Antimetastatic effect of sulfamate carbonic anhydrase IX inhibitors in breast carcinoma xenografts

Article abstract of DOI:10.1021/jm300529u

A panel of compounds belonging to the underexposed sulfamate class of carbonic anhydrase (CA, EC 4.2.1.1) inhibitors was generated that displayed high specificity at nanomolar levels for the tumor-associated CA IX/XII isoforms. Three of the specific CA IX

Full text of DOI:10.1021/jm300529u

Article
pubs.acs.org/jmc
Antimetastatic Effect of Sulfamate Carbonic Anhydrase IX Inhibitors
in Breast Carcinoma Xenografts
Roben G. Gieling,† Muhammad Babur,† Lupti Mamnani,† Natalie Burrows,† Brian A. Telfer,†
Fabrizio Carta,‡ Jean-Yves Winum,§ Andrea Scozzafava,‡ Claudiu T. Supuran,*,‡ and Kaye J. Williams*,†
†Hypoxia and Therapeutics Group, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester,
United Kingdom
‡Department of Chemistry, University of Florence, Florence, Italy
§Institut des Biomolec
Ecole Nationale Super
́
ules Max Mousseron (IBMM), UMR 5247 CNRS-UM1-UM2, Bat
̂
iment de Recherche Max Mousseron,
́
ieure de Chimie de Montpellier, 8 Rue de l’Ecole Normale, 34296 Montpellier Cedex, France
ABSTRACT: A panel of compounds belonging to the underexposed
sulfamate class of carbonic anhydrase (CA, EC 4.2.1.1) inhibitors was
generated that displayed high specificity at nanomolar levels for the
tumor-associated CA IX/XII isoforms. Three of the specific CA IX/XII
inhibitors showed a positive response in in vitro assays for tumor cell
migration and spreading. One of them, 4-(3′-(3″,5″-dimethylphenyl)-
ureido)phenyl sulfamate (S4), was taken forward into the orthotopic
MDA-MB-231 (breast carcinoma) model in mice. Treatment with a
10 mg/kg maintenance dosage of S4 given daily on a “5 days on, 2 days
off” regimen reduced metastatic tumor burden in the lung while not
affecting primary tumor growth or mouse condition. CA inhibitors of the sulfamate class specifically targeting the tumor-
associated isoforms are potential candidates in antimetastatic therapy.
selective inhibitors of CA IX/XII over the past few years has been
shown to be very promising in anticancer therapy. Sulfonamide-
based compounds appear very effective in inhibiting specifically
the tumor-associated isoform CA IX. Experiments with CA IX
selective inhibitors ureidosulfonamides 25 and 104 and glycosyl
coumarins 204 and 205 in mice showed that these are very
effective as inhibitors of breast cancer (4T1) metastasis.12,13 The
earliest studies on sulfamates showed that they have CA inhibitory
properties.14,15 However, these early compounds were not able to
distinguish between tumor-associated and more generally ex-
pressed CA isozymes (i.e., CA IX versus CA I, CA II).
INTRODUCTION
■
Membrane-associated carbonic anhydrase (CA, EC 4.2.1.1) IX
(CA IX) is strongly overexpressed in a broad range of tumor
types, and the expression of CA IX negatively correlates with
the prognosis of cancer patients.1−3 Hypoxia and hypoxia-
inducible factor (HIF) are important inducers of CA IX
expression in solid tumors.4,5 In normal tissues CA IX expression
is much more restricted with abundant expression mainly present
in the mucosa of the glandular stomach.6 On the basis of these
features, it is not surprising that CA IX is a pivotal target for
anticancer therapy.
Here we report the synthesis and enzyme inhibitory activity
of a panel of CA IX selective inhibitors belonging to the
sulfamate class, an underexplored class of CAIs. Validation in in
vitro studies identified the compounds that were selectively
inhibiting cell migration and spreading in the absence of
oxygen, a condition commonly found in solid tumors. From
this panel a lead compound (S4) was taken forward into in
vivo studies. Here we show that S4 was effectively inhibiting
the spontaneous metastasis formation in MDA-MB-231
xenografts.
CA IX expression is important in metastatic dissemination by
increasing the tumor cell survival and invasion in hypoxic envi-
ronments.7 CA IX supports tumor cell survival by catalysis, the
reversible hydration of carbon dioxide to bicarbonate and
protons, maintaining a more alkaline resting intracellular pH
(pHi 7.2−7.4) and thus enhancing tumor cell survival.8 As a
transmembrane enzyme, CA IX activity also supports the flow
of protons out of the cell, which contributes to the acidification
of the microenvironment in the tumor enhancing invasion.9
The catalytic properties of CA IX in lowering the extracellular/
interstitial pH (pHe) have experimentally been tested in
hypoxic tumors as for LS174Tr (colon adenocarcinoma) and
MDA-MB-231 and MCF-7 (breast carcinomas).9−11
RESULTS AND DISCUSSION
■
Chemistry. Using the sulfonamides reported by Pacchiano
et al. as lead molecules,13,16 we report here a series of sulfa-
mates incorporating urea/thiourea moieties of types 1A−10A.
Carbonic anhydrase inhibitors (CAIs) derived from acetazola-
mide, ethoxzolamide, and benzenesulfonamides are effective at
inhibiting tumor cell growth in vitro and in vivo.8,12,13 Inhibition of
the tumor-associated isoforms CA IX and CA XII explains the
antitumor activity of these CAIs.8 The development of more
Received: April 13, 2012
Published: May 24, 2012
© 2012 American Chemical Society
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Scheme 1. Preparation of Ureido/Thioureido Sulfamates 1A−10A
The chemistry for obtaining these new compounds is outlined
here (Scheme 1). Reaction of 4-aminophenol with isocyanates/
isothiocyanates afforded the key intermediates 1−10, which
were sulfamoylated by reaction with sulfamoyl chloride. The
corresponding sulfamates 1A−10A have been thus obtained
with high yield (see Experimental Protocols for details).
Alternatively a pyridnium sulfamate derivative (11A) was
obtained as outlined in Scheme 2, by reaction of 4-aminophenol
with 2,4,6-triphenylpyrylium perchlorate followed by sulfamoy-
lation (Scheme 2).
In Vitro CA Inhibition Data. The sulfamates 1A−11A have
been assayed as inhibitors of four physiologically relevant CA
isoforms, hCA I and II (cytosolic, off-target enzymes) and hCA
IX and XII (transmembrane, tumor-associated CAs). All
compounds inhibited hCA IX and XII at low nanomolar levels
but essentially not the hCA I and II isoforms (Table 1 with
individual KI values).
