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Experimental Therapeutics |
Cancer Research Laboratories, School of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom [M-S. C., T. D. B., M. F. G. S.], and Laboratory of Drug Discovery Research and Development, Developmental Therapeutic Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH, Frederick, Maryland 21702-1201 [E. K., S. F. S., E. A. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-naphthoflavone. Both -naphthoflavone and 6-OH
203 abrogated DF 203-induced growth inhibition. Microsomes from
genetically engineered human B-lymphoblastoid cells expressing CYP1A1,
CYP1B1, or CYP2D6 metabolized DF 203 to 6-OH 203. Immunoblot analysis
detected significantly enhanced CYP1A1 protein in a panel of sensitive
breast cancer cell lines after exposure to DF 203. Neither constitutive
expression nor induction of CYP1A1 protein was detected in
nonresponsive breast (HBL 100, MDA-MB-435, and MCF-7/ADR) and prostate
(PC 3 and DU 145) cancer cell lines. The expression of CYP1B1 was also
modulated by DF 203 in the same sensitive cell lines. However, of the
two isoforms, only CYP1A1 activity was irreversibly inhibited by DF 203
and significantly inhibited by 6-OH 203. In sensitive cell lines only,
[4C]DF 203-derived radioactivity bound covalently to a
Mr 50,000, protein which was
immunoprecipitated by CYP1A1 antiserum. The covalent binding of
[14C]DF 203 to recombinant CYP1A1 enzyme was
NADPH-dependent and reduced by 6-OH 203 and glutathione. CYP1A1 appears
essential for the metabolism of DF 203 and may have a pivotal, yet
undefined, role in its antitumor activity. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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substituted at the 3'-position with either a methyl group (DF 203) or
a halogen atom (Cl, Br, I) comprise a class of agent with
remarkably similar, highly selective profiles of antitumor activity
(1, 2, 3)
. Antitumor fingerprints are unique; agents failed
to COMPARE (4)
with any clinical class of chemotherapeutic
agent. No molecular target(s) have been identified.
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Rapid and extensive N-acetylation is the major metabolic route of unsubstituted 2-(4-aminophenyl)benzothiazole (CJM 126) in vitro and of DF 203 in vivo in rats (5) . However, the major in vitro biotransformation pathway of 3'-substituted analogues is C-oxidation within the benzothiazole nucleus; DF 203 was metabolized by sensitive MCF-7 cells to a major metabolite that cochromatographed with 6-OH 203 (6) . Homogenates prepared from untreated MCF-7 or T-47D cells failed to catalyze the 6-hydroxylation of DF 203; the ability to do so was induced only after pretreatment of cells with DF 203.
The main oxidizing enzymes in phase I metabolism are CYPs4 that catalyze the initial step in either detoxification or bioactivation of environmental toxins and xenobiotics (7, 8, 9) . CYP1 isoforms are capable of activating aromatic amines; therefore, extrahepatic and cell line-specific regulation of CYP1A1 and CYP1B1 (10) are major determinants of chemosensitivity or resistance.
Herein, we report identification of CYP isoforms responsible for
6-hydroxylation of DF 203 and specific inhibition by -NF
of 6-hydroxylase activity and growth arrest
in MCF-7 cells treated with DF 203. This novel antitumor agent
modulates the expression and activity of CYP1A1 and CYP1B1 in sensitive
cell lines, and the implications of these observations on its antitumor
properties are discussed. In addition, we present evidence to support
that CYP1A1 is the covalently bound Mr
50,000 protein in sensitive cell lines.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-NF and ß-NF were from Aldrich
(Milwaukee, WI), and furafylline was from Ultrafine Chemicals
(Manchester, United Kingdom). HPLC-grade acetonitrile was purchased
from Baker (Philipsburg, NJ). All chemicals were of the highest
commercial grade. Cell culture media and supplements were supplied by
Life Technologies, Inc. (Gaithersburg, MD; Paisley, United Kingdom) and
Costar (Cambridge, MA; Buckinghamshire, United Kingdom). Abs specific
for CYP1A1 (polyclonal antiserum for human CYP1A1/1A2) and CYP1B1
(polyclonal rabbit antihuman CYP1B1 primary Ab), as well as a panel of
CYP microsomes, were obtained from Gentest Corp. (Woburn, MA;
Cambridge, United Kingdom). ECL kits were purchased from Pierce, and
alkaline phosphatase kits were from Upstate Technology (San Diego, CA).
