A novel celecoxib analog UTX-121 inhibits HT1080 cell invasion by modulating membrane-type 1 matrix metalloproteinase

Article abstract of DOI:10.1016/j.bbrc.2019.10.092

We designed and synthesized a celecoxib derivative UTX-121 to enhance its anti-tumor activity. Similar to celecoxib, this compound could also inhibit matrix metalloproteinase (MMP)-9 activity. In addition, UTX-121 suppressed membrane-type 1 MMP (MT1-MMP)-

Full text of DOI:10.1016/j.bbrc.2019.10.092

Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
A novel celecoxib analog UTX-121 inhibits HT1080 cell invasion by
modulating membrane-type 1 matrix metalloproteinase
,
Hirari Yamahana a, Takahisa Takino b *, Yoshio Endo c, Hisatsugu Yamada a,
,
**
Takeshi Suzuki d, Yoshihiro Uto a
a Graduate School of Technology, Industrial and Social Science, Tokushima University, Tokushima, 770-8506, Japan
b Division of Education for Global Standard, Institute of Liberal Arts and Science, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan
c Central Research Resource Branch, Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan
d Division of Functional Genomics, Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed and synthesized a celecoxib derivative UTX-121 to enhance its anti-tumor activity. Similar
to celecoxib, this compound could also inhibit matrix metalloproteinase (MMP)-9 activity. In addition,
UTX-121 suppressed membrane-type 1 MMP (MT1-MMP)-mediated pro-MMP-2 activation by disturbing
the cell surface expression of MT1-MMP. UTX-121 also impeded the glycosylation of cell surface proteins,
resulting in the suppression of cell attachment to fibronectin. This inhibition by UTX-121 caused the
reduction of fibronectin-stimulated focal adhesion kinase activation, Akt activation, and cell migration.
Consequently, UTX-121 treatment significantly inhibited fibronectin-induced HT1080 cell invasion into
the Matrigel. UTX-121 may be a potent lead compound that can be used to develop a novel anti-tumor
drug.
Received 25 September 2019
Accepted 10 October 2019
Available online xxx
Keywords:
COX
MMP
MT1-MMP
Invasion
Cell adhesion
© 2019 Elsevier Inc. All rights reserved.
1. Introduction
cancer [1e3]. Deregulation of COX-2/PGE2 signaling activates
mitogen-activated protein kinase (MAPK) and phosphatidylinosi-
Celecoxib, a nonsteroidal anti-inflammatory drug, is a selective
cyclooxygenase (COX)-2 inhibitor. COX enzymes, COX-1 and COX-2,
are involved in the synthesis of prostaglandins (PG), which are
autocrine mediators of multiple cellular processes. COX-1 is
constitutively and ubiquitously expressed under basal conditions;
however, COX-2 is only rapidly and transiently expressed in
response to extracellular or intracellular stimuli, such as mitogens,
growth factors, cytokines, and infectious agents. Therefore, COX-2
has been implicated in various pathologies, such as inflammation
and carcinogenesis, by activating downstream PGE2 signaling [1].
Aberrant COX-2 expression has been observed in many cancers and
is associated with poor prognosis, particularly in colon and breast
tide 3-kinase (PI3K)/Akt pathways, which can induce COX-2
expression as a positive-feedback loop [1,4,5].
Many studies have reported on the anti-tumor activities of
celecoxib, including anti-angiogenesis activity, apoptosis-induction
activity, and anti-epithelialemesenchymal transition properties
[5]. Celecoxib treatment significantly reduces the number of colo-
rectal polyps in patients with familial adenomatous polyposis [6].
However, these anti-tumor activities of celecoxib are only observed
at higher concentrations than those needed to inhibit PG synthesis.
Moreover, celecoxib suppresses tumor growth in the absence of any
apparent COX-2 involvement [7]. These findings indicate that cel-
ecoxib exerts its anti-tumor activity through both COX-2-
dependent and COX-2-independent mechanisms [8]. However,
the COX-2-independent pharmacologic activities and mechanisms
of celecoxib are unclear thus far.
