissue.The basement membrane is a dense, fibrous, ECM-like structure that underlies epithelia and endothelia [5].In mammary glands, the basement membrane encapsulates the glands and interacts with the luminal epithelium.The motility, invasion, and metastatic potential of epithelial tumor cells are highly correlated with the degradation of ECM in the basement membrane [5].One enzyme involved in the degradation of ECM is matrix metalloproteinase-9 (MMP-9; also known as 92 kDa type IV collagenase or gelatinase-B), a protease that efficiently degrades denatured collagen.MMP-9 is involved in degradation of collagen IV, a major component of the basement membrane [6].As such, upregulation of MMP-9 is associated with promotion of invasion, metastasis, and angiogenesis [7,8].The ubiquitous transcription factor, nuclear factor kappa B (NF-B), plays an important role in the expression of multiple genes involved in the regulation of tumor invasion and metastasis [9,10] and controls MMP-9 expression in a variety of cell types [11][12][13][14].Flavonoids are polyphenolic compounds consisting of a C6-C3-C6 skeleton.Chalcones (1,3-diphenyl-2-propen-1ones), open-chain flavonoids in which the two aromatic rings are joined by a three-carbon ,-unsaturated carbonyl system, are metabolic precursors of some flavonoids [15].Previous studies showed that chalcones display multiple biological activities, including inhibition of cell cycle progression and induction of apoptosis, in a variety of cancer types [2,[15][16][17][18][19][20][21].However, the anti-invasion effects of naphtochalcone derivatives have not been well characterized.

Chemical Synthesis.
The procedure for synthesizing DK-512 has been described elsewhere [23].
The pRL-null plasmid, which encodes Renilla luciferase, was also purchased from Promega.

Cell Viability Assay.
Cell viability was determined using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Gaithersburg, MD, USA) according to the manufacturer's instructions.Briefly, exponentially growing cells were treated with different concentrations (0, 1, 5, 10, and 20 M) of DK-512.After 24 h, CCK-8 solution was added and the absorbance was measured at 450 nm after an additional 1 h using an Emax Endpoint ELISA Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

RT-PCR and Quantitative
Real-Time PCR.Total RNA was extracted using Isol-RNA lysis reagent (NucleoZOL; Clontech, Mountain View, CA, USA) and the synthesis of cDNA was carried out using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA).Reverse transcriptionpolymerase chain reaction (RT-PCR) and real-time PCR were performed as described previously [24].A TaqMan-iQ Supermix Kit (Bio-Rad) was used with the Bio-Rad iCycler iQ thermal cycler according to the manufacturer's instructions.The TaqMan fluorogenic probes and gene-specific PCR primers for MMP-9 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been described elsewhere [24].The relative fold changes were normalized to GAPDH mRNA in the same sample.The EC 50 value was calculated using MasterPlex QT software (Hitachi Software Engineering America, South San Francisco, CA, USA).

NF-𝜅B-Dependent Transcriptional Activity Assay.
The cis-acting 5x NFB-Luc plasmid, containing five repeats of NF-B binding sites, was obtained from Stratagene (La Jolla, CA, USA).MDA-MB-231 cells were transfected with 0.1 g 5x NFB-Luc plasmid and treated with 10 ng/mL TNF in the absence and presence of DK-512, as described previously [25].The luciferase activities were measured with a Centro LB960 luminometer (Berthold Technologies).
2.9.Statistical Analysis.Statistical analysis was performed by one-way ANOVA followed by Sidak's multiple comparisons test using GraphPad Prism version 7.0 software (GraphPad Software Inc., La Jolla, CA).A  value < 0.05 was considered statistically significant.

Results and Discussion
3.1.Cytotoxic Effect of DK-512.We first tested the cytotoxic effect of DK-512 on MDA-MB-231 cells (Figure 2(a)).Exponentially growing cells were treated with 1 and 5 M DK-512 for 24 h.The cell viability was not affected by DK-512 at concentrations up to 10 M but significantly decreased to approximately 80% at 20 M ( = 0.015 by Sidak's test,  = 3), suggesting that DK-512 is not toxic at concentrations below 10 M. ), along with 50 ng pRL-null.After 48 h, cells were pretreated with 5 M DK-512 for 30 min before the addition of 10 ng/mL TNF.After 8 h, cells were harvested and luciferase activities were measured.Firefly luciferase activity was normalized to Renilla luciferase activity.The data shown represent the mean ± SD ( = 3). value was analyzed by Sidak's test.(b) Promoter reporter assay.MDA-MB-231 cells were transfected with wild-type construct, pMMP9-Luc(-925/+13), or NF-B site-mutant construct, pMMP9-Luc(-925/+13)mtNFB, along with 50 ng pRL-null.After 48 h, cells were treated and luciferase activities were measured as in (a).The data shown represent the mean ± SD ( = 3); * * * *  < 0.0001. value was analyzed by Sidak's test.

