# 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 TNFα-Induced NF-κB/MMP-9 Axis in MDA-MB-231 Breast Cancer Cells

Authors: Soon Young Shin; Chang Gun Kim; Seunghyun Ahn; You Jung Jung; Dongsoo Koh; Young Han Lee; Yoongho Lim
Journal: Journal of Chemistry
 (2016)

---

## Abstract

Tumor invasion is a critical step in tumor metastasis. In this study, we synthesized a novel benzochalcone derivative, (E)-1-(2-hydroxy-6-methoxyphenyl)-3-(naphthalen-2-yl) prop-2-en-1-one (DK-512), and characterized its effects on tumor invasion and its mechanism of action. We found that DK-512 strongly inhibited invasion of metastatic MDA-MB-231 breast cancer cells as revealed by a three-dimensional spheroid culture system. Tumor invasion and metastasis require disruption of the extracellular matrix. Matrix metalloproteinase-9 (MMP-9) is an endopeptidase that degrades extracellular matrix components. DK-512 significantly reduced tumor necrosis factor-α- (TNFα-) induced MMP-9 mRNA expression through the inhibition of RelA nuclear factor- (NF-) κB transcription factor. As our study was assessedin vitro, further works aboutin vivo efficacy of DK-512 are needed to gain further insights into whether DK-512 could be utilized as a scaffold for the development of antimetastatic agents for breast cancer.


---

## Body

## 1. 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-κB), 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 [11–14].Flavonoids are polyphenolic compounds consisting of a C6-C3-C6 skeleton. Chalcones (1,3-diphenyl-2-propen-1-ones), open-chain flavonoids in which the two aromatic rings are joined by a three-carbonα,β-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, 15–21]. However, the anti-invasion effects of naphtochalcone derivatives have not been well characterized.Previously, we analyzed the structure-activity relationships of flavonoids and found that methoxy-chalcones (Figure1(a)) efficiently inhibit NF-κB activity in HCT116 human colon cancer cells [22]. Further study showed the antitumor activity of a hydroxy-methoxy-benzochalcone with an additional benzene ring (Figure 1(b)) [21]. Based on these reports, we synthesized a novel hydroxy-methoxy-benzochalcone derivative, (E)-1-(2-hydroxy-6-methoxyphenyl)-3-(naphthalen-2-yl) prop-2-en-1-one (DK-512) (Figure 1(c)), and evaluated its biological effect on MMP-9 expression and invasion in highly metastatic MDA-MB-231 human breast cancer cells.Figure 1
The structures of methoxy-chalcone (a), hydroxy-methoxy-benzochalcone (b), and (E)-1-(2-hydroxy-6-methoxyphenyl)-3-(naphthalen-2-yl)prop-2-en-1-one (c).
(a)
(b)
(c)


## 2. Materials and Methods

### 2.1. Chemical Synthesis

The procedure for synthesizing DK-512 has been described elsewhere [23].


### 2.2. Cells and Chemicals

MDA-MB-231 human breast carcinoma cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Cellgro, Manassas, VA, USA). Human TNFα was purchased from Sigma-Aldrich (Cat number H8916; Saint Louis, MO, USA). The firefly andRenilla Dual-Glo™ Luciferase Assay System was purchased from Promega (Madison, WI, USA). The pRL-null plasmid, which encodesRenilla luciferase, was also purchased from Promega.


### 2.3. Three-Dimensional Spheroid Culture and Invasion Assay

A three-dimensional (3D) invasion assay was performed using a Cultrex 3D Spheroid Cell Invasion Assay kit (Trevigen, Inc., Gaithersburg, MD, USA), as described previously [16]. Invasive spreads were visualized using an EVOS f1 fluorescence microscope (Advanced Microscopy Group, Bothell, WA, USA).


### 2.4. 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 20μM) 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).


### 2.5. 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 transcription-polymerase 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 EC50 value was calculated using MasterPlex QT software (Hitachi Software Engineering America, South San Francisco, CA, USA).


### 2.6. MMP-9 Promoter Reporter Assay

The human MMP-9 promoter constructs, including wild-type pMMP9-Luc(–925/+13) and NF-κB binding site-mutated reporter pMMP9-Luc(–925/+13)mtNFκB, have been described previously [11]. Transfection of promoter reporters and luciferase reporter assays was performed as described previously [11]. Luminescence was measured using a Centro LB960 dual luminometer (Berthold Technologies, Bad Wildbad, Germany).