Abbreviations: COX, cyclooxygenase; DMEM, Dulbecco's modified Eagle's me-
dium; ECM, extracellular matrix; FAK, focal adhesion kinase; FBS, fetal bovine
serum; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase;
MT1-MMP, membrane-type 1 MMP; PBS, phosphate-buffered saline; PG, prosta-
glandin; PI3K, phosphatidylinositide 3- kinase.
* Corresponding author.
** Corresponding author.
Matrix metalloproteinases (MMPs), a family of Zn2þ-dependent
enzymes that are essential for extracellular matrix (ECM) remod-
eling, are involved in many biological processes such as angio-
genesis, inflammation, and cancer [9]. Membrane-type 1 MMP
(MT1-MMP) is considered to play a crucial role in tumor progres-
sion as its expression most closely correlates with the invasive
E-mail addresses: ttakino@staff.kanazawa-u.ac.jp (T. Takino), uto.yoshihiro@
tokushima-u.ac.jp (Y. Uto).
https://doi.org/10.1016/j.bbrc.2019.10.092
0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: H. Yamahana et al., A novel celecoxib analog UTX-121 inhibits HT1080 cell invasion by modulating membrane-type 1
matrix metalloproteinase, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.092

2
H. Yamahana et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
phenotype of malignant tumors among MMPs. Furthermore, MT1-
MMP inhibition suppresses tumor cell invasion both in vitro and
in vivo. Originally, MT1-MMP was identified as a tumor-specific
latent MMP-2 (pro-MMP-2) activator [10]. It activates pro-MMP-9
and pro-MMP-13 and degrade a variety of ECM components,
including type I, II, and III collagen and fibronectin. Besides ECM
degrading activity, this enzyme cleavages cell adhesion molecules,
cytokines, and others [11].
Celecoxib reduces PGE2 and MMP expression in many types of
cells, including macrophages, fibroblasts, and tumor cells [12,13].
This study aimed to evaluate whether the anti-metastatic activity of
the newly designed and synthesized celecoxib analog UTX-121 can
enhance COX-2-independent anti-tumor activity.
Fluor 680 (Molecular Probes). The gels were processed and moni-
tored using an Odyssey infrared imaging system (LI-COR, Lincoln,
NE, USA).
2.6. Western blotting
Cells were lysed in 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM
EDTA, 2 mM Na3VO4, 2 mM NaF, 1% SDS, and a protease inhibitor
cocktail (Nacalai Tesque, Kyoto, Japan). A bicinchoninic acid assay
(Pierce, Rockford, IL, USA) was used to determine the protein con-
centration. The secreted proteins were concentrated by adding
StrataClean Resin (Stratagene, La Jolla, CA, USA) to an appropriate
volume of CM and were centrifuged for 2 h. After centrifugation,
the supernatant was removed and a sample buffer was added to the
precipitated resin. The samples were separated by electrophoresis
on SDS-polyacrylamide gels and transferred to a nitrocellulose
membrane.
2. Materials and methods
2.1. Synthesis of UTX-121
(Supplementary methods).
2.2. COX-2 inhibition assay
(Supplementary methods).
2.3. Cell culture and materials
2.7. Cell surface biotinylation
Cells were washed with ice-cold PBS and incubated with 0.5 mg/
ml sulfoeNHSebiotin (Pierce) in PBS at 4 ꢀC for 30 min. After
washing, the cells were homogenized in a buffer containing 50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM phenyl-
methylsulfonyl fluoride (PMSF), 1 mM Na3VO4, 1 mM NaF, 1% Triton
X-100, and a protease inhibitor cocktail. The cell lysates were
precipitated with Streptavidin Agarose Resin (Thermo Fisher Sci-
entific, Rockford, IL, USA) after centrifugation. The precipitates were
subjected to immunoblotting.