Antimetastatic Effects of CA IX Inhibitors. In Vitro.
Carbonic anhydrase IX (CA IX) is important in cell migration
and adhesion, and experiments with cells expressing an inactive
CA IX variant failed to support cell migration and adhesion.17
CA IX effects cell migration and adhesion by acidification in
hypoxic tumors.18 We evaluated the ability of a panel of CA IX
inhibitors to inhibit migration in MDA-MB-231 cells.
Specifically we aimed to identify compounds that inhibited
migration only under hypoxic conditions, in line with the
physiological response to hypoxia in increased CA IX activity in
the MDA-MB-231 cell line. We chose invasive eGFP-MDA-
MB-231 cells that express large amounts of CA IX in response
to anoxia (Figure 1). With the up-regulation of CA IX
expression in eGFP-MDA-MB-231 cells in anoxia, we selected
Scheme 2. Preparation of the Pyridinium Sulfamate 11A
Table 1. CA Inhibition Data with the Sulfamates 1A−11A
a
KI (nM)
compd
R
hCA I
hCA II
hCA IX
hCA XII
KI(CA II)/KI(CA IX)
1A
2A
3A
4A
5A
6A
7A
8A
9A
10A
11A
4-F-C6H4
2800
2870
2350
4360
3180
5460
1230
4370
5600
287
291
413
319
145
213
450
348
546
13
12
15
27
6
9
22.1
24.3
27.5
11.8
24.2
11.8
75
4-Cl-C6H4
5
4-MeO-C6H4
4-PhO-C6H4
C6F5
3
8
1
4-PhCH2CH2
4-O2N-C6H4
4-Me2N-C6H4
3,5-Me2C6H3
18
6
7
4
9
2
42.7
78
7
2
b
b
b
6125
569
12
19
29
47.4
54.3
12500
760
14
a
b
Mean from three different assays, with errors in the range of 5−10% of the reported values. From Dubois et al.et al. Radiother. Oncol. 2011, 99,
424−431.
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Figure 1. MDA-MB-231 cells express CA IX under anoxic conditions. (A) MDA-MB-231 cells (blue stained nuclei) express CA IX (red stain) after
exposure to anoxia for 24 h. (B) Representative blot from three independent experiments with whole cell extracts from MDA-MB-231 cells in anoxia
for 24 h probed for CA IX, HIF-1α, and β-actin.
Figure 2. Sulfamate CA IX inhibitors 4A, 5A, and S4 inhibit tumor cell
migration. (A) Representative fluorescence images of confluent
monolayers of eGFP-MDA-MB-231 cells with scratch wounds after
24 h treatment with DMSO (control) or CA IX inhibitor 4A, 5A, or
S4. (B) Quantification of the difference in scratch-wound width before
and after 24 h exposure to CA IX inhibitor 4A, 5A, or S4: n = 3 per
compound. The inhibition for the different compounds in anoxia is as
follows: 4A, 49%; 5A, 32%; S4,50%.
for CA IX inhibitors that inhibited cell migration in anoxia only.
A range of phenotypes were observed. 3A, 8A, 10A, and 11A
did not inhibit migration in anoxia, and 1A, 2A, and 7A
inhibited cell migration in both normoxia and anoxia. CA IX
inhibitors that showed inhibition of eGFP-MDA-MB-231 cell
migration in anoxia only were 4A (49% decreased migration;
p = 0.0005, 4A versus DMSO), 5A (32%, p = 0.0266), and S4
(50%; p = 0.0018) (Figure 2). These CA IX inhibitors
significantly inhibited eGFP-MDA-MB-231 cell migration when
used at 33 μM. As we observed the largest inhibition in cell
migration with S4, this CA IX inhibitor was tested in more
detail in in vitro assays to validate the effectiveness on tumor
cell activity. S4 was inhibiting migration in the 3.3−33 μM
range with signs of excessive cytotoxicity in eGFP-MDA-MB-
231 cells when used in concentrations of around 100 μM.
There was a strong correlation between the concentration of S4
and inhibition of migration in eGFP-MDA-MB-231 cells: 3.3 μM
(5%, p = 0.1663), 10 μM (21%, p = 0.5177), and 33 μM (59%, p =
0.0105) (Figure 3A,B). The effectiveness of S4 on cell migration
was confirmed in WRO and FTC-133 thyroid carcinoma cell lines
which are both positive for CA IX as reported by Burrows et al19
(WRO, 41%; p = 0.0277) (Figure 3C). In two cell lines that do not
express CA IX in anoxia, S4 either had no effect (RT112, bladder
carcinoma) or stimulated cell migration (HCT116, colon
carcinoma, 44%; p = 0.0381) (Figure 3C). However, S4 treatment
of mice with primary HCT116 tumors had no effect on the
number of lung metastasis compared to control mice (data not
shown).
Figure 3. S4 inhibits tumor cell migration in a CA IX-dependent
manner. (A) Representative fluorescence images of confluent mono-
layers of eGFP-MDA-MB-231 cells treated with DMSO or 33 μM
S4. (B) Quantification of the inhibition by S4 at 3.3, 10, or 33 μM
of migration of eGFP-MDA-MB-231 cells. (C) S4 inhibits
migration of WRO and FTC-133 thyroid carcinoma cells which
express CA IX in response to anoxia. S4 either had no effect
(RT112) or increased migration (HCT116) of cell lines which do
not express CA IX in response to anoxia. For each cell line, n = 3
per concentration of S4.
