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Discovery of Inhibitors of MCF-7 Tumor Cell Adhesion to Endothelial Cells and Investigation on their Mode of Action.

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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 687−694
Thomas Bild,
Joachim Jose*,
Rolf W. Hartmann
Pharmaceutical and
Medicinal Chemistry,
Saarland University,
Saarbrücken, Germany
Inhibitors of MCF-7 Tumor Cell Adhesion 687
Discovery of Inhibitors of MCF-7 Tumor Cell
Adhesion to Endothelial Cells and Investigation
on their Mode of Action
Metastasis, the main reason for high mortality of cancer, is a multistep process.
One important step in this process is the adhesion of tumor cells to vascular
endothelium at sites distant from primary tumors during hematogenous dissemination. In order to investigate and quantify the adhesion of tumor cells to endothelial cells we developed an in vitro model using MCF-7 breast cancer cells
and monolayers of human umbilical vein endothelial cells (HUVEC). The tumor
cells were specifically labeled with a fluorescent dye for quantification; for increasing the amount of adherent cells, HUVEC monolayers were stimulated with
phorbol ester before the addition of the tumor cells. Due to previous reports that
products of several P450 enzymes contribute to the progression of certain kinds
of cancer, inhibitors of CYP5 (thromboxane A2 synthase), CYP17 (17α-hydroxylase-C17,20-lyase), and CYP19 (aromatase) were tested in this in vitro
model for their potency to reduce cancer cell adhesion. Within each series of
P450 inhibitors, compounds with high inhibitory activity on tumor cell adhesion
were identified. At an initial concentration of 100 µM, BW26, a potent inhibitor
of CYP5, reduced tumor cell adhesion of MCF-7 to HUVECs to 15 %, BW40
(CYP17) to 29 %, and SU5a (CYP19) to 11 % of the corresponding controls (no
inhibitor). Reduction of tumor cell adhesion was shown to occur in a concentration-dependent manner. In addition to these inhibitors of CYP5, CYP17, and
CYP19, liarozole, known to be a potent inhibitor of CYP26 (retinoic acid-4-hydroxylase) and ATRA (all-trans-retinoic acid) metabolism, was able to reduce
tumor cell adhesion to 51 % of the initial rate. Experiments elucidating the mode
of action of these compounds revealed that inhibition of the mentioned CYP
enzymes is not responsible for their ability to reduce tumor cell adhesion.
Keywords: Cancer metastasis; Tumor cell adhesion; MCF-7; Inhibitors of
CYP5, CYP17, CYP19, CYP26; ATRA metabolism
Received: August 30, 2004; Accepted: November 3, 2004 [FP922b]
DOI 10.1002/ardp.200400622
Introduction
Metastases, rather than primary tumors, are responsible for most cancer deaths [1], since the primary
tumor can mostly be excised by surgery. Hence, the
prophylaxis of cancer metastasis would be the best
therapeutic approach to reduce the mortality rate. For
developing a corresponding strategy it is important to
take into account that the formation of metastasis is a
multistep process [2]. Within this process the change
in the adhesive potential of tumor cells plays an essential role. The loss of intercellular adhesion of single
tumor cells from the solid primary tumor is the first step
essential for the formation of metastasis [3]. SubCorrespondence: Rolf W. Hartmann, Pharmaceutical and
Medicinal Chemistry, Saarland University, P.O. Box 151150,
D-66041 Saarbrücken, Germany. Phone: +49 681 302-2424,
Fax: +49 681 302-4386, e-mail: rwh@mx.uni-saarland.de,
web site: http://www.uni-saarland.de/fak8/hartmann/index.htm
sequently, new cell-to-cell or cell-to-extracellular matrix interactions are required for the development of
micrometastasis. Interaction of tumor cells to the basement membran is another important step during the
intra- and extravasation processes. Recently, we analyzed this step by developing a new in vitro model with
Matrigel® as mimicry of the basement membrane [4].
Using this model we elucidated the role of thromboxane A2 (TxA2) and CYP5 following several lines of evidence which strongly support the concept that this
metabolite of the arachidonic acid cascade contributes
to the formation of metastasis [5⫺7]. The results, however, indicated that inhibitors of CYP5 can not decrease tumor cell adhesion to Matrigel® [8].