### 2.7. NF-κB-Dependent Transcriptional Activity Assay

Thecis-acting 5x NFκB-Luc plasmid, containing five repeats of NF-κB binding sites, was obtained from Stratagene (La Jolla, CA, USA). MDA-MB-231 cells were transfected with 0.1 μg 5x NFκB-Luc plasmid and treated with 10 ng/mL TNFα in the absence and presence of DK-512, as described previously [25]. The luciferase activities were measured with a Centro LB960 luminometer (Berthold Technologies).


### 2.8. Immunoblot Analysis

Cells were lysed in 20 mM HEPES (pH 7.2), 1% (v/v) Triton X-100, 10% (v/v) glycerol, 150 mM NaCl, 10μg/mL leupeptin, and 1 mM PMSF. Protein extracts (20 μg per sample) were separated via 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and incubated with appropriate primary and secondary antibodies. Primary antibodies against phospho-IκB (Ser32) and phospho-RelA/p65 NF-κB (Ser536) were obtained from Cell Signaling Technology (Beverly, MA, USA) and an antibody against GAPDH was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The blots were developed using an enhanced chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA). The relative protein band intensities were determined using ImageJ software.


### 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). AP value < 0.05 was considered statistically significant.


## 2.1. Chemical Synthesis

The procedure for synthesizing DK-512 has been described elsewhere [23].


## 2.2. Cells and Chemicals

MDA-MB-231 human breast carcinoma cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Cellgro, Manassas, VA, USA). Human TNFα was purchased from Sigma-Aldrich (Cat number H8916; Saint Louis, MO, USA). The firefly andRenilla Dual-Glo™ Luciferase Assay System was purchased from Promega (Madison, WI, USA). The pRL-null plasmid, which encodesRenilla luciferase, was also purchased from Promega.


## 2.3. Three-Dimensional Spheroid Culture and Invasion Assay

A three-dimensional (3D) invasion assay was performed using a Cultrex 3D Spheroid Cell Invasion Assay kit (Trevigen, Inc., Gaithersburg, MD, USA), as described previously [16]. Invasive spreads were visualized using an EVOS f1 fluorescence microscope (Advanced Microscopy Group, Bothell, WA, USA).


## 2.4. 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 20μM) 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).


## 2.5. 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 transcription-polymerase 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 EC50 value was calculated using MasterPlex QT software (Hitachi Software Engineering America, South San Francisco, CA, USA).


## 2.6. MMP-9 Promoter Reporter Assay

The human MMP-9 promoter constructs, including wild-type pMMP9-Luc(–925/+13) and NF-κB binding site-mutated reporter pMMP9-Luc(–925/+13)mtNFκB, have been described previously [11]. Transfection of promoter reporters and luciferase reporter assays was performed as described previously [11]. Luminescence was measured using a Centro LB960 dual luminometer (Berthold Technologies, Bad Wildbad, Germany).


## 2.7. NF-κB-Dependent Transcriptional Activity Assay

Thecis-acting 5x NFκB-Luc plasmid, containing five repeats of NF-κB binding sites, was obtained from Stratagene (La Jolla, CA, USA). MDA-MB-231 cells were transfected with 0.1 μg 5x NFκB-Luc plasmid and treated with 10 ng/mL TNFα in the absence and presence of DK-512, as described previously [25]. The luciferase activities were measured with a Centro LB960 luminometer (Berthold Technologies).


## 2.8. Immunoblot Analysis

Cells were lysed in 20 mM HEPES (pH 7.2), 1% (v/v) Triton X-100, 10% (v/v) glycerol, 150 mM NaCl, 10μg/mL leupeptin, and 1 mM PMSF. Protein extracts (20 μg per sample) were separated via 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and incubated with appropriate primary and secondary antibodies. Primary antibodies against phospho-IκB (Ser32) and phospho-RelA/p65 NF-κB (Ser536) were obtained from Cell Signaling Technology (Beverly, MA, USA) and an antibody against GAPDH was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The blots were developed using an enhanced chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA). The relative protein band intensities were determined using ImageJ software.