It is well established that CA IX is enhancing cell migration
by acidification of extracellular pH and destabilization of
intercellular contacts.20,21 CA IX is interacting with bicarbonate
transporters in lamellipodia which facilitates ion transport and
controls pH, allowing cells to move.17 It is for this reason that
we tested the effectiveness of S4 in inhibiting CA IX dependent
cell movement. We validated the cell spreading in vitro after
seeding MDA-MB-231 cells (wild type) in dishes on coverslips
either uncoated or fibronectin-coated. Cells were fixed in
formalin and stained with phalloidin-594, and average cell
surface area was measured at the end of each test. Data from a
time−response test (0, 15, 30, 45, and 60 min) revealed that at
the 60 min point optimal mesenchymal spreading in control
cell population was reached (data not shown). The 60 min time
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Figure 4. Inhibition of tumor cell spreading and survival by S4 in anoxia. (A) Representative images of MDA-MB-231 cells grown on fibronectin-
coated coverslips stained with phalloidin (red) for each experimental condition. (B) Graphs on cell area measurements of MDA-MB-231 cells grown
on glass coverslips coated with or without fibronectin. (C) MTT assay to validate the proliferation of MDA-MB-231, HCT116, and HT29 cells 4
days after incubation with increasing concentrations of S4 for 24 h.
point was repeated for the different conditions in three
independent tests. Representative images of phalloidin-594
stained MDA-MB-231 cells on fibronectin-coated coverslips are
shown for each experimental condition (Figure 4A). Cell
spreading of MDA-MB-231 cells increased in anoxia compared
to that in normoxia (Figure 4B). S4 treatment delayed the cell
spreading of MDA-MB-231 cells in anoxia but essentially not in
normoxia. The effects of S4 on cell spreading were enhanced by
a supportive layer of fibronectin (Figure 4B).
Cell proliferation was detected by MTT assay, and the results
show that S4 inhibited the proliferation of MDA-MB-231 cells
in a dose-dependent manner (Figure 4C). S4 also inhibited
proliferation of other cells lines, i.e., HCT116 and HT29
cells. The IC50 values from the dose−response curves were for
MDA-MB-231 (481 μM S4), HCT116 (>1000 μM S4), and
HT29 (20 μM S4) (Figure 4C). The level of inhibition of
proliferation, by increasing concentrations of S4, correlates well
with the level of CA IX expression, with MDA-MB-231
expression low, HCT116 expression not detectable, and HT29
expressing high amounts of CA IX in normoxia (Figure 1B,
Figure 4D).
and was taken forward into in vivo studies. The general health
of the mice was inspected daily with no signs of intolerance for
S4 at 10 mg/kg (n = 8 per group). The average body weights
between vehicle and S4 treated mice were similar throughout
the experiments (Figure 5A). The average growth rate of the
primary orthotopic tumors of eGFP-MDA-MB-231 was not
affected by a daily dose of S4 (Figure 5B). We noticed an
increase in CA IX activity expressed in MDA-MB-231 tumors
near areas of necrosis, indicating that these areas were under
hypoxic stress (Figure 5C). The effect of S4 on metastatic
dissemination was based on counting the number of tumor cell
colonies growing out of the cell suspension isolated from lung
(Figure 5D). Treatment with S4 significantly reduced the
metastatic tumor burden in lungs of mice bearing orthotopic
eGFP-MDA-MB-231 tumors (Figure 5D, p = 0.0284).
CONCLUSIONS
■
Sulfamates 4-(3′-(4″-phenoxyphenyl)ureido)phenyl sulfamate
(4A), 4-(3′-(perfluorophenyl)ureido)phenyl sulfamate (5A),
and 4-(3′-(3″,5″-dimethylphenyl)ureido)phenyl sulfamate
(9A = S4) inhibited migration and spreading of tumor cells
under oxygen-decreased conditions as found in solid tumors.
In addition, low dose maintenance therapy with S4 strongly
inhibited the development of MDA-MB-231 metastases in
lung with no signs of toxicity. S4 had no effect on the primary
In Vivo. Orthotopic xenograft tumors of the human breast
cancer line MDA-MB-231 are widely used for the ability to
study spontaneous metastases development to the lung.22 From
the panel of CA IX inhibitors we validated in vitro, S4 appeared
to be very effective in inhibiting cell migration and spreading
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used as eluent. Melting points (mp) were measured in open capillary
tubes and are uncorrected. All compounds were >99% pure as assessed
by HPLC.
General Procedure for the Synthesis of Compounds 1−
10.13,16,23 A 1.0 M solution of the appropriate isocyanate (1.0 equiv)
was dissolved in dry ACN and treated with 4-aminophenol (1.0 equiv).
The reaction mixture was stirred vigorously at room temperature until
no starting material was left (TLC monitoring). The solid formed was
separated by filtration, washed with H2O, dried under vacuum, and
purified to produce the corresponding ureidophenols 1−10.
Freshly prepared chlorosulfanilamide (2.0 equiv) was added to a 2.0 M
solution of ureidophenols 1−10 in dry DMA under a nitrogen atmo-
sphere until no starting material was left (TLC monitoring). Then the
solution was quenched with slush and extracted with ethyl acetate (3 ×
20 mL). The combined organic layers were washed with H2O (4 × 20 mL),
brine (3 × 20 mL), dried over Na2SO4, filtered, and concentrated under
vacuum to give a sticky residue that was purified to produce the desired
sulfamates 1A−10A.
Synthesis of 4-(3′-(4″-Fluorophenyl)ureido)phenyl Sulfa-
mate (1A).
4-Aminophenol (0.2 g, 1.0 equiv) and 4-fluorophenyl isocyanate
(1.0 equiv) in dry ACN (10 mL) were treated according to the general
procedure previously described. The residue obtained was purified by
silica gel column chromatography, eluting with 50% ethyl acetate/
n-hexane to afford 1 as a white solid which was treated with freshly
prepared sulfamyl chloride in dry DMA. The crude residue was
purified by silica gel column chromatography, eluting with 50% ethyl
acetate/n-hexane to produce 1A as a white solid.
Figure 5. S4 inhibits metastatic tumor burden in MDA-MB-231 model
while having no effect on primary tumor growth or mouse condition.
(A) Table with the average body weights after treatment with S4. (B)
Growth curve of the primary tumor volumes from the day of
treatment. (C) Representative image of section from primary MDA-
MB-231 tumor stained for human carbonic anhydrase IX (CA IX)
(N = necrosis). (D) Assessment of the metastatic tumor burden in
lung by calculating numbers of colonies per gram of lung established
after 10 days in vitro (N = 8 per group).
1-(4″-Fluorophenyl)-3′-(4-hydroxyphenyl)urea (1): 92% yield; silica
gel TLC Rf = 0.24 (ethyl acetate/n-hexane, 50% v/v); δH (400 MHz,
DMSO-d6) 6.71 (2H, d, J = 8.8, Ar-H), 7.13 (2H, t, J = 8.8, Ar-H),
7.23 (2H, d, J = 8.8, Ar-H), 7.46 (2H, m, Ar-H), 8.33 (1H, s, exchange
with D2O, NH), 8.59 (1H, s, exchange with D2O, NH), 9.08 (1H, s,
exchange with D2O, OH); δC (100 MHz, DMSO-d6) 158.1 (d, J1
=
C−F
236.2, C-4″), 153.8, 153.5, 137.2 (d, J4
= 2.3, C-1″), 132.0, 121.5,
C−F
120.7 (d, J3
= 7.6, C-2″), 116.1 (d, J2
= 22.0, C-3″), 116.0; δF
C−F
C−F
MDA-MB-231 tumor growth. S4 is an excellent new candidate
as an antimetastatic drug in breast cancer therapy.