* Current address: Department of Bioanalytics, Institute for
Pharmaceutical
Chemistry,
Heinrich-Heine-University,
Düsseldorf, Germany
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
688 Hartmann et al.
Another important step in the multistep process of
metastasis is the attachment of tumor cells to endothelial cells [9], called homing. It is the critical move
during hematogenous dissemination and escape from
the immunological response. Interestingly, the theories
about cancer cell homing differ widely. The mechanical
capture of tumor cells from the blood stream through
size restriction in small capillaries is discussed as one
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 687−694
possibility, resulting in subsequent adhesion to the
vessel wall [2]. Secondly, the adhesion of tumor cells
to the vessel wall in a way similar to leukocytes is proposed and called “docking and locking” hypothesis
[10]. In addition, cytokines can promote tumor cell adhesion by inducing the expression of cell adhesion
molecules on the endothelial surface [11]. In more recent investigations the importance of the tumor cell in-
Figure 1. Structures of the test compounds, synthesized in our group.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 687−694
Inhibitors of MCF-7 Tumor Cell Adhesion 689
teraction with endothelial cells has been demonstrated. It was proposed that tumor progress is dependent on the attachment of tumor cells to the endothelium followed by intravascular homotypic cancer
cell aggregation [12] or intravascular proliferation of attached tumor cells [13]. All these findings and hypotheses prompted us to develop an in vitro assay for
studying the effects of different enzyme inhibitors on
the adhesion process. For this purpose we used MCF7 breast cancer cells and phorbol ester-stimulated
HUVEC monolayers. The attached tumor cells were
quantified by a specific fluorescence labeling of the
MCF-7 tumor cells with CellTracker GreenTM. This derivative of fluorescein allows fast fluorimetric detection
in a manner similar to the well known method using
Calcein AM [14].
Results
For the development of an in vitro adhesion assay we
used fluorescence labeled MCF-7 breast cancer cells
and HUVECs as confluent monolayer. To quantify the
number of attached tumor cells on the endothelial
monolayer, MCF-7 cells were labeled with CellTracker
GreenTM before starting the adhesion assays. This reagent is converted by intracellular esterases to a fluorescent dye and captured inside the cell by enzymatic
coupling to soluble glutathion and other thiol-containing proteins. This method allows a permanent labeling
of living cells. Preincubation (1 h) of endothelial cells
with 10 ng/mL TPA (12-O-Tetradecanoylphorbol-13acetate) increased the adhesion of MCF-7 tumor cells
by a factor of more than two (data not shown). Using
the new assay we investigated compounds available
in our group (Figure 1) on their potential to inhibit
tumor cell adhesion to the endothelium. These compounds had been synthesized as inhibitors of CYP enzymes like thromboxane A2 synthase (CYP5), 17α-hydroxylase-C17, 20-lyase (CYP17), or aromatase
(CYP19). The MF series of compounds and BW26 are
potent inhibitors of CYP5 [15], whereas BW compounds with a biphenyl structure are highly active inhibitors of CYP17 [16]. More recently, SU compounds
with pyridylmethylene-tetrahydronaphthalene/ -indane
structures have been synthesized in our group and
identified as highly potent inhibitors of aldosterone
synthase (CYP11B2). Some of the latter compounds
Figure 2. Structures of the reference compounds.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
690 Hartmann et al.
were also shown to be potent inhibitors of CYP19
(Ulmschneider et al., submitted).
In addition, we added several well known inhibitors of
these enzymes and cyclooxygenase (COX, Figure 2)
to our in vitro study on the adhesion of tumor cells to
the endothelium. The COX inhibitors ASS and
indometacin were chosen because COX catalyzes the
step immediately before CYP5 in the metabolic pathway of arachidonic acid.