## 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). AP value < 0.05 was considered statistically significant.


## 3. Results and Discussion

### 3.1. Cytotoxic Effect of DK-512

We first tested the cytotoxic effect of DK-512 on MDA-MB-231 cells (Figure2(a)). Exponentially growing cells were treated with 1 and 5 μM DK-512 for 24 h. The cell viability was not affected by DK-512 at concentrations up to 10 μM but significantly decreased to approximately 80% at 20 μM (P
=
0.015 by Sidak’s test, n
=
3), suggesting that DK-512 is not toxic at concentrations below 10 μM.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 20μM) for 24 h. Cell viability was determined using the Cell Counting Kit-8 (CCK-8). The data shown represent the mean ± SD (n
=
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 TNFα (10 ng/mL) in the absence or presence of 5 μM DK-512. Morphology of invasive spreads was captured with an EVOS FL Auto Cell Imaging System.
(a)
(b)


### 3.2. Effect of DK-512 on Tumor Invasion

TNFα is an inflammatory cytokine that plays an important role in tumor invasion and metastasis [26–30]. 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 TNFα stimulation, invasive spreads into the surrounding matrix were substantially increased compared to the control spheroid. However, when the spheroids were preexposed to 5 μM DK-512, TNFα-induced invasive spreads were markedly inhibited. These data suggest that DK-512 has the ability to prevent the TNFα-induced invasive spread of breast cancer cells.


### 3.3. Effect of DK-512 on TNFα-Induced MMP-9 mRNA Expression

MMP-9 contributes to the invasion of epithelial tumor cells through degradation of the basement membrane [31]. TNFα is known to promote the progression of invasion and metastasis through the induction of MMP-9 in a variety of cancer cells [26–30]. We also observed that MMP-9 mRNA levels accumulated within 12 h of TNFα treatment (Figure 3(a)). However, this accumulation of MMP-9 mRNA was significantly reduced by preexposure to 5 μM DK-512 (P
=
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 TNFα treatment as compared to that observed in vehicle-treated 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 μM DK-512, respectively (EC50 = 2.27 μM calculated by MasterPlex software). These data suggest that DK-512 inhibits TNFα-induced MMP-9 mRNA expression.Figure 3
Effect of DK-512 on the inhibition of TNFα-induced MMP-9 mRNA expression. (a) RT-PCR analysis. MDA-MB-231 cells were treated with 10 ng/mL TNFα 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 (n
=
3). P
∗
∗
∗
<
0.001.P value was analyzed by Sidak’s test. (b) RT-PCR analysis. MDA-MB-231 cells were pretreated with 5 μM DK-512 for 30 min before the addition of 10 ng/mL TNFα. 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 (n
=
3). P
∗
∗
∗
<
0.001. P 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 TNFα. 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 (n
=
3). ns: not significant compared to TNFα-only treatment. P
∗
∗
∗
<
0.001.P value was analyzed by Sidak’s test.
(a)
(b)
(c)


### 3.4. Effect of DK-512 on TNFα-Induced MMP-9 Promoter Activity

To investigate whether DK-512 inhibits MMP-9 mRNA expression at the transcriptional level, MMP-9 promoter reporter plasmid, pMMP9-Luc(–925/+13), was transfected into MDA-MB-231 cells. As shown in Figure4(a), treatment with 5 μM DK-512 significantly reduced TNFα-induced MMP-9 gene promoter activity (P
=
0.0002 by Sidak’s test). NF-κB participates in TNFα-induced MMP-9 gene expression in various cancer cells [16, 33]. We also observed that TNFα-induced upregulation of MMP-9 promoter activity was significantly attenuated due to mutation of the NF-κB binding site in the MMP-9 gene promoter (P
<
0.0001 by Sidak’s test; Figure 4(b)). These data suggest that DK-512 may target NF-κB to prevent TNFα-induced MMP-9 expression.Figure 4
Effect of DK-512 and NF-κB on the inhibition of TNFα-induced MMP-9 gene promoter activity. (a) Promoter reporter assay. MDA-MB-231 cells were transfected with 0.2 μg of MMP-9 gene promoter reporter, pMMP9-Luc(–925/+13), along with 50 ng pRL-null. After 48 h, cells were pretreated with 5 μM DK-512 for 30 min before the addition of 10 ng/mL TNFα. 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 (n
=
3). P 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-κB site-mutant construct, pMMP9-Luc(–925/+13)mtNFκB, 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 (n
=
3); P
∗
∗
∗
∗
<
0.0001. P value was analyzed by Sidak’s test.
(a)
(b)