HT1080 human fibrosarcoma cells were obtained from the
American Type Culture Collection (Manassas, VA, USA) and were
maintained in Dulbecco's modified Eagle medium (DMEM; Sigma-
Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine
serum (FBS). Type I collagen was purchased from Nitta Gelatin
(Osaka, Japan). Fibronectin (FN) was obtained from Asahi Techno
Glass (Tokyo, Japan). The synthetic MMP inhibitor BB94 was gifted
by Kotobuki Pharmaceutical Co. (Nagano, Japan). Anti-MT1-MMP,
anti-MMP-2, anti-MMP-9, and tissue inhibitors of metal-
loproteinase (TIMP)-2 antibodies were gifted by Daiichi Fine
Chemicals (Toyama, Japan). The following antibodies were used:
anti-FLAG, anti-FN, and anti-tubulin antibodies (Sigma-Aldrich);
anti-paxillin, anti-focal adhesion kinase (FAK), and anti-N-cadherin
2.8. Pulldown of glycoproteins on concanavalin A beads
Cells were washed twice with ice-cold PBS and homogenized in
a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM
CaCl2, 2 mM MnCl2, 1 mM EGTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM
NaF, 1% Triton X-100, and protease inhibitor cocktail. After centri-
fugation, the supernatants were used for pulldown with conca-
navalin
A
(Con A)-Sepharose beads (Sigma-Aldrich). The
precipitates were subjected to immunoblotting.
(BD Biosciences, Lexington, KY, USA); anti-phospho-Akt (Ser473
and anti-Akt antibodies (Cell Signaling Technology, Danvers, MA,
)
2.9. Cell adhesion assay
USA); anti-integrin b1 antibody (Santa Cruz Biotechnology, Santa
Cruz, CA, USA); and anti-phosphorylated FAK (pTyr397) antibody
(BioSource, Camarillo, CA, USA). Hoechst 33342, rhodamine-
labeled phalloidin, Alexa Fluor 680-conjugated wheat germ
agglutinin (WGA), and Alexa Fluor-labeled secondary antibodies
were purchased from Molecular Probes (Eugene, OR, USA).
Cell adhesion assay was performed as described previously [10].
In brief, 96-well plates were coated with 10
mg/ml of FN, 30 mg/ml of
type I collagen, or 100
m
g/ml of poly-L-lysine (PLL) overnight at 4 ꢀC
and blocked with 1 mg/ml bovine serum albumin (BSA). HT1080
cells were serum starved with 0.5% FBS/DMEM overnight and
treated with the inhibitors in Opti-MEM for 24 h. Cells were har-
vested as single cell suspensions and suspended for 30 min at 37 ꢀC
in Opti-MEM. The cells were allowed to adhere to the plate for
2 h at 37 ꢀC and stained with 1% crystal violet. The crystal violet
bound to the cells was eluted with 10% acetic acid and measured at
an absorption of 590 nm.
2.4. Expression plasmids
FLAG epitope-tagged MT1-MMP (MT1F) and MT1-MMP fused
with FLAG-tagged mCherry (MT1-mCherry) were constructed ac-
cording to the methods described in a previous study [14].
2.5. Gelatin zymography
2.10. Immunofluorescence staining
HT1080 cells were serum starved with 0.5% FBS/DMEM over-
night and treated with the inhibitors in Opti-MEM for 24 h. The
conditioned medium (CM) was analyzed by gelatin zymography, as
previously described [14]. MMP-2 and MMP-9 levels in CM were
measured by adding an equal volume of sample buffer. To detect
cell-bound MMP-2 and MMP-9, cells were washed twice with
phosphate-buffered saline (PBS) and dissolved in sample buffer
using sonication. The samples were separated by electrophoresis on
a SDS-polyacrylamide gel containing gelatin labeled with Alexa
HT1080 cells were transfected with MT1F plasmid, serum
starved, and treated with the inhibitors for 24 h. For cell surface
staining, the cells were incubated for 30 min at 37 ꢀC with anti-
FLAG antibody. After washing, the cells were fixed with 4% para-
formaldehyde for 15 min. Alternatively, the cells were fixed and
permeabilized with 0.5% Triton X-100 and reacted with anti-FLAG
antibody. The cells were visualized with Alexa™488-conjugated
goat anti-mouse antibody, Hoechst 33342, and rhodamine-
labeled phalloidin.