In Vitro Cell Culture.
Monolayer cells were cultured at 37°C in an atmosphere of 5%
CO2 in RPMI 1640 containing 2 mM
L-glutamine and supplemented with 10% FCS, 100 IU/ml
penicillin, and 100 µg/ml streptomycin. Cells were maintained in
continuous logarithmic growth by routine subculturing twice weekly.
Cell lines used include human breast carcinoma cell lines MCF-7, MDA
468, MDA-MB-435, T-47D, SKBR3, ZR 75, and MCF-7/ADR; human prostate
carcinoma cell lines PC 3 and DU 145; and a nonmalignant human breast
cell line HBL 100.
MTT Colorimetric Assay.
Cells were seeded onto 96-well microtiter plates at a density of
5 x 103
per well and allowed
24 h to adhere before drugs were added over a concentration range
of 0.1 nM to 100 µM (n = 8). Serial dilutions were prepared in media prior to each assay,
with final DMSO concentration <0.25%. After 72-h exposure, MTT was
added to each well (final concentration, 400 µg/ml) and incubated for
4 h to allow metabolism of MTT by mitochondrial dehydrogenase to
an insoluble formazan product. Medium was then aspirated, and formazan
was solubilized by the addition of 125 ml of DMSO:glycine buffer (pH
10.5; 4:1). Cell viability was determined as absorbance at 550 nm, read
on an Anthos Labtec systems plate reader. An MTT assay performed at the
time of drug treatment determined an initial absorbance from which cell
growth or drug toxicity could be assessed.
Metabolism of DF 203 by Cell Homogenates.
Homogenates were prepared from cells pretreated for 24 h with DF
203, 3-MC (1 µM), ß-NF (10 µM), or
vehicle control (0.1% DMSO). Metabolites of DF 203 were detected by
HPLC. A typical reaction mixture consisted of 100 µM DF
203, 100 mM Tris-HCl (pH 7.4), 5 mM
MgCl2, 0.5 mM NADH, NADPH-generating
system (1 mM NADP+, 5 mM
glucose 6-phosphate, and 1 unit/ml glucose 6-phosphate dehydrogenase)
and MCF-7 homogenate (1 mg/ml) in a final volume of 0.2 ml. DF 203 was
dissolved in acetonitrile and added to the incubation mixture at a
final acetonitrile concentration of 1%. The reaction mixture was
preincubated at 37°C for 5 min, and the reaction was started by the
addition of NADH and NADPH-generating system. After 30 min incubation
at 37°C, 0.6 ml of ice-cold acetonitrile was added to stop the
reaction. The reaction mixture was centrifuged at 14,000 rpm for 10
min, and supernatant (100 µl) was analyzed by HPLC.
The HPLC system consisted of a Hewlett Packard 1050 Series Module
(solvent delivery pump, autosampler, and multiple wavelength detector;
Hewlett Packard, Palo alto, CA) and a Hewlett Packard 1046A
fluorescence detector. Compounds were separated at room temperature on
a C18 reversed-phase column (YMC-ODS-AQ, 150 x 4.6 mm
inside diameter, S-5 µm; YMC Inc., Wilmington, NC). The mobile phase
composition was changed linearly during 40 min from 10:90:1 to 80:20:1
(acetonitrile:water:acetic acid). Mobile phase was continuously
degassed with nitrogen, and the flow rate through the column was 1
ml/min. UV detection was at 338 nm, and fluorescence detection was at
ex 344 nm and em 434
nm.
Metabolism of DF 203 by Specific Human P450 Isoforms.
Microsomes from human B-lymphoblastoid cells expressing human CYP
isoforms 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9-Arg, 2C9-Cys, 2C19, 2D6,
2E1, and 3A4 (11)
were tested for their ability to
metabolize DF 203. Microsomes from cells carrying the expression vector
without P450 cDNA were used as a negative control. Reactions were
conducted as described above but with incubation periods of 2 h.
Inhibition of DF 203-induced Metabolism.