As shown in Figure 3, ASS, indometacin, dazoxiben,
and ridogrel had no effect on tumor cell adhesion to
endothelial cells when compared to the controls. In
contrast, BW26 tested in a concentration of 100 µM
reduced tumor cell adhesion to residual 15 % of the
initial adhesion rate. All other inhibitors of CYP5 had
no significant effect on adhesion. The evaluation of the
CYP17 and CYP19 inhibitors synthesized in our group
together with reference compounds like aminoglutethimide, fadrozole, and liarozole led to interesting results. The CYP17 inhibitor BW40 reduced tumor cell
adhesion to 29 % and the CYP19 inhibitor SU5a reduced tumor cell adhesion to 11 % compared to the
corresponding controls (Figure 3). Although other inhibitors of CYP17 and CYP19 were also capable to
reduce the adhesion of MCF-7 cells to the endothelium (BW39: 53 % reduction, SU7a: 28 % reduction,
SU15a: 34 % reduction), we were not able to correlate
this inhibitory effect on tumor cell adhesion with the
potency of the corresponding compounds on enzyme
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 687−694
inhibition. Aminoglutethimide and fadrozole, for example, two potent CYP19 inhibitors, as well as SU5b,
did not affect adhesion of MCF-7 cells at all. A similar
result was obtained with the MF series of compounds,
dazoxiben and ridogrel (CYP5 inhibitors) as well as
with BW51, BW56, BW60 (CYP17 inhibitors), and the
COX inhibitors ASS and indometacin. Surprisingly, the
CYP19 inhibitor liarozole reduced MCF-7 tumor cell
adhesion by almost 50 % in a concentration of 100 µM.
To find out whether these effects were indeed due to
the compound added, tumor cell adhesion was monitored with increasing inhibitor concentrations. As can
be seen in Figure 4, reduction of tumor cell adhesion
was a concentration-dependent effect for liarozole as
well as for SU5a, the most potent of the compounds.
Besides, the reduced attachment of MCF-7 tumor cells
to HUVECs could be observed and documented by
phase-contrast and fluorescence microscopy (Figure
5).
For all compounds reducing cell-to-cell adhesion cytotoxicity was determined with a commercially available
cytotoxity detection kit, measuring LDH (lactic dehydrogenase) activity. No effect on cell viability was
detectable under conditions applied in the adhesion
assays (data not shown) and no damages of the
endothelial monolayers could be detected after the
adhesion assays with these compounds (routinely
monitored by phase-contrast microscopy). A typical
example is shown in Figure 5.
Figure 3. Adhesion of MCF-7 cells to HUVEC monolayer, quantified by fluorescence labeling as described in the
experimental section. Compounds were tested at an initial concentration of 100 µM. Results are shown as percentage tumor cell adhesion in comparison to controls without inhibitor and represent the mean (± SD) of three
independent assays (except BW26 and BW40: two assays). Within each assay values were determined as quadruplicates for each sample.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 687−694
Inhibitors of MCF-7 Tumor Cell Adhesion 691
Figure 4. Inhibition of MCF-7 tumor cell adhesion to HUVECs by liarozole and SU5a. Both compounds were
tested at increasing concentrations. Results are shown as percentage tumor cell adhesion in comparison to the
controls without inhibitor and represent the mean (± SD) of three independent assays. Within each assay values
were determined as quadruplicates for each sample.
Figure 5. Phase-contrast microscopy and fluorescence microscopy of MCF-7 cells attached to HUVEC monolayers. HUVEC monolayers were preincubated with 10 ng/mL TPA for 60 min at 37 °C. MCF-7 tumor cells were
labeled with 2.5 µM CellTracker GreenTM (Molecular Probes) for 30 min before being added to the endothelial
cells. For each of the three samples the visual field is identical in the phase-contrast and the fluorescence view.
In addition to its inhibitory effect on CYP26, liarozole
is known to be an inhibitor of CYP17 and CYP19 [17].
As our results indicated that the capacity to inhibit
CYP17 and CYP19 was not a sufficient prerequisite
for reducing tumor cell adhesion, we focused on the
role of CYP26. If inhibition of CYP26 was the cause of
reduced tumor cell adhesion, not only liarozole, but
also SU5a should be able to inhibit CYP26. Consequently, we established an enzyme activity assay for
CYP26 and investigated the effect of SU5a. For this
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
692 Hartmann et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 687−694
purpose the conversion of ATRA to polar metabolites
in MCF-7 breast cancer cells was investigated. The
formation of metabolites was time-dependent and
could be monitored by using radiolabeled substrate
[18]. In contrast to liarozole which blocks the ATRA
conversion at a concentration of 100 µM completely,
SU5a has no influence on CYP26 activity at all.
was described that structural remodelling in human
breast carcinoma MCF-7 cells occurred after treatment with liarozole [30] and severe alterations in the
expression of cell adhesion molecules by ATRA were
observed [31, 32]. Hence, we established an assay for
CYP26 and investigated the inhibitory potency of
SU5a. Although SU5a is the most active compound in
the adhesion assay, SU5a failed to inhibit the conversion of ATRA.