Please cite this article as: H. Yamahana et al., A novel celecoxib analog UTX-121 inhibits HT1080 cell invasion by modulating membrane-type 1
matrix metalloproteinase, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.092

H. Yamahana et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
3
2.11. FN-induced FAK and Akt activation
pro-MMP-2, and its enzymatic activity can be inhibited by TIMP-2,
but not by TIMP-1 [10]. Celecoxib and UTX-121 treatment reduced
the amount of secreted TIMP-2, suggesting that the suppression of
MT1-MMP-mediated pro-MMP-2 activation by UTX-121 is not
caused by TIMP-2 induction. To examine the effect of UTX-121 on
ectopically expressed MT1-MMP, HT1080 cells stably expressing
FLAG-mCherry-tagged MT1-MMP (HT1080-MT1mCherry) were
established. When HT1080-MT1mCherry cells were exposed to the
MMP inhibitor BB94, pro-MMP-2 activation was completely sup-
pressed and MT1-MMP protein levels were increased due to
inhibited autodegradation (Fig. 1C). UTX-121 treatment suppressed
pro-MMP-2 activation in a dose-dependent manner; however, the
ectopically expressed MT1-MMP levels remained unchanged, sug-
gesting that UTX-121 suppresses MT1-MMP-mediated pro-MMP-2
activation without affecting MT1-MMP expression. Since pro-
MMP-2 activation was executed by MT1-MMP expressed on the
cell surface, the cell-bound MMP-2 activity was examined. Cell-
bound MMP-2 activity and pro-MMP-2 activation were mostly
inhibited in the cells treated with UTX-121 (Fig. 1D). These results
indicate that UTX-121 may influence the level of cell surface MT1-
MMP, which functions as a receptor for and activator of pro-MMP-2.
The cells were detached and kept in suspension in DMEM
containing 2 mg/ml BSA for 30 min after treatment with 0.5 mg/ml
trypsin inhibitor. Subsequently, they were replated on culture
dishes coated with 10 mg/ml FN for 20 min. The cell lysates were
analyzed by western blotting.
2.12. Wound healing assay
Serum-starved HT1080 cells were treated with the inhibitors in
Opti-MEM for 24 h and allowed to adhere to FN-coated dishes for
2 h at 37 ꢀC. Subsequently, confluent cell monolayers were scraped
off. After adding Opti-MEM containing the inhibitors, wound
closure was monitored for 15 h.
2.13. Cell invasion assay
Cell invasion was assayed using a modified Boyden chamber
consisting Transwell membrane filters (Corning Costar, Cambridge,
MA, USA). The upper surfaces of the membranes were coated with
1 mg/ml Matrigel matrix (BD Bioscience) and placed in 24-well
tissue culture plates filled with 600
m
l Opti-MEM with inhibitors.
3.3. Effect of UTX-121 on MT1-MMP cell surface expression
Serum-starved HT1080 cells (2 ꢁ 105 cells) were suspended in
100
ml Opti-MEM, added to each Transwell chamber, and cultured
Latent MT1-MMP (63 kDa) is activated in a post-Goldi
for 6 h as pretreatment. Subsequently, FN was added to the lower
chambers (final concentration: 10 g/ml). After further cultivation
for 16 h, the membranes were fixed and stained. The number of
crystal violet-stained cells on the lower surface was counted under
a microscope.
compartment by proteolytic cleavage of the pro-peptide. The
active 57-kDa MT1-MMP protein on the cell surface usually un-
dergoes autocatalytic processing, yielding a membrane-tethering
and a catalytic domain-lacking 44-kDa specie [15]. Western blot-
ting analysis revealed that the 44-kDa degraded form of MT1-MMP
was decreased by UTX-121 treatment in a dose-dependent manner,
while both latent and active forms remained unchanged (Fig. 2A).