(376 MHz, DMSO-d6) −122.0 (1F, s).
4-(3′-(4″-Fluorophenyl)ureido)phenyl sulfamate (1A): 90% yield;
silica gel TLC Rf = 0.12 (ethyl acetate/n-hexane, 50% v/v); δH (400 MHz,
DMSO-d6) 7.12 (2H, t, J = 8.8, Ar-H), 7.19 (2H, d, J = 8.8, Ar-H),
7.47 (4H, m, Ar-H), 7.89 (2H, s, exchange with D2O, SO2NH2), 8.71
(2H, brs, exchange with D2O, NH), 8.78 (2H, brs, exchange with D2O,
NH); δC (100 MHz, DMSO-d6) 158.0 (d, J1C−F = 236.6, C-4″), 153.5,
145.4, 139.0, 136.8 (d, J4C−F = 2.7, C-1″), 123.4, 120.9 (d, J 3C−F = 7.5,
EXPERIMENTAL PROTOCOLS
■
Chemistry. Anhydrous solvents and all reagents were purchased
from Sigma-Aldrich, Alfa Aesar, and TCI. All reactions involving air- or
moisture-sensitive compounds were performed under a nitrogen atmo-
sphere using dried glassware and syringe techniques to transfer
solutions. NaH, 60% in oil dispersion, was washed with n-hexane until
a homogeneous white solid was obtained, which was dried and stored
under a nitrogen atmosphere prior to use. Infrared (IR) spectra were
recorded as KBr plates and are expressed in ν (cm−1). Nuclear
magnetic resonance (1H NMR, 13C NMR, DEPT-135, DEPT-90,
HSQC, HMBC) spectra were recorded using a Bruker Avance III
400 MHz spectrometer with CDCl3, MeOH-d4, or DMSO-d6.
Chemical shifts are reported in parts per million (ppm), and the
coupling constants (J) are expressed in hertz (Hz). Splitting patterns
are designated as follows: s, singlet; d, doublet; sept, septet; t, triplet; q,
quadruplet; m, multiplet; brs, broad singlet; dd, double of doubles;
appt, apparent triplet; appq, apparent quartet. The assignment of
exchangeable protons (OH and NH) was confirmed by the addition
of D2O. Analytical thin-layer chromatography (TLC) was carried out
on Merck silica gel F-254 plates. Flash chromatography purifications
were performed on Merck silica gel 60 (230−400 mesh ASTM) as the
stationary phase, and ethyl acetate/n-hexane or MeOH/DCM was
C-2″), 120.1, 116.2 (d, J2
−121.4 (1F, s).
= 22.2, C-3″); δF (376 MHz, DMSO-d6)
C−F
Synthesis of 4-(3′-(4″-Chlorophenyl)ureido)phenyl Sulfa-
mate (2A).
4-Aminophenol (0.2 g, 1.0 equiv) and 4-chlorophenyl isocyanate
(1.0 equiv) in dry ACN (10 mL) were treated according to the
general procedure previously described. The residue obtained was
purified by silica gel column chromatography, eluting with 50% ethyl
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acetate/n-hexane to afford 2 as a white solid which was treated with
freshly prepared sulfamyl chloride in dry DMA. The crude residue was
purified by silica gel column chromatography, eluting with 50% ethyl
acetate/n-hexane to produce 2A as a white solid.
1-(4-Hydroxyphenyl)-3′-(4″-phenoxyphenyl)urea (4): 88% yield;
silica gel TLC Rf = 0.59 (ethyl acetate/n-hexane, 70% v/v); δH (400
MHz, DMSO-d6) 6.71 (2H, d, J = 6.8, Ar-H), 6.98 (4H, m, Ar-H),
7.12 (1H, t, J = 6.8, Ar-H), 7.24 (2H, d, J = 6.8, Ar-H), 7.41 (2H, t, J =
6.8, Ar-H), 7.47 (2H, d, J = 6.8, Ar-H), 8.33 (1H, s, exchange with
D2O, NH), 8.56 (1H, s, exchange with D2O, NH), 9.08 (1H, s,
exchange with D2O, OH); δC (100 MHz, DMSO-d6) 158.6, 153.8,
153.5, 151.3, 137.0, 132.0, 130.8, 123.6, 121.3, 120.7, 120.6, 118.4,
116.1.
4-(3′-(4″-Phenoxyphenyl)ureido)phenyl sulfamate (4A): 90% yield;
silica gel TLC Rf = 0.65 (ethyl acetate/n-hexane, 70% v/v); δH (400
MHz, DMSO-d6) 6.98 (4H, m, Ar-H), 7.12 (1H, t, J = 7.2, Ar-H), 7.23
(2H, d, J = 7.2, Ar-H), 7.39 (2H, d, J = 7.2, Ar-H), 7.52 (4H, m, Ar-H),
7.93 (2H, s, exchange with D2O, SO2NH2), 8.72 (1H, s, exchange with
D2O, NH), 8.79 (1H, s, exchange with D2O, NH); δC (100 MHz,
DMSO-d6) 158.5, 153.5, 151.7, 145.4, 139.0, 136.5, 130.8, 123.7,
123.5, 120.9, 120.7, 120.0, 118.5.
1-(4″-Chlorophenyl)-3′-(4-hydroxyphenyl)urea (2): 89% yield;
silica gel TLC Rf = 0.28 (ethyl acetate/n-hexane, 50% v/v); δH (400
MHz, DMSO-d6) 6.71 (2H, d, J = 8.8, Ar-H), 7.24 (2H, d, J = 8.8,
Ar-H), 7.32 (2H, d, J = 8.8, Ar-H), 7.49 (2H, d, J = 8.8, Ar-H), 8.47
(1H, s, exchange with D2O, NH), 8.81 (1H, s, exchange with D2O,
NH), 9.09 (1H, s, exchange with D2O, OH); δC (100 MHz, DMSO-
d6) 153.6, 140.0, 131.8, 129.5, 125.9, 123.1, 121.5, 120.4, 116.1.