### 3.5. Effect of DK-512 on TNFα-Induced NF-κB Activity

To investigate whether DK-512 inhibits NF-κB-dependent transcriptional activity, MDA-MB-231 cells were transfected withcis-acting reporter plasmid 5x NFκB-Luc, which contains five repeats of the NF-κB binding element. As shown in Figure 5(a), TNFα increased NF-κB-dependent transcriptional activity 7.7
±
0.71-fold as compared to vehicle-treated control. The increased NF-κB-dependent transcriptional activity was significantly reduced 3.8
±
0.31- (P
=
0.0007 by Sidak’s test) and 1.4
±
0.38-fold (P
<
0.0001 by Sidak’s test) by 5 and 10 μM DK-512, respectively. Thus, it seems likely that DK-512 exhibits inhibition of TNFα-induced NF-κB-dependent transcriptional activity.Figure 5
Effect of DK-512 on the inhibition of TNFα-induced NF-κB activation. (a) MDA-MB-231 cells were transfected withcis-acting 5x NFκB-Luc plasmid along with 50 ng pRL-null. After 48 h, cells were treated with or without 10 ng/mL TNFα in the absence or presence of 5 or 10 μM DK-512. The data shown represent the mean ± SD. P
∗
∗
∗
<
0.0001 (n
=
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 μM DK-512 for 30 min before stimulation with 10 ng/mL TNFα. After 30 min, whole-cell lysates were prepared and immunoblotting was performed using the phosphospecific antibody against IκBα (Ser32) or RelA NF-κB (Ser536). The anti-GAPDH antibody was used as an internal control. The relative band intensities of p-IκB (c) and p-RelA (d) in (b) were measured using ImageJ software. The data shown represent the mean ± SD (n
=
3). P
∗
∗
∗
<
0.001.P value was analyzed by Sidak’s test.
(a)
(b)
(c)
(d)To further address the effect of DK-512 on NF-κB inhibition, MDA-MB-231 cells were treated with 10 ng/mL TNFα in the presence or absence of 5 or 10 μM DK-512. Representative immunoblot (Figure 5(b)) and quantitative densitometry (Figure 5(c)) showed that DK-512 significantly reduced the phosphorylation of both IκB on serine-32 (at 5 μM, P
=
0.0007 by Sidak’s test) and RelA NF-κB on serine-536 (at 5 μM, P
=
0.0036 by Sidak’s test).


## 3.1. Cytotoxic Effect of DK-512

We first tested the cytotoxic effect of DK-512 on MDA-MB-231 cells (Figure2(a)). Exponentially growing cells were treated with 1 and 5 μM DK-512 for 24 h. The cell viability was not affected by DK-512 at concentrations up to 10 μM but significantly decreased to approximately 80% at 20 μM (P
=
0.015 by Sidak’s test, n
=
3), suggesting that DK-512 is not toxic at concentrations below 10 μM.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 20μM) for 24 h. Cell viability was determined using the Cell Counting Kit-8 (CCK-8). The data shown represent the mean ± SD (n
=
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 TNFα (10 ng/mL) in the absence or presence of 5 μM DK-512. Morphology of invasive spreads was captured with an EVOS FL Auto Cell Imaging System.
(a)
(b)


## 3.2. Effect of DK-512 on Tumor Invasion

TNFα is an inflammatory cytokine that plays an important role in tumor invasion and metastasis [26–30]. 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 TNFα stimulation, invasive spreads into the surrounding matrix were substantially increased compared to the control spheroid. However, when the spheroids were preexposed to 5 μM DK-512, TNFα-induced invasive spreads were markedly inhibited. These data suggest that DK-512 has the ability to prevent the TNFα-induced invasive spread of breast cancer cells.


## 3.3. Effect of DK-512 on TNFα-Induced MMP-9 mRNA Expression

MMP-9 contributes to the invasion of epithelial tumor cells through degradation of the basement membrane [31]. TNFα is known to promote the progression of invasion and metastasis through the induction of MMP-9 in a variety of cancer cells [26–30]. We also observed that MMP-9 mRNA levels accumulated within 12 h of TNFα treatment (Figure 3(a)). However, this accumulation of MMP-9 mRNA was significantly reduced by preexposure to 5 μM DK-512 (P
=
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 TNFα treatment as compared to that observed in vehicle-treated 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 μM DK-512, respectively (EC50 = 2.27 μM calculated by MasterPlex software). These data suggest that DK-512 inhibits TNFα-induced MMP-9 mRNA expression.Figure 3
Effect of DK-512 on the inhibition of TNFα-induced MMP-9 mRNA expression. (a) RT-PCR analysis. MDA-MB-231 cells were treated with 10 ng/mL TNFα 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 (n
=
3). P
∗
∗
∗
<
0.001.P value was analyzed by Sidak’s test. (b) RT-PCR analysis. MDA-MB-231 cells were pretreated with 5 μM DK-512 for 30 min before the addition of 10 ng/mL TNFα. 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 (n
=
3). P
∗
∗
∗
<
0.001. P 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 TNFα. 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 (n
=
3). ns: not significant compared to TNFα-only treatment. P
∗
∗
∗
<
0.001.P value was analyzed by Sidak’s test.
(a)
(b)
(c)