We hypothesized that UTX-121 disrupts the cell surface trafficking
of MT1-MMP. To examine this possibility, HT1080 cells were tran-
siently transfected with FLAG-tagged MT1-MMP (MT1F), and cell
surface MT1F levels were examined by immunofluorescence
staining. Compared with DMSO treatment, Cell surface staining of
MT1F in nonpermeabilized cells was significantly reduced by UTX-
121 treatment and increased by BB94 treatment. When the cells
were permeabilized, the level of MT1F signals were almost the
same in DMSO- and UTX-121-treated cells (Fig. 2B). The reduction
of cell surface MT1-MMP levels by UTX-121 was confirmed by
pulldown of biotinylated proteins. Only biotinylated cell surface
MT1-MMP, the active 57-kDa and degraded 45-kDa forms, were
pulled down with streptavidin beads (Fig. 2C). When cells were
treated with BB94, the active form of cell surface MT1-MMP was
increased and the degraded form was not pulled down. UTX-121
treatment downregulated both forms of cell surface MT1-MMP.
This was not only observed in the case of MT1-MMP but also with
UTX-121 treatment, which reduced cell surface N-cadherin but not
integrin b1. These results suggest that UTX-121 treatment perturbs
the trafficking of some cell surface proteins, including MT1-MMP.
m
3. Results
3.1. Design, synthesis, and COX-2 inhibitory activity of UTX-121
The design of novel analog was based on the structural modi-
fication of celecoxib as the lead compound by the bioisosterism
principle. The sulfonamide of celecoxib and the carboxylic acid of
compound 2 have a bioisosteric relationship, but carboxylic acid
has high polarity and poor cell permeability. We, therefore,
designed compound 3 (UTX-121) to protect compound 2 with
methyl ester (Fig. 1A). In the synthesis process, a simple synthetic
route was selected (Fig. 1A). The synthesized products were
analyzed by 1H NMR and MS. When the COX-2 inhibitory activity of
celecoxib and UTX-121 was evaluated using the COX-2 (human)
Inhibitor Screening Assay Kit as described in supplemental
methods, the IC50 values were 0.02
and UTX-121, respectively.
mM and 237 mM for celecoxib
3.2. UTX-121 suppressing MMP-9 activity and pro-MMP-2
activation
In order to study the effect of UTX-121 in tumor progression
other than COX-2/PGE2 pathway, HT1080 cells, which expressed
little COX-2 at basal condition, were used in this study. The effect of
UTX-121 on MMP activity was examined by gelatin zymography
and western blotting. Serum-starved HT1080 cells were treated
3.4. Effect of UTX-121 on cell surface proteins
Nearly all proteins that are expressed in the plasma membrane
or secreted are glycoproteins [16]. Glycosylation modifies the
structures and functions of glycoproteins, and many MMPs contain
N- or O-linked oligosaccharides [17]. Aberrant glycosylation is a
common feature of malignant change and is suspected to
contribute to the tumor progression. COX-2 has been implicated in
aberrant glycosylation in tumor cells [2]. To examine the effect of
UTX-121 on glycosylation, Con A precipitation assay was per-
formed. Treatment of cells with N-linked glycosylation inhibitor
tunicamycin significantly decreased the amount of N-cadherin and
with either celecoxib or UTX-121 at 12.5
following which the conditioned media were analyzed. Treatment