Discussion
In conclusion, we identified several compounds capable of blocking the in vitro MCF-7 tumor cell adhesion
to endothelium. However, our data indicate that inhibition of CYP5, CYP17, CYP19, and CYP26 appear to
be not the only cause for the reduction of tumor cell
adhesion. Therefore, additional investigations are
necessary to identify the mode of action of these compounds in reducing tumor cell adhesion.
A critical step in the formation of metastasis is the adhesion of tumor cells to vascular endothelium. Several
research groups have shown that this tumor cell/endothelial cell interaction can be increased by cytokines
[19, 20], but can also be reduced by several compounds like cimetidine [21], lovastatin [22], or nordihydroguiaretic acid [23]. The findings that lipoxygenase
and its products could be involved in this step of metastasis [23, 24] and several indications that CYP5
could also play an essential role therein [6, 7] encouraged us to develop a new, fast in vitro assay for the
evaluation of compounds synthesized in our group.
Similar to earlier results obtained with an in vitro model
on tumor cell adhesion to Matrigel® [8], most CYP5
inhibitors failed to reduce adhesion, with a single exception. BW26 diminished both, tumor cell adhesion
to HUVECs (Figure 3) as well as to Matrigel® [8]. In
order to clarify the mode of action of BW26, additional
compounds and enzyme inhibitors including inhibitors
of various CYP enzymes were tested. CYP17 inhibitors and CYP19 inhibitors were chosen because the
corresponding enzymes CYP17 [25] and CYP19 [26,
27] have been reported to be expressed in breast cancer tissue. It is well known that the proliferation of
MCF-7 tumor cells is increased in the presence of estrogens [28] as well as androgens [29]. These findings
indicate the possibility that these enzymes might also
be involved in metastatic processes of cancer cells.
Although some of the compounds reduced the tumor
cell adhesion to endothelial cells, this property did not
correlate with the inhibitory activities of the compounds
towards one of the two enzymes. Interestingly, in contrast to aminoglutethimide and fadrozole, liarozole
blocked the attachment of MCF-7 to HUVEC significantly. Liarozole is known to inhibit CYP17 and
CYP19, but is also a potent inhibitor of CYP26. CYP26
catalyzes the conversion of all-trans-retinoic acid
(ATRA) to polar metabolites and is an enzyme with
inducible activity in MCF-7 cells. As liarozole can inhibit the enzymatic conversion of ATRA and ATRA
plays an important role in several cellular processes
like differentiation and proliferation we took into consideration that CYP26 participates in the process of
MCF-7 cell adhesion to the endothelium. Recently, it
Experimental
Cells
Human umbilical vein endothelial cells were isolated from
fresh human umbilical cords by treatment with 0.5 mg/mL collagenase (Serva, Heidelberg, Germany) and cultured on
gelatine-coated culture dishes in a 1:1 mixture of endothelial
cell growth medium 2 kit with supplement mix (PromoCell,
Heidelberg, Germany) and MCDB 131 supplemented with 2
mM L-glutamine, 1 mM pyruvate, penicillin and streptomycin
(100 µg/mL), and 10 % fetal bovine serum (FBS). HUVECs,
between passage 2 and 5, were used in this study. HUVECs
obtained from several unrelated donors were assessed, but
HUVECs from a single donor were used within each experiment. No significant differences in the evaluation of control
substances were observed for cells from different donors.
MCF-7 is an adherent tumor cell line which was isolated
from a pleural effusion of a breast cancer patient and was
kindly provided by Prof. Dr. M. Schneider (Schering AG,
Berlin, Germany). This cell line was maintained in standard
MEM supplemented with 0.01 mg/mL insulin, 2 mM L-glutamine, 1 mM pyruvate, penicillin and streptomycin (100 µg/
mL), and 10 % FBS. Tissue culture reagents were from
c.c.pro GmbH (Neustadt/Weinstr, Germany).
Reagents
CellTracker GreenTM was provided by Molecular Probes
(Leiden, Netherlands). The reference compounds were gifts
of the following companies: dazoxiben (Pfizer Central
Research, Sandwich, UK), ridogrel (Bayer AG, Wuppertal,
Germany), aminoglutethimide (Bachem, Bubendorf, Switzerland), fadrozole (Novartis, Basel, Switzerland), and liarozole
(Janssen Pharmaceutics, Beerse, Belgium). [3H]-ATRA was
purchased from PerkinElmer Life Sciences (Boston, MA,
USA). The ATRA metabolite 4-oxo-retinoic acid was a gift
from Dr. E.-M. Gutknecht and A. Perrin (Roche, Basel,
Switzerland). All other chemicals were obtained from SigmaAldrich (Deisenhofen, Germany).