4-(3′-(4″-Chlorophenyl)ureido)phenyl sulfamate (2A): 95% yield;
silica gel TLC Rf = 0.17 (ethyl acetate/n-hexane, 50% v/v); δH (400
MHz, DMSO-d6) 7.22 (2H, d, J = 8.8, Ar-H), 7.37 (2H, d, J = 8.8, Ar-
H), 7.53 (4H, m, Ar-H), 7.94 (2H, s, exchange with D2O, SO2NH2),
8.86 (2H, brs, exchange with D2O, NH); δC (100 MHz, DMSO-d6)
153.3, 145.6, 139.5, 138.8, 129.5, 126.3, 123.5, 120.7, 120.2.
Synthesis of 4-(3′-(4″-Methoxyphenyl)ureido)phenyl Sulfa-
mate (3A).
Synthesis of 4-(3′-(Perfluorophenyl)ureido)phenyl Sulfa-
mate (5A).
4-Aminophenol (0.2 g, 1.0 equiv) and pentafluorophenyl isocyanate
(1.0 equiv) in dry ACN (10 mL) were treated according to the general
procedure previously described. The obtained residue was purified by
silica gel column chromatography, eluting with 50% ethyl acetate/
n-hexane to produce 5 as a white solid which was treated with freshly
prepared sulfamyl chloride in dry DMA. The crude product was
purified by silica gel column chromatography, eluting with 50% ethyl
acetate/n-hexane to produce 5A as a pale yellow solid.
1-(4-Hydroxyphenyl)-3′-(perfluorophenyl)urea (5): 94% yield;
silica gel TLC Rf = 0.38 (50% v/v ethyl acetate/n-hexane); δH (400
MHz, DMSO-d6) 6.72 (2H, d, J = 8.7, Ar-H), 7.24 (2H, d, J = 8.7,
Ar-H), 8.38 (1H, s, exchange with D2O, NH), 8.73 (1H, s, exchange
with D2O, NH), 9.15 (1H, s, exchange with D2O, OH); m/z (ESI)
341 [M + Na].
4-Aminophenol (0.2 g, 1.0 equiv) and 4-methoxyphenyl isocyanate
(1.0 equiv) in dry ACN (10 mL) were treated according to the general
procedure previously described. The residue obtained was purified by
silica gel column chromatography, eluting with ethyl acetate to
produce 3, a white solid that was treated with freshly prepared sulfamyl
chloride in dry DMA. The crude residue was purified by silica gel
column chromatography, eluting with ethyl acetate to produce 3A as a
white solid.
1-(4-Hydroxyphenyl)-3′-(4″-methoxyphenyl)urea (3): 85% yield;
silica gel TLC Rf = 0.76 (ethyl acetate); δH (400 MHz, DMSO-d6)
3.74 (2H, s, CH3), 6.70 (2H, d, J = 8.8, Ar-H), 6.88 (2H, t, J = 8.8,
Ar-H), 7.23 (2H, d, J = 8.8, Ar-H), 7.36 (2H, m, Ar-H), 8.27 (1H, s,
exchange with D2O, NH), 8.38 (1H, s, exchange with D2O, NH), 9.04
(1H, s, exchange with D2O, OH); δC (100 MHz, DMSO-d6) 155.2,
153.9, 153.3, 134.0, 132.2, 121.2, 120.7, 116.1, 114.9, 56.1.
4-(3′-(4″-Methoxyphenyl)ureido)phenyl sulfamate (3A): 86% yield;
silica gel TLC Rf = 0.88 (ethyl acetate); δH (400 MHz, DMSO-d6)
3.75 (3H, s, CH3), 6.90 (2H, d, J = 8.8, Ar-H), 7.21 (2H, d, J = 8.8,
Ar-H), 7.38 (2H, d, J = 8.8, Ar-H), 7.52 (2H, d, J = 8.8, Ar-H), 7.92
(2H, s, exchange with D2O, SO2NH2), 8.51 (1H, s, exchange with
D2O, NH), 8.73 (1H, s, exchange with D2O, NH); δC (100 MHz,
DMSO-d6) 153.6, 139.2, 133.5, 128.2, 123.5, 121.0, 119.9, 114.9, 55.7.
Synthesis of 4-(3′-(4″-Phenoxyphenyl)ureido)phenyl Sulfa-
mate (4A).
4-(3′-(Perfluorophenyl)ureido)phenyl sulfamate (5A): 78% yield;
silica gel TLC Rf = 0.25 (50% v/v ethyl acetate/n-hexane); δH (400
MHz, DMSO-d6) 7.23 (2H, d, J = 9.1, Ar-H), 7.53 (2H, d, J = 9.1,
Ar-H), 7.94 (2H, s, exchange with D2O, SO2NH2), 8.56 (1H, s,
exchange with D2O, NH), 9.20 (1H, s, exchange with D2O, NH); δC
(100 MHz, DMSO-d6) 153.0, 145.9, 143.9 (dt, J1CF = 238, J3CF = 15),
139.4 (dt, J1 = 247, J3 = 15), 138.2, 137.8 (dt, J1 = 247, J3
=
CF
CF
CF
CF
15), 123.5, 120.5, 115.0 (t, J3 = 15, C-1″),
CF
Synthesis of 4-(3′-Phenethylureido)phenyl Sulfamate (6A).
4-Aminophenol (0.2 g, 1.0 equiv) and phenethyl isocyanate
(1.0 equiv) in dry ACN (10 mL) were treated according to the
general procedure previously described. The residue obtained was
purified by silica gel column chromatography, eluting with ethyl
acetate to produce 6 as a white solid which was treated with freshly
prepared sulfamyl chloride in dry DMA. The crude residue was
triturated from DCM to produce 6A as a white solid.
4-Aminophenol (0.2 g, 1.0 equiv) and 4-phenoxyphenyl isocyanate
(1.0 equiv) in dry ACN (10 mL) were treated according to the general
procedure previously described. The residue obtained was purified by
silica gel column chromatography, eluting with 50% ethyl acetate/
n-hexane to produce 4 as a white solid which was treated with freshly
prepared sulfamyl chloride in dry DMA. The crude residue was
purified by silica gel column chromatography, eluting with 70% v/v
ethyl acetate/n-hexane to produce 4A as a white solid.