with 25 M celecoxib slightly suppressed MMP-9 production and
mM or 25 mM for 24 h,
m
pro-MMP-2 activation (Fig. 1B). UTX-121 augmented this inhibitory
effect of celecoxib on the activity of MMP-9 and MMP-2 since the
inhibition of MMP-9 production and pro-MMP-2 activation was
observed in cells treated with 12.5
mM UTX-121, which was
enhanced by treatment with 25 M UTX-121. MT1-MMP activates
m
Please cite this article as: H. Yamahana et al., A novel celecoxib analog UTX-121 inhibits HT1080 cell invasion by modulating membrane-type 1
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H. Yamahana et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 1. UTX-121 inhibits pro-MMP-2 activation. (A) Design and synthesis of UTX-121. (B) Serum-starved HT1080 cells were treated with either UTX-121 or celecoxib (CXB) at
indicated concentrations for 24 h. The conditioned media (CM) were analyzed by gelatin zymography and immunoblotted with anti-MMP-9, anti-MMP-2, and anti-TIMP-2 anti-
bodies. L, latent form; I, intermediate form; A, active form of MMP-2. (C) Serum-starved HT1080-MT1mCherry cells were treated with either UTX-121 or BB94 (2
were analyzed by gelatin zymography. The cell lysates were immunoblotted with anti-FLAG and anti-tubulin antibodies. (D) Serum-starved HT1080 cells were treated with
indicated inhibitors (25 M) for 24 h. The cells were homogenized and analyzed by gelatin zymography.
mM) for 24 h. CM
m
Please cite this article as: H. Yamahana et al., A novel celecoxib analog UTX-121 inhibits HT1080 cell invasion by modulating membrane-type 1
matrix metalloproteinase, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.092

H. Yamahana et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
5
Fig. 2. UTX-121 suppresses cell surface expression of MT1-MMP. (A) HT1080 cells were treated with UTX-121 at indicated concentrations for 24 h. The cell lysates were immu-
noblotted with anti-MT1-MMP and anti-paxillin antibodies. L, latent form; A, active form; D, degraded form of MT1-MMP. (B) HT1080 cells were transfected with MT1F. The cells
were treated with either UTX-121 (25
performed under either permeabilized (total) or nonpermeabilized (cell surface) conditions. Bar, 20
mM) or BB94 (2 m
M) for 12 h and incubated with anti-FLAG antibody for 30 min at 37 ꢀC. After washing, immunofluorescence staining was
m
m. (C) Cell surface biotinylation and immunoprecipitation. The whole-cell
lysates were immunoblotted with anti-MT1-MMP and anti-tubulin antibodies. Biotinylated cell surface proteins were precipitated with streptavidin agarose. The precipitates
(SA prep) were immunoblotted with anti-MT1-MMP, anti-N-cadherin, and anti-integrin b1 antibodies.
integrin b1, which were precipitated with Con A-Sepharose beads
(Fig. 3A). This decrease was also observed in cells treated with UTX-
121. WGA binding revealed that UTX-121 reduces N-linked glyco-
sylation in Con A-precipitates similar to tunicamycin. MT-MMP is
known to be O-glycosylated [17]. Latent and active form of MT1-
MMP were precipitated with Con A-Sepharose beads, which were
not affected by UTX-121 treatment. Tunicamycin treatment resul-
ted in the suppression of pro-MMP-2 activation, autodegradation of
MT1-MMP, and accumulation of latent MT1-MMP.