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 687−694
Inhibitors of MCF-7 Tumor Cell Adhesion 693
Selected CYP inhibitors synthesized in our group have the
following structures and IC50 values:
MF15- 5-(1-Imidazolylcarbonyloxy)-5,6,7,8-tetrahydroquinoline, IC50 (CYP5): 1.6 µM, [33]
MF20- 1-[2-(Imidazol-1-yl)ethyl]-1,2,3,4-tetrahydronaphthalene, IC50 (CYP5): 3.4 µM, [34]
MF27- 7-[(Imidazol-1-yl)methyl]isoquinoline, IC50 (CYP5):
5.4 µM, [15]
BW26- 1-[(1,2-Dihydro-4-ethyl-7-methoxynaphthalin-3-yl)methyl]-1H-imidazole, IC50 (CYP5): 3.7 µM, [35]
BW37- 4⬘-Methoxy-4-(imidazol-1-yl-methyl)-biphenyl, IC50
(CYP17): 3.7 µM, [16]
BW39- 4⬘-Chloro-4-(imidazol-1-yl-methyl)-biphenyl, IC50
(CYP17): 5.8 µM, [16]
BW40- 4⬘-Methyl-4-(imidazol-1-yl-methyl)-biphenyl, IC50
(CYP17): 4.2 µM, [16]
BW51- 4⬘-Fluoro-3-(imidazol-1-yl-methyl)-biphenyl, IC50
(CYP17): 2.9 µM, [16]
BW56- 4⬘-Cyano-4-(imidazol-1-yl-methyl)-biphenyl, IC50
(CYP17): 2.5 µM, [16]
BW60- 4⬘-Hydroxy-4-(imidazol-1-yl-methyl)-biphenyl, IC50
(CYP17): 0.31 µM, [16]
SU5a- 3-[(E)-(6-methoxy-3,4-dihydronaphthalen-1(2H)-ylidene)methyl]pyridine, IC50 (CYP19): 1.13 µM
SU5b- 3-[(Z)-(6-methoxy-3,4-dihydronaphthalen-1(2H)-ylidene)methyl]pyridine, IC50 (CYP19): 4.00 µM
SU7a- 3-[(E)-(5-methoxy-2,3-dihydro-1H-inden-1-ylidene)methyl]pyridine, IC50 (CYP19): 4.31 µM
SU15a- 3-[(E)-(6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)ylidene)methyl]pyridine, IC50 (CYP19): 0.98 µM
Fluorescence labeling of tumor cells
the test compounds for 30 min at 37 °C under shaking at 120
rpm. Endothelial monolayers were washed with medium to
remove TPA and 2.5 ⫻ 105 tumor cells were added per well
in a final volume of 500 µL serum-free MEM. Endothelial cells
were incubated with tumor cells for 60 min in a humidified
atmosphere at 37 °C without shaking. Following this incubation, monolayers were washed 4x with MEM containing
0.5 % BSA to remove nonadherent cells. Phase-contrast microscopy was performed to check the HUVEC monolayers
for consistency and fluorescence microscopy was performed
to ascertain tumor cell labeling. Subsequently, all attached
cells were solubilized with 1 % SDS in buffer (pH 12) and the
well plate was frozen over night at ⫺20 °C. The following day,
plates were thawed at 30 °C and the fluorescence of each
well was determined using a multilabel counter (Wallac 1420
Victor2, PerkinElmer Life Sciences) with an excitation filter at
485 nm and an emission filter at 535 nm. All compound
samples and the controls containing DMSO as solvent in the
same concentration were tested in quadruplicates within
each experiment. Percent inhibition in comparison to the
DMSO control values was determined. Every test row was
usually repeated at least twice.
Cytotoxicity assay
Cytotoxicity of the compounds was determined using the
cytotoxicity detection kit (LDH) from Roche Diagnostics
GmbH (Mannheim, Germany). All assays and calculations
were performed according to the instruction manual of Roche
Diagnostics. HUVECs were tested as adherent monolayers
for 60 min and MCF-7 cells as cell suspension for 2 h. All
compounds were tested in quadruplicate for each experiment
and complete experiments were repeated twice.
