Biochemical and Biophysical Research Communications 485 (2017) 76e81

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Biochemical and Biophysical Research Communications

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Inhibition of MMP-2-mediated cellular invasion by NF-kB inhibitor

DHMEQ in 3D culture of breast carcinoma MDA-MB-231 cells: A
model for early phase of metastasis
Tamami Ukaji a, Yinzhi Lin a, Shoshiro Okada b, Kazuo Umezawa a, *
a
Department of Molecular Target Medicine, Aichi Medical University School of Medicine, 1-1 Yazako-Karimata, Nagakute 480-1195, Japan
b
Department of Pharmacology, Aichi Medical University School of Medicine, 1-1 Yazako-Karimata, Nagakute 480-1195, Japan

a r t i c l e i n f o a b s t r a c t

Article history: The three-dimensional (3D) culture of cancer cells provides an environmental condition closely related
Received 27 January 2017 to the condition in vivo. It would especially be an ideal model for the early phase of metastasis, including
Accepted 5 February 2017 the detachment and invasion of cancer cells from the primary tumor. In one hand, dehydrox-
Available online 8 February 2017
ymethylepoxyquinomicin (DHMEQ), an NF-kB inhibitor, is known to inhibit cancer progression and late
phase metastasis in animal experiments. In the present research, we studied the inhibitory activity on
Keywords:
the 3D invasion of breast carcinoma cells. Breast carcinoma MDA-MB-231 cells showed the most active
3D culture
invasion from spheroid among the cell lines tested. DHMEQ inhibited the 3D invasion of cells at the 3D-
Invasion
MDA-MB-231 cells
nontoxic concentrations. The PCR array analysis using RNA isolated from the 3D on-top cultured cells
DHMEQ indicated that matrix metalloproteinase (MMP)-2 expression is lowered by DHMEQ. Knockdown of
MMP-2 MMP-2 and an MMP inhibitor, GM6001, both inhibited the invasion. DHMEQ was shown to inhibit the
promoter activity of MMP-2 in the reporter assay. Thus, DHMEQ was shown to inhibit NF-kB/MMP-2-
dependent cellular invasion in 3D-cultured MDA-MB-231 cells, suggesting that DHMEQ would inhibit
the early phase of metastasis.
© 2017 Elsevier Inc. All rights reserved.

1. Introduction stages, such as attachment of cells and formation of secondary

Metastasis inhibitors without cellular toxicity should be useful
for anticancer agents with few side effects. Until recently, the cell-
based study of cancer growth and metastasis has been carried out
mainly in monolayer 2D cultured cells, as with the wound healing
assay and Matrix chamber assay [1]. However, cancer cells grow
and metastasize in the body with the 3D organization interacting
with neighboring cancer cells. Recently, invasion of cancer cells has
become available for study in the 3D condition [2]. In the 3D in-
vasion assay, firstly, multicellular spheroids are prepared. Then,
they are embedded in the extracellular matrix, such as the base-
ment membrane extract (BME) or collagen I [3].
The process of metastasis includes detachment of the cells from
the primary tumor, migration and invasion, transportation to a
remote place through the vessels, attachment and growth of the
secondary tumor. There exist many in vivo models of the later
* Corresponding author.
E-mail address: umezawa@aichi-med-u.ac.jp (K. Umezawa).
tumor. However, earlier metastasis models which study the
detachment from the primary tumor and invasion are very rare.
The 3D culture of cancer cells should be an ideal model of these
earlier processes of metastasis (Fig. 1).
On one hand, we previously discovered DHMEQ (Fig. 2A) by
molecular design as an inhibitor of NF-kB [4]. It binds to the specific
cysteine residues of NF-kB components, including p65, RelB, cRel,
and p50, to inhibit their DNA binding [5,6]. It was shown to
ameliorate various animal models of cancer and inflammation
without showing any side effect [7]. Previously, it was shown to
inhibit Matrigel invasion in cultured ovarian clear cell carcinoma
RMG1 cells [8]. Intraperitoneal administration of DHMEQ inhibited
liver metastasis of pancreatic carcinoma AsPC-1 cells injected via
the portal vein [9]. However, the effect of DHMEQ on the earlier
metastatic process, on cell detachment from the primary tumor and
on invasion, has never been tested.
In the present research we studied the effect of DHMEQ on the
invasion of 3D-cultured cancer cells. DHMEQ inhibited the 3D-in-
vasion of breast carcinoma MDA-MB-231 cells without toxicity, by
inhibition of MMP-2 expressions.

http://dx.doi.org/10.1016/j.bbrc.2017.02.022
0006-291X/© 2017 Elsevier Inc. All rights reserved.
 T. Ukaji et al. / Biochemical and Biophysical Research Communications 485 (2017) 76e81 77

Fig. 1. Time-course of 3D invasion in MDA-MB-231 cells. The cells were cultured in the 96-well round bottom well, and treated as in illustration.