## 3.4. Effect of DK-512 on TNFα-Induced MMP-9 Promoter Activity

To investigate whether DK-512 inhibits MMP-9 mRNA expression at the transcriptional level, MMP-9 promoter reporter plasmid, pMMP9-Luc(–925/+13), was transfected into MDA-MB-231 cells. As shown in Figure4(a), treatment with 5 μM DK-512 significantly reduced TNFα-induced MMP-9 gene promoter activity (P
=
0.0002 by Sidak’s test). NF-κB participates in TNFα-induced MMP-9 gene expression in various cancer cells [16, 33]. We also observed that TNFα-induced upregulation of MMP-9 promoter activity was significantly attenuated due to mutation of the NF-κB binding site in the MMP-9 gene promoter (P
<
0.0001 by Sidak’s test; Figure 4(b)). These data suggest that DK-512 may target NF-κB to prevent TNFα-induced MMP-9 expression.Figure 4
Effect of DK-512 and NF-κB on the inhibition of TNFα-induced MMP-9 gene promoter activity. (a) Promoter reporter assay. MDA-MB-231 cells were transfected with 0.2 μg of MMP-9 gene promoter reporter, pMMP9-Luc(–925/+13), along with 50 ng pRL-null. After 48 h, cells were pretreated with 5 μM DK-512 for 30 min before the addition of 10 ng/mL TNFα. 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 (n
=
3). P 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-κB site-mutant construct, pMMP9-Luc(–925/+13)mtNFκB, 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 (n
=
3); P
∗
∗
∗
∗
<
0.0001. P value was analyzed by Sidak’s test.
(a)
(b)


## 3.5. Effect of DK-512 on TNFα-Induced NF-κB Activity

To investigate whether DK-512 inhibits NF-κB-dependent transcriptional activity, MDA-MB-231 cells were transfected withcis-acting reporter plasmid 5x NFκB-Luc, which contains five repeats of the NF-κB binding element. As shown in Figure 5(a), TNFα increased NF-κB-dependent transcriptional activity 7.7
±
0.71-fold as compared to vehicle-treated control. The increased NF-κB-dependent transcriptional activity was significantly reduced 3.8
±
0.31- (P
=
0.0007 by Sidak’s test) and 1.4
±
0.38-fold (P
<
0.0001 by Sidak’s test) by 5 and 10 μM DK-512, respectively. Thus, it seems likely that DK-512 exhibits inhibition of TNFα-induced NF-κB-dependent transcriptional activity.Figure 5
Effect of DK-512 on the inhibition of TNFα-induced NF-κB activation. (a) MDA-MB-231 cells were transfected withcis-acting 5x NFκB-Luc plasmid along with 50 ng pRL-null. After 48 h, cells were treated with or without 10 ng/mL TNFα in the absence or presence of 5 or 10 μM DK-512. The data shown represent the mean ± SD. P
∗
∗
∗
<
0.0001 (n
=
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 μM DK-512 for 30 min before stimulation with 10 ng/mL TNFα. After 30 min, whole-cell lysates were prepared and immunoblotting was performed using the phosphospecific antibody against IκBα (Ser32) or RelA NF-κB (Ser536). The anti-GAPDH antibody was used as an internal control. The relative band intensities of p-IκB (c) and p-RelA (d) in (b) were measured using ImageJ software. The data shown represent the mean ± SD (n
=
3). P
∗
∗
∗
<
0.001.P value was analyzed by Sidak’s test.
(a)
(b)
(c)
(d)To further address the effect of DK-512 on NF-κB inhibition, MDA-MB-231 cells were treated with 10 ng/mL TNFα in the presence or absence of 5 or 10 μM DK-512. Representative immunoblot (Figure 5(b)) and quantitative densitometry (Figure 5(c)) showed that DK-512 significantly reduced the phosphorylation of both IκB on serine-32 (at 5 μM, P
=
0.0007 by Sidak’s test) and RelA NF-κB on serine-536 (at 5 μM, P
=
0.0036 by Sidak’s test).


## 4. Conclusion

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



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*Source: 4921717-2016-10-11.xml*