1-(4-Hydroxyphenyl)-3′-phenethylurea (6): 91% yield; silica gel
TLC Rf = 0.63 (ethyl acetate); δH (400 MHz, DMSO-d6) 2.76 (2H, t,
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J = 7.2, 5′-H2), 3.35 (2H, q, J = 7.2, 6′-H2), 5.98 (1H, t, J = 7.2,
exchange with D2O, 3′-NH), 6.65 (2H, d, J = 8.8, Ar-H), 7.16 (2H, d,
J = 8.8, Ar-H), 7.28 (5H, m, Ar-H), 8.15 (1H, s, exchange with
D2O, NH), 8.95 (1H, s, exchange with D2O, OH); δC (100 MHz,
DMSO-d6) 156.4, 152.8, 140.6, 132.9, 129.6, 129.2, 126.9, 120.7,
116.0, 41.6, 36.9.
4-(3′-Phenethylureido)phenyl sulfamate (6A): 92% yield; silica gel
TLC Rf = 0.75 (ethyl acetate); δH (400 MHz, DMSO-d6) 2.80 (2H, t,
J = 6.4, 5′-H2), 3.38 (2H, q, J = 6.4, 4′-H2), 6.15 (1H, t, J = 6.4, exchange
with D2O, 3′-NH), 7.15 (2H, d, J = 8.8, Ar-H), 7.27 (3H, m, Ar-H), 7.35
(2H, d, J = 8.8, Ar-H), 7.47 (2H, d, J = 8.8, Ar-H), 7.88 (2H, s, exchange
with D2O, SO2NH2), 8.62 (1H, s, exchange with D2O, NH), 10.31
(1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 156.0,
144.9, 140.4, 139.9, 129.6, 129.3, 127.0, 123.4, 119.3, 41.6, 36.7.
Synthesis of 4-(3′-(4″-Nitrophenyl)ureido)phenyl Sulfamate
(7A).
J = 8.8, Ar-H), 7.22 (2H, d, J = 8.8, Ar-H), 7.26 (2H, d, J = 8.8, Ar-H),
8.19 (1H, s, exchange with D2O, NH), 8.21 (1H, s, exchange with
D2O, NH), 9.03 (1H, s, exchange with D2O, OH); δC (100 MHz,
DMSO-d6) 154.0, 153.2, 147.2, 132.4, 130.8, 121.1, 120.9, 116.1,
114.1, 41.7.
4-(3′-(4″-(Dimethylamino)phenyl)ureido)phenyl sulfamate (8A):
88% yield; silica gel TLC Rf = 0.65 (ethyl acetate); δH (400 MHz,
DMSO-d6) 2.87 (6H, s, 2 × CH3), 6.73 (2H, d, J = 8.8, Ar-H), 7.20
(2H, d, J = 8.8, Ar-H), 7.28 (2H, d, J = 8.8, Ar-H), 7.51 (2H, d, J = 8.8,
Ar-H), 7.93 (2H, s, exchange with D2O, SO2NH2), 8.30 (2H, brs,
exchange with D2O, NH), 8.66 (2H, brs, exchange with D2O, NH); δC
(100 MHz, DMSO-d6) 153.2, 147.1, 145.6, 130.2, 126.1, 122.9, 122.1,
117.0, 115.3, 39.6.
Synthesis of 4-(3′-(3″,5″-Dimethylphenyl)ureido)phenyl Sul-
famate (9A = S4).
4-Aminophenol (0.2 g, 1.0 equiv) and 3,5-dimethylphenylphenyl
isocyanate (1.0 equiv) in dry ACN (10 mL) were treated according to
the general procedure previously described. The residue was purified
by silica gel column chromatography, eluting with 50% ethyl acetate/
n-hexane to produce 9 as a white solid which was treated with freshly
prepared sulfamyl chloride in dry DMA. The crude residue was
purified by silica gel column chromatography, eluting with 50% ethyl
acetate/n-hexane to produce 9A (=S4) as a white solid.
1-(3,5-Dimethylphenyl)-3-(4-hydroxyphenyl)urea (9): 89% yield;
silica gel TLC Rf = 0.30 (ethyl acetate/n-hexane, 50% v/v); δH (400
MHz, DMSO-d6) 2.23 (6H, s, 2 × CH3), 6.61 (1H, s, 4-H), 6.72 (2H,
d, J = 7.2, Ar-H), 7.08 (2H, s, 2 × 2-H), 7.23 (2H, d, J = 7.2, Ar-H),
8.31 (1H, s, exchange with D2O, NH), 8.37 (1H, s, exchange with
D2O, NH), 9.09 (1H, s, exchange with D2O, OH); δC (100 MHz,
DMSO-d6) 153.7, 153.4, 140.7, 138.5, 132.1, 124.0, 121.2, 116.7,
116.1, 22.0.
4-Aminophenol (0.2 g, 1.0 equiv) and 4-nitrophenyl isocyanate
(1.0 equiv) in dry ACN (10 mL) were treated according to the general
procedure previously described. The residue obtained was purified by
silica gel column chromatography, eluting with 50% ethyl acetate/
n-hexane to produce 7 as a white solid which was treated with freshly
prepared sulfamyl chloride in dry DMA. The residue was filtered-off,
washed several times with water, and dried under vacuum to produce
7A as a yellow solid.
1-(4-Hydroxyphenyl)-3′-(4″-nitrophenyl)urea (7): 90% yield; silica
gel TLC Rf = 0.18 (ethyl acetate/n-hexane, 50% v/v); δH (400 MHz,
DMSO-d6) 6.74 (2H, d, J = 8.8, Ar-H), 7.28 (2H, d, J = 8.8, Ar-H),
7.70 (2H, d, J = 8.8, Ar-H), 8.20 (2H, d, J = 8.8, Ar-H), 8.66 (1H, s,
exchange with D2O, OH), 9.09 (2H, s, exchange with D2O, 2 x NH);
δC (100 MHz, DMSO-d6) 154.0, 153.0, 147.6, 141.7, 131.2, 126.0,
121.9, 118.1, 116.2.
4-(3′-(4″-Nitrophenyl)ureido)phenyl sulfamate (7A): 88% yield;
silica gel TLC Rf = 0.12 (ethyl acetate/n-hexane, 50% v/v); δH (400
MHz, DMSO-d6) 7.26 (2H, d, J = 8.8, Ar-H), 7.56 (2H, d, J = 8.8,
Ar-H), 7.75 (2H, d, J = 8.8, Ar-H), 7.96 (2H, s, exchange with D2O,
SO2NH2), 8.23 (2H, d, J = 8.8, Ar-H), 9.70 (2H, brs, exchange with
D2O, NH), 10.20 (2H, brs, exchange with D2O, NH); δC (100 MHz,
DMSO-d6) 153.0, 147.3, 146.0, 141.9, 138.4, 126.0, 123.6, 120.2,
118.1.