Fig. 2. Inhibition of invasion and NF-kB by DHMEQ in 2D-cultured MDA-MB-231 cells. (A) Structure of NF-kB inhibitor ()-DHMEQ. Racemic DHMEQ was employed in the present
research. (B) The cells were incubated with DHMEQ for 24 h and the viability was assessed by MTT. (C) Inhibition of cellular invasion in 2D cultured MDA-MB-231 cells. (D) In-
hibition of cellular NF-kB activity by DHMEQ in MDA-MB-231 cells. (E) Inhibition of IL-6 secretion by DHMEQ in MDA-MB-231 cells. The 2D-cultured MDA-MB-231 cells were
incubated with DHMEQ for 6 h *, P < 0.05. **, P<0.01. (n ¼ 3).

2. Materials and methods 2.2. Cell culture

2.1. Materials
DHMEQ was synthesized in our laboratory, as previously
described [10]. MMP inhibitor GM6001 was purchased from Abcam
(Cambridge, United Kingdom). Phorbol 12-myristate 13-acetate
(PMA) was purchased from Sigma-Aldrich (St. Louis, MO). The
ELISA kit for IL-6 was purchased from R&D Systems (Minneapolis,
MN).
Human breast cancer MDA-MB-231 cells were purchased from
RIKEN Cell Bank (Tsukuba, Japan). The cells were cultured in Dul-
becco's modified Eagle's medium (DMEM), supplemented with 10%
(v/v) fetal bovine serum and penicillin/streptomycin at 37
C in a
humidified incubator with 5% CO2.
2.3. Cell viability assay