Effect of DK-512 on Tumor Invasion.
TNF is an inflammatory cytokine that plays an important role in tumor invasion and metastasis [26][27][28][29][30].To determine the effect of DK-512 on the invasive capability of MDA-MB-231 cells, we used a 3D spheroid culture system (Figure 2(b)).In basal conditions, cells were aggregated in a compact multicellular spheroid.After 5 d, invasive protrusion out of the spheroid was detectable.Upon TNF stimulation, invasive spreads into the surrounding matrix were substantially increased compared to the control spheroid.However, when the spheroids were preexposed to 5 M DK-512, TNF-induced invasive spreads were markedly inhibited.These data suggest that DK-512 has the ability to prevent the TNF-induced invasive spread of breast cancer cells.[31].TNF is known to promote the progression of invasion and metastasis through the induction of MMP-9 in a variety of cancer cells [26][27][28][29][30].We also observed that MMP-9 mRNA levels accumulated within 12 h of TNF treatment (Figure 3(a)).However, this accumulation of MMP-9 mRNA was significantly reduced by preexposure to 5 M DK-512 ( = 0.0002 by Sidak's test), while GAPDH mRNA level, an internal control, was not affected (Figure 3(b)).To more precisely measure the change in MMP-9 mRNA levels due to DK-512 treatment, quantitative real-time PCR analysis was carried out [32]. Figure 3(c) shows that the MMP-9 mRNA level was increased 28.7 ± 4.04-fold by 10 ng/mL TNF treatment as compared to that observed in vehicletreated control.This increase was dose-dependently reduced by 25.3 ± 3.79-, 21.7 ± 4.73-, 18.7 ± 5.03-, 8.67 ± 2.09-, 5.33 ± 1.53-, and 3.00 ± 1.00-fold in the presence of 0.1, 0.5, 1, 5, 10, and 20 M DK-512, respectively (EC 50 = 2.27 M calculated by MasterPlex software).These data suggest that DK-512 inhibits TNF-induced MMP-9 mRNA expression.

Conclusion
DK-512, a novel hydroxy-methoxy-benzochalcone derivative, displayed inhibition of tumor invasion against metastatic MDA-MB-231 cells.DK-512 inhibited TNF-induced MMP-9 gene expression through downregulation of NF-Bdependent transcriptional activity.These results suggest that DK-512 can be considered a potential scaffold for the development of antimetastatic agents against breast cancer cells.

Figure 2 :
Figure 2: Effect of DK-512 on cell viability.(a) Cytotoxicity of DK-512.MDA-MB-231 cells were treated with different concentrations of DK-512 (0, 1, 5, 10, and 20 M) for 24 h.Cell viability was determined using the Cell Counting Kit-8 (CCK-8).The data shown represent the mean ± SD ( = 3).ns: not significant; P value was analyzed by Sidak's test.(b) 3D invasion assay.MDA-MB-231 cell spheroid in the extracellular matrix was either left untreated or treated with TNF (10 ng/mL) in the absence or presence of 5 M DK-512.Morphology of invasive spreads was captured with an EVOS FL Auto Cell Imaging System.

Figure 3 :Figure 4 :
Figure3: Effect of DK-512 on the inhibition of TNF-induced MMP-9 mRNA expression.(a) RT-PCR analysis.MDA-MB-231 cells were treated with 10 ng/mL TNF for 12 and 18 h.Total RNA was isolated and RT-PCR was performed.GAPDH mRNA was used as an internal control.Band intensities were measured using ImageJ software.The data shown represent the mean ± SD ( = 3).* * *  < 0.001.P value was analyzed by Sidak's test.(b) RT-PCR analysis.MDA-MB-231 cells were pretreated with 5 M DK-512 for 30 min before the addition of 10 ng/mL TNF.After 12 h, total RNA was isolated and RT-PCR was performed.GAPDH mRNA was used as an internal control.Band intensities were measured using ImageJ software.The data shown represent the mean ± SD ( = 3).* * *  < 0.001. value was analyzed by Sidak's test.(c) Real-time PCR.MDA-MB-231 cells were pretreated with different concentrations of DK-512 before the addition of 10 ng/mL TNF.After 12 h, total RNA was isolated and MMP-9 mRNA levels were measured by quantitative real-time PCR.The relative fold changes were normalized to GAPDH mRNA in the same sample.The data shown represent the mean ± SD ( = 3).ns: not significant compared to TNF-only treatment.* * *  < 0.001.P value was analyzed by Sidak's test.

Figure 5 :
Figure5: Effect of DK-512 on the inhibition of TNF-induced NF-B activation.(a) MDA-MB-231 cells were transfected with cis-acting 5x NFB-Luc plasmid along with 50 ng pRL-null.After 48 h, cells were treated with or without 10 ng/mL TNF in the absence or presence of 5 or 10 M DK-512.The data shown represent the mean ± SD. * * *  < 0.0001 ( = 3).P value was analyzed by Sidak's test.(b) MDA-MB-231 cells were incubated with 0.5% FBS for 24 h, followed by pretreatment with 5 or 10 M DK-512 for 30 min before stimulation with 10 ng/mL TNF.After 30 min, whole-cell lysates were prepared and immunoblotting was performed using the phosphospecific antibody against IB (Ser32) or RelA NF-B (Ser536).The anti-GAPDH antibody was used as an internal control.The relative band intensities of p-IB (c) and p-RelA (d) in (b) were measured using ImageJ software.The data shown represent the mean ± SD ( = 3).* * *  < 0.001.P value was analyzed by Sidak's test.