As glycosylation of integrins affects cell adhesion and migration
[18], the effect of UTX-121 on cell adhesion and spreading was
examined. Treatment of cells with UTX-121 impaired the adhesion
of cells to FN but not to PLL and type I collagen (Fig. 3B). Thus, cell
spreading on FN was impeded in the UTX-121-treated cells
compared with DMSO-treated cells (Fig. 3C). FAK is a widely
expressed nonreceptor protein tyrosine kinase localized in focal
adhesions. FAK becomes activated and phosphorylates tyrosine in
response to adhesion of cells to ECM. Activated FAK undergoes
autophosphorylation at Tyr-397 and, thereby, associates a number
of signaling molecules and structural proteins. PI3K is one of the
FAK-activated proteins, which recruits and activates its down-
stream target Akt [19]. The PI3K/Akt pathway is the most frequently
activated in human cancer tissues and plays a pivotal role in pro-
liferation, survival, and metastasis of cancer cells. The effect of UTX-
121 on FN-induced FAK and Akt activation was examined. HT1080
cells treated with inhibitors were either held in suspension or
Please cite this article as: H. Yamahana et al., A novel celecoxib analog UTX-121 inhibits HT1080 cell invasion by modulating membrane-type 1
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Fig. 3. UTX-121 reduces N-glycosylation. (A) HT1080 cells were treated with either UTX-121 or tunicamycin (TM: 25 mM) for 24 h. The conditioned media (CM) were analyzed by
gelatin zymography. Cell lysates were immunoblotted with anti-MT1-MMP and anti-tubulin antibodies. Alternatively, glycoproteins were pulled down with Con A-Sepharose beads.
The precipitates (Con A prep) were immunoblotted with anti-MT1-MMP, anti-N-cadherin, and anti-integrin b1 antibodies, and wheat germ agglutinin (WGA). (B) Cell adhesion
assay. Error bars, S.D.; *, P < 0.01 versus DMSO; PLL, poly-L-lysine; Coll, type I collagen; FN, fibronectin. (C) HT1080 cells were treated with UTX-121 (25
detached, kept in suspension, and replated on FN-coated cover slips for 30 min or 2 h. Cells were stained with Hoechst 33342 (Hoechst) and rhodamine-labeled phalloidin (F-actin).
Bar, 20 m. (D) HT1080 cells were treated with either UTX-121 (25 M) or CXB (25 M) for 24 h. The cells were detached, kept in suspension, and replated on FN-coated dishes for
20 min. Cell lysates were immunoblotted with anti-phospho-FAK (Blot: pTyr397-FAK), anti-FAK, anti-phospho-Akt (Blot: pS473-Akt), anti-Akt, and anti-tubulin antibodies.
mM) for 24 h. The cells were
m
m
m
attached to FN. When the cells were treated with DMSO, FAK
autophosphorylation at Tyr-397 was effectively induced in the cells
stimulated with FN, but not in the suspended cells (Fig. 3D). This
FN-induced FAK phosphorylation was attenuated by either UTX-
121 or celecoxib treatment, which caused the reduction of Akt
activation. These results suggest that glycosylation of cell surface
proteins is impeded by celecoxib and its derivative UTX-121, which
results in the suppression of cell attachment to FN, FAK auto-
phosphorylation, and Akt activation.
3.5. Effect of UTX-121 on cell invasion
The FAK/Akt signaling pathway plays a crucial role in cell
migration and invasion [19]. The effect of UTX-121 on HT1080 cell
Please cite this article as: H. Yamahana et al., A novel celecoxib analog UTX-121 inhibits HT1080 cell invasion by modulating membrane-type 1
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H. Yamahana et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
7
migration and invasion was tested. As expected, wound closure
assay revealed that FN-stimulated cell migration was suppressed by
UTX-121 treatment (Fig. 4A). FN-stimulated invasion of
HT1080 cells through Matrigel matrix was reduced to 57.9% and
29.9% by celecoxib treatment and UTX-121 treatment, respectively
(Fig. 4B). These results strongly suggest that downregulation of
glycosylation in cell adhesion molecules and inhibition of MT1-
MMP activity by UTX-121 treatment impede the invasion of
HT1080 cells.
which was suppressed by celecoxib, suggesting that COX-2 might
affect the glycosylation of tumor cells [2]. Similarly, UTX-121 and
tunicamycin attenuated N-linked glycosylation in HT1080 cells
(Fig. 3). MT1-MMP trafficking inhibition in the cell surface by UTX-
121 can be possibly attributed to the perturbation of N-linked
glycosylation, which may affect the cell surface trafficking of MT1-
MMP. This may be supported by our results, which show that
tunicamycin inhibits MT1-MMP-mediated pro-MMP-2 activation,
and the fact that tunicamycin suppresses proliferation and migra-
tion of hepatocellular carcinoma cells through CD44 and MAPK
pathways [22], which regulates MT1-MMP activity.