Cell viability was evaluated by MTT assay performed as

78 T. Ukaji et al. / Biochemical and Biophysical Research Communications 485 (2017) 76e81
previously reported [1].
2.4. NF-kB binding assay
NF-kB binding activity was measured as previously reported
[11] with slight modifications. The 2D-cultured cells were grown in
100 mm dishes. The following day, the cells were treated with
DHMEQ for 2 h.
2.5. Wound healing assay and cell invasion assay
Wound healing and Matrigel cell invasion assays were per-
formed as previously reported [1].
2.6. RNA isolation and semi-quantitative RT-PCR analysis
These assays were performed as previously reported [1]. The
primers used for semi-quantitative RT-PCR, the number of cycles,
and the annealing temperature, as follows: MMP-2, 50 - AAG TCT
GGA GCG ATG TGA CC -3’ (forward) and 50 - GAG TCC GTC CTT ACC
GTC AA -3’ (reverse), 34 cycles, 58
C; and b-actin, 50 - CTT CTA CAA
TGA GCT GCG TG-3’ (forward) and 50 - TCA TGA GGT AGT CAG TCA
GG-3’ (reverse), 21 cycles, 58
C. PCR products were electro-
phoresed on 2% agarose gels, stained with ethidium bromide, and
visualized with a UV illuminator.
2.7. 3D spheroid proliferation assay
The 3D spheroid proliferation assay kit was purchased from
Trevigen Inc. (Gaithersburg, MD) and used according to the man-
ufacturer's protocol. After incubation with chemical for several
days, proliferation was assessed by MTT.
2.8. 3D spheroid cell invasion assay
3D spheroid cell invasion assay kit was purchased from Trevigen
Inc. and used according to the manufacturer's protocol. For analysis,
spheroids were photographed in each well every 24 h. Spheroid
Area were measured using Image J (National Institute of Health).
2.9. On-top 3D cell culture and harvest
The 3D on-top method was followed as previously reported [12].
3D culture Matrix Reduced Growth Factor Basement Membrane
Extract (BME) was purchased from Trevigen Inc. Cells were cultured
for 4 days and the medium was changed every 2 days. Then the
medium was replaced to the new one including DHMEQ and in-
cubation was for 24 h. Cell supernatants were used for the ELISA
assay. The 3D culture matrix cell harvesting kit (Trevigen Inc.) was
used according to the manufacturer's protocol, and harvested cell
lysates were used for array analysis and RT-PCR.
2.10. PCR array
Total RNA was extracted from 3D-cultured MDA-MB-231 cells
using RNeasy mini (Qiagen, Hilden, Germany). The cDNA was added
to the qPCR Master Mix and the aliquot mixture across the Human
Tumor Metastasis PCR Array (Qiagen). Data analysis was carried out
by the comparative Ct method.
2.11. Knockdown of MMP-2 by siRNA treatment
siMMP-2 for human (sc-29398) and small interfering RNA-A as
control (sc-37007) were purchased from Santa Cruz Biotechnology
Inc. (Santa Cruz, CA). Transfection of cells with siRNAs was carried
out as previously reported [1]. The efficiency of transfection for 96 h
was determined by mRNA expression.
2.12. Plasmid construction
The DNA fragment (bp 1259 to þ57) upstream from the
transcription initiation site of human MMP-2 was amplified by PCR
with KOD plus DNA polymerase (Toyobo, Tokyo, Japan). A human
MMP-2 promoter was a gift from Dr. Etty Benveniste (Addgene
plasmid # 53432) and used as the template [13]. The pGL3-Basic
vector (Promega, Madison, WI) was used to construct expression
vectors by subcloning PCR-amplified DNA of human MMP-2 pro-
moter into the XhoI/HindIII site of the pGL3-Basic vector. The clone
was confirmed by DNA sequencing.
2.13. DNA transfection and luciferase assay
MDA-MB-231 cells were seeded at a density of 4  105/6-well
plate and transiently transfected with pGL3-Basic or pGL3-MMP-
2 promoter vector and pRL-TK vector (2 mg and 100 ng, respec-
tively) following the standard Lipofectamine LTX (Life Technolo-
gies, Carlsbad, CA) transfection protocols. Reporter assays for
promoter activity were carried out using the Dual-Glo Luciferase
Reporter Assay System (Promega). Firefly luciferase activity was
normalized to Renilla luciferase in each well.
2.14. Statistical analysis
All experiments were performed independently at least three
times. Differences between groups were examined for significance
with ANOVA and/or the Student's t-test where appropriate.
3. Results
3.1. Selection of cell line and time-course of 3D invasion
We evaluated 5 highly malignant cell lines including human
breast carcinoma MDA-MB-231, human fibrosarcoma HT-1080,
human pancreatic carcinoma AsPC-1 [9], human ovarian clear cell
carcinoma ES-2 [1], and mouse melanoma B16-F10 [14] cells.
Among them, ES-2, AsPC-1, and B16-F10 cells showed no invasion,
and HT-1080 cells weakly showed the invasion. MDA-MB-231
shows 3D detachment/invasion clearly, and we mainly employed
MDA-MB-231 cells for further experimentation. Fig. 1 shows the
time-course of 3D invasion in MDA-MB-231 cells.
3.2. Inhibition of invasion and NF-kB in 2D-cultured MDA-MB-231
cells
DHMEQ (Fig. 2A) is not toxic below 10 mg/ml (Fig. 2B) in 2D-
cultured MDA-MB-231 cells. It inhibited invasion in the Matrigel
chamber at 1e10 mg/ml (Fig. 2C). MDA-MB-231 cells possess
constitutively activated NF-kB [14], which was inhibited by DHMEQ
(Fig. 2D). IL-6 is dependent on NF-kB, and its secretion was also
inhibited by DHMEQ (Fig. 2E).
3.3. Inhibition of invasion and MMP-2 expression in 3D-cultured
MDA-MB-231 cells
Firstly, we measured the toxicity of DHMEQ in 3D-cultured cells.
As shown in the illustration in Fig. 3A, DHMEQ was added after
spheroid formation. DHMEQ did not lower the viability even at
50 mg/ml (Fig. 3A). Next, DHMEQ was shown to inhibit 3D-invasion
at 10e30 mg/ml, as shown in Fig. 3B.
Next, we studied the mechanism of inhibition by DHMEQ
 T. Ukaji et al. / Biochemical and Biophysical Research Communications 485 (2017) 76e81 79

Fig. 3. DHMEQ inhibits 3D invasion, MMP-2 expression and NF-kB-dependent IL-6 secretion. (A) Effect of DHMEQ on the proliferation in 3D-cultured MDA-MB-231 cells. The
experimental procedure is shown in the illustration. (B) Inhibition of 3D invasion by DHMEQ in MDA-MB-231 cells. Spheroids were incubated with DHMEQ for 72 h *, P < 0.05.
(n ¼ 3) (Scale bar, 500 mm) (C) Preparation of 3D-cultured cells for the experiments with 3D on-top method. (D) Inhibition of MMP-2 mRNA expression by DHMEQ in 3D-cultured
cells. (E) Inhibition of IL-6 secretion by DHMEQ in 3D-cultured cells. The 3D-cultured cells were incubated with DHMEQ for 24 h *, P < 0.05. **, P<0.01. (n ¼ 3).