4-(3-(3,5-Dimethylphenyl)ureido)phenyl sulfamate (9A): 89%
yield; silica gel TLC Rf = 0.25 (ethyl acetate/n-hexane, 50% v/v);
δH (400 MHz, DMSO-d6) 2.27 (6H, s, 2 × CH3), 6.65 (1H, s, 4-H),
7.11 (2H, s, 2 × 2-H), 7.22 (2H, d, J = 7.2, Ar-H), 7.53 (2H, d, J = 7.2,
Ar-H), 7.93 (2H, s, exchange with D2O, SO2NH2), 8.54 (1H, s,
exchange with D2O, NH), 8.77 (1H, s, exchange with D2O, NH); δC
(100 MHz, DMSO-d6) 153.4, 145.4, 140.3, 139.1, 138.6, 124.4, 123.5,
119.9, 116.9, 22.0.
Synthesis of 2-(3-Hydroxy-6-oxo-6H-xanthen-9-yl)-5-(3-(4-
(sulfamoyloxy)phenyl)thioureido)benzoic Acid (FC11-489A
bis = 10A). The synthesis of 10A has been reported by Dubois et al.24
2-(3-Hydroxy-6-oxo-6H-xanthen-9-yl)-5-(3-(4-hydroxyphenyl)-
thioureido)benzoic acid (10): 57% yield; silica gel TLC Rf = 0.11
(ethyl acetate); δH (400 MHz, DMSO-d6) 6.49 (1H, dd, J = 7.5, 6′-H),
6.65 (4H, brs), 6.70 (2H, s), 6.80 (2H, d, J = 8.8), 7.22 (2H, d, J =
8.8), 8.83 (1H, d, J = 7.5, 5′-H), 8.18 (1H, s, 2′-H), 9.49 (1H, s,
exchange with D2O, OH), 9.87 (1H, s, exchange with D2O, NH), 9.95
(1H, s, exchange with D2O, NH), 10.18 (1H, s, exchange with D2O,
OH); δC (100 MHz, DMSO-d6) 180.9 (CS), 169.6, 160.5, 156.2,
152.9, 148.6, 142.5, 131.9, 131.2, 131.0, 130.1, 127.4, 127.38, 125.0,
118.7, 116.7, 116.6, 116.2, 113.7, 110.7, 103.3, 84.1.
2-(3-Hydroxy-6-oxo-6H-xanthen-9-yl)-5-(3-(4-(sulfamoyloxy)-
phenyl)thioureido)benzoic acid (10A): 56% yield; silica gel TLC Rf =
0.07 (ethyl acetate); δH (400 MHz, DMSO-d6) 7.06−7.38 (8H, m),
7.40 (2H, s), 7.62 (2H, d, J = 8.8), 7.93 (1H, d, J = 7.5), 8.10 (2H, s,
exchange with D2O, SO2NH2), 8.26 (4H, s), 8.32 (1H, s), 10.20 (1H,
s, exchange with D2O, NH), 10.31 (1H, s, exchange with D2O, NH);
δC (100 MHz, DMSO-d6) 180.8 (CS), 169.1, 162.0, 152.5, 151.7,
148.3, 147.8, 145.8, 142.7, 138.9, 138.3, 131.8, 130.5, 127.5, 126.6,
126.1, 125.0, 123.7, 123.3, 119.8, 119.7, 118.8, 117.9, 111.5, 81.6.
Synthesis of 4-(3′-(4″-(Dimethylamino)phenyl)ureido)-
phenyl Sulfamate (8A).
4-Aminophenol (0.2 g, 1.0 equiv) and 4-(dimethylamino)phenyl
isocyanate (1.0 equiv) in dry ACN (10 mL) were treated according to
the general procedure previously described. The residue obtained was
purified by silica gel column chromatography, eluting with ethyl
acetate to produce 8 as a white solid which was treated with freshly
prepared sulfamyl chloride in dry DMA. The crude residue was
purified by silica gel column chromatography, eluting with ethyl
acetate to produce 8A as a white solid.
1-(4″-(Dimethylamino)phenyl)-3′-(4-hydroxyphenyl)urea (8): 87%
yield; silica gel TLC Rf = 0.59 (ethyl acetate); δH (400 MHz, DMSO-
d6) 2.89 (6H, s, 2 × CH3), 6.69 (2H, d, J = 8.8, Ar-H), 6.71 (2H, d,
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1
1-(4-Hydroxyphenyl)-2,4,6-triphenylpyridinium 11: 70% yield; H
NMR (400 MHz, DMSO) δ ppm 9.89 (s, 1H), 8.62 (s, 2H), 8.33 (d,
2H, J = 4 Hz), 7.67 (m, 3H), 7.40 (m, 10H), 7.19 (d, 2H, J = 8 Hz),
6.48 (d, 2H, J = 8 Hz). MS ESI+: m/z 400.00 (M)+.
(length × breadth × height) was measured 3 times a week using
callipers. eGFP-MDA-MB-231 (150 mm3) carrying mice were treated
with S4 (10 mg/kg made up in saline with 4% DMSO) daily
intraperitoneally on a “5 days on, 2 days off” dosing regimen. Mice
were culled when tumor volume reached 1000 mm3. Ex vivo lung
clonogenic assays were performed as previously described.30
Experiments were ethically approved and carried out according
to the Scientific Procedure Act 1986 and Guidelines for the
Welfare and Use of Animals in Cancer 2010 under the Home
Office License PPL40/3112 (held by K.J.W.).
1-(4-O-Sulfamoylphenyl)-2,4,6-triphenylpyridinium 11A: 80%
1
yield; H NMR (400 MHz, DMSO) δ ppm 8.70 (s, 2H), 8.4 (d,
2H, J = 7 Hz), 8.2 (s, 2H), 7.70 (m, 3H), 7.55 (d, 2H, J = 9 Hz),
7.40 (m, HH), 7.15 (d, 2H, J = 9 Hz), 5.75 (s, 2H). MS ESI+: m/z
479.18 (M)+.