4. Discussion
Tumor cell invasion is a complex process that includes cell
adhesion to ECM, ECM degradation, and cell migration [23]. Cell
adhesion to ECM governs cellular functions, including gene
expression, proliferation, survival, and motility. The engagement of
integrins by attachment of cells to ECM stimulates the activation of
FAK, which functions as a scaffold to activate various signals [19].
When macrophages were attached to rat aortic smooth muscle cell-
derived ECM, COX-2 and MMP-9 expressions was induced, which
was inhibited by celecoxib [13]. When HT1080 cells were treated
with UTX-121, cell adhesion to FN was reduced, which resulted in
the suppression of cell spreading, migration, and FAK/Akt activation
(Fig. 3). This is probably due to the inhibition of integrin b1 glyco-
sylation by UTX-121 since the cellular receptors for FN are integrins
In this study, we designed and synthesized UTX-121 to augment
the COX-2-independent pharmacologic activities of celecoxib. UTX-
121 treatment suppressed MMP-9 production, MT1-MMP-medi-
ated MMP-2 activation, cell adhesion, and cell migration, resulting
in the inhibition of HT1080 cell invasion into the Matrigel.
MMP-2 and MMP-9 are type IV collagenase/gelatinase, and their
expression is often associated with tumor malignancy [20]. Cele-
coxib suppressed MMP-9 production by inhibiting nuclear factor-
k
B and activator protein 1 [12,13,21]. UTX-121 was more effective in
reducing the MMP-9 production in HT1080 cells than celecoxib
(Fig. 1). Activation of pro-MMP-2 by MT1-MMP contributes to the
malignant conversion of various tumors [9e11]. We found here that
UTX-121 suppresses pro-MMP-2 activation by disrupting cell sur-
face expression of MT1-MMP (Fig. 2). Aberrant glycosylation
occurring in cancer cells alters cell adhesion, molecular trafficking,
and signal transduction, and thus appears to induce tumor cell
invasion and metastasis [16e18]. COX-2/PGE2 pathway induced
sialyltransferase-3 expression in MDA-MB-231 breast cancer cells,
a5b1, a9b1, and a4b1 and av-integrins. Together with MMP inhibi-
tion, UTX-121 significantly decreased FN-stimulated migration and
invasion of HT1080 cells (Fig. 4). As UTX-121 had little inhibitory
effect on COX-2, its anti-tumor activities seemed to underlie a COX-
2-independent mechanism. However, we cannot rule out the effect
of UTX-121 on COX-1 and COX-3 [24]. Hence, further studies are
required to examine the molecular mechanism by which UTX-121
perturb the trafficking of membrane proteins to the cell surface.
In conclusion, we succeeded in the development of the cele-
coxib derivative UTX-121 possessing the inhibitory activities of
MMPs, cell migration, and cell invasion. UTX-121 may be a potent
lead compound that can be used to develop a novel anti-tumor
drug, which improves the COX-2-independent pharmacologic ac-
tivities of celecoxib.
Declaration of competing interest
None.
Acknowledgement
This work was supported partly by Extramural Collaborative
Research Grant of Cancer Research Institute, Kanazawa University.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2019.10.092.
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Please cite this article as: H. Yamahana et al., A novel celecoxib analog UTX-121 inhibits HT1080 cell invasion by modulating membrane-type 1
matrix metalloproteinase, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.092