employing a PCR array analysis targeting tumor metastasis. As
shown in the illustration in Fig. 3C, we employed the BME gel and
cultured cells in the 3D on-top method [12]; then, DHMEQ was
added for 24 h. Only a few gene expressions were found to be
lowered in the array analysis. Among them, DHMEQ inhibited the
expression of MMP-2 about 60%. Then, we confirmed the decrease
independently by PCR employing the mRNA from the 3D-cultured
cells. As a result, DHMEQ inhibited the MMP-2 expression (Fig. 3D).
Since the amount of nuclei from the 3D-cultured cells was not
enough for NF-kB analysis, we measured the downstream IL-6
secretion. DHMEQ lowered the secretion of IL-6 (Fig. 3E).
3.4. Inhibition of 3D-invasion by MMP-2 siRNA or MMP inhibitor
To confirm that MMP-2 is the functional target for the inhibition
of invasion, we have knocked down MMP-2 by siRNA. Expression of
MMP-2 was suppressed even after the formation of the spheroid
(Fig. 4A). Knockdown of MMP-2 did not affect 3D cell proliferation
(Fig. 4B), but the 3D invasion was inhibited by knockdown of MMP-
2 (Fig. 4C). Furthermore, DHMEQ did not lower the 3D invasion in
siMMP-2-treated cells (Fig. 4D). GM6001 is an inhibitor of several
MMPs, including MMP-2. It showed no toxicity even at 30 mM
(Fig. 4E), and also inhibited 3D invasion at 3e10 mM (Fig. 4F).
Moreover, DHMEQ did not further lower the 3D invasion in
GM6001-treated cells, as shown in Fig. 4G. Thus, DHMEQ inhibited
3D invasion by down-regulation of MMP-2 expression via NF-kB
inhibition.
Finally, to show the inhibition of promoter activity by DHMEQ,
we constructed the reporter plasmid containing the MMP-2
promoter sequence and luciferase gene. Using this plasmid,
DHMEQ was shown to inhibit the promoter activity, as shown in
Fig. 4H.
4. Discussion
These 3D culture systems are useful for the mechanistic study of
the early steps in metastasis and for the screening of metastasis
inhibitors. Fig. 1 clearly shows relation of the 3D invasion experi-
ment and early phase of metastasis, detachment and invasion.
DHMEQ inhibited this early stage of metastasis. Moreover, it also
inhibited the 3D invasion of HT-1080 cells (data not shown).
NF-kB is often constitutively activated in cancer cells [15]
including human breast cancer MDA-MB-231 cells [16]. NF-kB of
tumor cells should activate the detachment and invasion via the
activation of MMP-2 from the primary tumor. Secretion of MMP-2
by tumor cells degrades the tissue matrix to facilitate invasion.
Moreover, the cell surface proteins that are essential for the cell-cell
attachment should be degraded by MMP-2 to facilitate the
detachment of tumor cells.
We demonstrated that DHMEQ inhibited the MMP-2 promoter
activity using the reporter plasmid (Fig. 4H). Epanchintsev et al.
also reported that NF-kB inhibitor BAY 11e7082 inhibited the
promoter activity of MMP-2, as well as migration, in MDA-MB-231
cells [17]. Thus, DHMEQ is likely to inhibit NF-kB dependent MMP-2
expression via direct transcript inhibitory activity [18].
DHMEQ has been reported to inhibit various disease models in
animal experiments thus far without any toxicity. It may be a non-
toxic anti-metastasis agent acting both at the early and late phases
80 T. Ukaji et al. / Biochemical and Biophysical Research Communications 485 (2017) 76e81

Fig. 4. DHMEQ inhibits 3D invasion via MMP-2 suppression. (A) The cells were treated with siRNA for 96 h. Expression of MMP-2 mRNA was assessed by RT-PCR. (B) Effect of siRNA
on 3D proliferation. siRNA were transfected to the cells for 24 h. Then, the cells were seeded into the plate to form spheroids. Proliferation of spheroid for 72 h was assessed by MTT.
(C) Inhibition of 3D invasion by MMP-2 siRNA. Cells were observed by phase-contrast microscopy. (D) Effect of DHMEQ on invasion siMMP-2-treated cells. The siRNA-treated
spheroids were cultured with or without DHMEQ for 48 h *, P < 0.05. (n ¼ 3) (E) Effect of GM6001 on cell viability in 3D-cultured cells. (F) Inhibition of 3D-invasion by
GM6001. (G) Effect of DHMEQ on invasion in GM6001-treated cells. *, P < 0.05. (n ¼ 3). (H) Reporter assays were carried out using the MMP-2 promoter-Luc containing plasmid in
MDA-MB-231 cells. DHMEQ or PMA was added to the transfected cells for 24 h. PMA was used for the positive control. *, P < 0.05. (n ¼ 3) “n.s.” indicates not significant. All scale bars
showed 500 mm.

of metastasis.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported financially by JSPS Kakenhi Grant
Number 26350975 and 23310163 and the MEXT-Supported Pro-
gram for the Strategic Research Foundation at Private Universities,
which is for Aichi Medical University 2011e2015 (S1101027).
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.bbrc.2017.02.022.
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