In Vitro CA Inhibition Data. An Applied Photophysics stop-flow
instrument was used for assaying the CA catalyzed CO2 hydration
activity.25 Phenol red (at 0.2 mM) was used as indicator, working at
the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as
buffer and 20 mM Na2SO4 (for maintaining constant ionic strength),
following the initial rates of the CA-catalyzed CO2 hydration reaction
for a period of 10−100 s. The CO2 concentrations ranged from 1.7
to 17 mM for the determination of the kinetic parameters and
inhibition constants. For each inhibitor at least six traces of the initial
5−10% of the reaction were used for determining the initial velocity.
The uncatalyzed rates were determined in the same manner and
subtracted from the total observed rates. Stock solutions of inhibitor
(0.1 mM) were prepared in distilled−deionized water, and dilutions
up to 0.01 nM were made thereafter with distilled−deionized water.
Inhibitor and enzyme solutions were preincubated together for
15 min at room temperature prior to assay in order to allow the
formation of the E−I complex. The inhibition constants were
obtained by nonlinear least-squares methods using PRISM 3, as
reported earlier,26 and represent the mean from at least three
different determinations. All CA isoforms were recombinant ones
obtained in house as reported earlier.27
Immunohistochemistry. Sections (2 μm) of paraffin-embedded
tissue samples were deparaffinized and rehydrated. Heat-induced
antigen retrieval with citrate buffer (pH 6.0) was used. Sections were
incubated in mouse monoclonal antibody M75 (human specific CA IX
antibody, gift from Dr. Pastorek, Bratislava, Slovakia) in the ARK
(Animal Research Kit) peroxidase assay according to the manufac-
turer’s instructions (Dako, Ely, U.K.). The protein was visualized with
3,3′-diaminobenzidine (DAB), and sections were counterstained with
Mayer’s hematoxylin.
Western Blotting. Western blotting was performed as previously
described.19 Antibodies against human CA IX, HIF-1α (BD Trans-
duction Laboratories, Oxford, U.K.), and β-actin (Sigma, Gillingham,
U.K.) were used.
AUTHOR INFORMATION
Corresponding Author
■
*For C.T.S.: phone, +39-055-4573005; e-mail, claudiu.supuran@
unifi.it. For K.J.W.: phone, +44 (0) 161 275 2428; e-mail, kaye.
williams@manchester.ac.uk.
Cell Culture. MDA-MB-231 (breast carcinoma), HCT116 and
HT29 (colon carcinomas), WRO and FTC-133, (thyroid carcinomas),
and RT112 (bladder carcinoma) cells were maintained in RMPI 1640
medium with the addition of 10% fetal calf serum (Biosera, East
Sussex, U.K.) and 2 mM glutamine (Gibco, Invitrogen Ltd., U.K.).
MDA-MB-231, WRO, and FTC-133 cells were tagged with enhanced
green fluorescence protein (eGFP). The eGFP MDA-MB-231 stable
cell line was generated by cloning the coding sequence of eGFP into
pEF IRESp28 and transfecting the construct into MDA-MB-231 cells
using the method previously described.29 Data are plotted as average
values standard error of the mean (SEM) and analyzed with the
Student’s t test (GraphPad Software, CA, U.S.). In the graphs, the
symbols ∗, ∗∗, and ∗∗∗ denote P < 0.05, P < 0.01, and P < 0.001,
respectively.
Notes
The authors declare the following competing financial
interest(s): Drs. Supuran and Williams are authors of a patent
(Carbonic Anhydrase Inhibitors, PCT/EP2011/052156).
ACKNOWLEDGMENTS
■
We thank the European Commission for support (EU FP7
Metoxia, Grant Agreement No. 222741). The Bioimaging Facility
microscopes used in this study were purchased with grants from
BBSRC, Wellcome, and the University of Manchester Strategic
Fund, U.K. Special thanks go to Dr. Peter March for his help with
the microscopy.
Scratch-Wound Migration Assay. The procedure was performed
as previously described.30 Cells were incubated under normoxia or
anoxia for 24 h with or without increasing concentrations of the
carbonic anhydrase inhibitors 1A−11A.
ABBREVIATIONS USED
■
aq, aqueous; Ar-H, aromatic proton; bs, broad singlet; °C,
temperature in degrees Celsius; DCC, dicyclohexylcarbodii-
mide; DCM, dichloromethane; dec, decomposition; DMA,
dimethylacetamide; DMAP, N,N-dimethyl-4-aminopyridine;
DMF, N,N-dimethylformamide; EDCI·HCl, N-(3-dimethyla-
minopropyl)-N′-ethylcarbodiimide hydrochloride; equiv, equiv-
alent; g, gram; h, hour; HOBt, 1-hydroxybenzotriazole; Hz,
hertz; IR, infrared; J, coupling constant in Hz; νmax, wave-
number; min, minute; MHz, megahertz; ppm, parts per million;
Pyr, pyridine; rt, room temperature; δc, 13C chemical shift
reported in ppm; δF, 19F chemical shift reported in ppm; δH, 1H
chemical shift reported in ppm; THF, tetrahydrofuran; TLC,
thin layer chromatography
Cell Spreading Assay. Cells (2.5 × 104) were seeded in six-well
plates on top of glass coverslips uncoated or coated with 10 μg/mL
human fibronectin (Millipore, Watford, U.K.). Cells in normoxia or
anoxia were incubated in medium containing 33 μM S4 or DMSO for
0, 15, 30, 45, or 60 min. The cell spreading was analyzed as previously
described.30
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bro-
mide (MTT) Assay. Cells (7500 cells per well for HT29, 1800
cells per well for HCT116 cells, and 2250 cells per well for
MDA-MB-231) were seeded in 96-well plates, and 24 h later the
medium was replaced with medium containing S4 (1−100 μM) or
DMSO. Medium was replaced again 24 h later with fresh medium,
and the cells were allowed to grow for an additional 96 h after
which cell proliferation was assessed in MTT assay as previously
described.31
Spontaneous Metastasis Model. eGFP-MDA-MB-231 cells
were prepared at 5 × 107 cells/mL in a 1:1 mix of serum-
free RPMI, and Matrigel (BD Biosciences, Erembodegem, Belgium)
eGFP-MDA-MB-231 cells were injected into the fourth inguinal area
nipple (orthotopic) of female nu/nu CBA mice aged 10−12 weeks old
(8 mice per group). eGFP-MDA-MB-231 tumors formed 5−7 days
after implantation, and the eGFP-MDA-MB-231 tumor volume
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