Retinoic acid metabolism
CellTracker GreenTM from Molecular Probes was used for the
specific labeling of the tumor cells. This reagent freely diffuses into the cells, where cytosolic esterases cleave the
acetate groups of this fluorescein derivative releasing the fluorescent product. Besides this enzymatic cleavage the fluorescent dye is captured inside the cells by enzymatic coupling
to thiol-containing proteins like glutathione, i.e. this reagent
causes a cell-specific, long-term labeling. Semiconfluent
MCF-7 tumor cells were washed twice with PBS and incubated with serum-free MEM containing 2.5 µM CellTracker
GreenTM for 30 min at 37 °C in a CO2 incubator, followed by
30 min incubation with serum-free medium alone to enable
the complete conversion of the fluorescent label.
Adhesion assay
24-well plates (Greiner Bio-one, Frickenhausen, Germany)
were covered with 0.1 % gelatine solution in PBS buffer and
aspirated after 30 min incubation at 37 °C. 7.5 ⫻ 104 HUVECs
were seeded per well and cultured in the 1:1 mixture of endothelial cell growth medium 2 kit with supplement mix and
MCDB 131 supplemented with 2 mM L-glutamine, 1 mM pyruvate, penicillin and streptomycin, and 10 % FBS over night.
Prior to each set of experiments, HUVECs were checked for
confluency using phase-contrast microscopy. Confluent
monolayers were subsequently incubated with MEM containing 0.5 % bovine serum albumin (BSA) and 10 ng/mL phorbol
ester TPA for 60 min at 37 °C to increase the adhesive potential of the endothelium. During this period, the fluorescencelabeled tumor cells were harvested by adding nonenzymatic
cell dissociation solution for 15 min at 37 °C. Subsequently,
MCF-7 cells were incubated in the presence or absence of
To quantify the CYP26 activity in MCF-7 cells, the metabolism
of retinoic acid was determined. Therefore, the conversion of
all-trans-retinoic acid (ATRA) was measured similar as described by Wouters et al. [36]. Semiconfluent MCF-7 cell cultures were incubated for 16 h with 1 µM ATRA in MEM medium containing 5 % DCC-treated (dextran-coated charcoal),
heat-inactivated FBS [37]. Subsequently, cells were washed
twice with PBS, trypsinated, and resuspended to a final concentration of 4 ⫻ 106 cells/mL. Aliquots (450 µL) of this cell
suspension were supplied with 25 µL of test compound solution or solvent and incubated in black reaction tubes (1.5 mL)
for 10 min at 37 °C with shaking (150 rpm). Then, 25 µL of a
mixture of [3H]-ATRA (200 nCi/tube) and unlabeled substrate
(final concentration: 0.1 µM) was added and incubation was
continued for another 90 min (37 °C, 150 rpm). The enzymatic
reaction was stopped by addition of 10 µL formic acid (5 % in
water) and 600 µL of dichloromethane-methanol (2:1, v/v),
containing 50 µg/mL of the antioxidant 2,6-Di-tert-butyl-4methylphenol (BHT). The mixture was vortexed for 1 min and
centrifuged for 5 min at 5000g to separate the phases. The
organic phase was transferred into a new black reaction tube
and the aqueous phase was extracted again with 600 µL of
dichloromethane-methanol (2:1, v/v), containing 50 µg/mL
BHT. The organic phases were collected, evaporated to dryness, and stored overnight at ⫺20 °C for subsequent HPTLC
analysis. All experiments with retinoids were performed in a
dark room with yellow illumination.
TLC analysis of retinoid metabolites
The dried samples were dissolved in 25 µL dichloromethane
containing 50 µg/mL BHT and applied to 20 ⫻ 10 cm HPTLC
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
694 Hartmann et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 687−694
plates (Silicagel 60 F254 with concentration zone; Merck,
Darmstadt, Germany). TLC plates were developed twice in a
glass chamber equilibrated for 1 h with 150 mL hexane/ether/
acetic acid (90:60:2, v/v/v) [38] and containing one sheet of
filter paper (Schleicher und Schüll, Dassel, Germany). ATRA
conversion was analyzed after exposition of TLC plates to
a BAS-TR2040 imaging plate for 48 h. Conversion of cells
incubated without inhibitor was set as 100 %. The imaging
plates were finally scanned by the FLA 3000 phosphoimager
system (raytest GmbH, Straubenhardt, Germany) for the detection of ATRA and its metabolites.
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