A typical incubation mixture contained 100 mM Tris-HCl
buffer (pH 7.4), 5 mM MgCl2,
NADPH-generating system, and 1 mg/ml MCF-7 cell homogenate (pretreated
for 24 h with 1 µM DF 203). -NF, furafylline,
coumarin, 7-ethoxycoumarin, sulfaphenazole, quinidine, DEDC, or TAO
were each added to the incubation mixture (100 µM in DMSO
vehicle; final DMSO concentration, 0.5%) as relatively specific
inhibitors of P450 isoforms. Each inhibitor was preincubated for 15 min
with the reaction mixture before the reaction was initiated by addition
of DF 203.
For immuno-inhibition studies, homogenates of DF 203-treated MCF-7 cells were preincubated with varying concentrations of human CYP1A1-selective polyclonal goat antirat CYP1A1 serum or normal goat serum at room temperature for 30 min before incubations were carried out as described.
Assay of EROD Activity.
A sensitive and rapid HPLC method was used to measure EROD activity
(12)
. The incubation mixture consisted of 100
mM Tris-HCl (pH 7.4), 5 mM
MgCl2, NADPH-generating system, and either MCF-7
cell homogenate (1 mg/ml) or microsomes expressing recombinant CYP
isoforms (0.1 mg/ml) and 80 µM 7-ethoxyresorufin in a
final volume of 0.2 ml. The mixture was preincubated for 5 min at
37°C, and the reaction was initiated by addition of the
NADPH-generating system. After incubation (15 min for recombinant
CYP1A1 and 30 min for recombinant CYP1B1 and MCF-7 homogenate), 0.6 ml
of ice-cold acetonitrile was added to stop the reaction. The reaction
mixture was centrifuged at 14,000 rpm for 10 min, and supernatant (100
µl) was analyzed by HPLC using a mobile phase of 25 mM
phosphate buffer (pH 7.0):acetonitrile (75%:25%, v/v) at a constant
flow rate of 1 ml/min. Resorufin fluorescence was detected at
ex 530 nm and em
580 nm.
Inactivation of EROD Activity.
Microsomes from human B-lymphoblastoid cells expressing human CYP1A1 or
CYP1B1 were incubated with NADPH-generating system in the presence or
absence of 100 µM DF 203 or concentrations of 6-OH 203 at
37°C for 30 min before EROD activity was measured. To investigate
EROD inactivation by DF 203, microsomes were incubated with
NADPH-generating system in the presence or absence of 100
µM DF 203 (37°C for 30 min) and 0.8 ml of hypotonic
buffer added. The reaction mixtures were ultracentrifuged at
100,000 x g for 30 min, and supernatant was
removed. The microsome precipitate was resuspended in 1 ml of hypotonic
buffer and ultracentrifuged again. The resulting microsome precipitate
was then assayed for EROD activity.
Western Blot Protocol.
Whole-cell lysates were prepared for examination of CYP1A1 and CYP1B1
expression. After appropriate treatment, cells were detached by
trypsinization, washed in PBS, and counted. Cells were pelleted by
centrifugation (5 min at 12,000 rpm) and lysed by the addition of lysis
buffer containing 20 mM Tris-HCl (pH 7.4), 2 mM
EDTA (pH 7.4), 2 mM EGTA (pH 7.4), 6 mM
ß-mercaptoethanol, 10 µg/ml leupeptin, 2 µg/ml aprotinin, and 1%
v/v NP40. Cell lysates were sonicated (3 x 10-s bursts,
setting 25 of MSE Soniprep 150 sonicator, United Kingdom), protein
content was estimated by the Bradford assay (13)
, and
sample buffer was added. Samples were boiled at 95°C for 5 min, and
solubilized proteins (50 µg) were separated on 10% gels by SDS-PAGE
before electroblotting to polyvinylidene difluoride membranes
(Bio-Rad, United Kingdom). Membranes were blocked for 1 h
in PBS-0.1% Tween 20 (T), 10% dried milk and then washed 3 x 5 min with PBS 0.1% before incubation (2 h) with goat
antirabbit polyclonal primary Ab (1:500 in PBS 0.02% T, 1% milk).
After washing, membranes were incubated for 1 h with alkaline
phosphatase-conjugated rabbit antigoat secondary Ab (1:5000 in PBS
0.02% T, 1% milk). CYP1A1 was detected after 10 min incubation with
substrates (bromochloroindolyl phosphate and nitro blue tetrazolium) in
alkaline phosphatase buffer.
For CYP1B1 immunoblots, nonspecific binding was blocked with 5% dried skimmed milk in TBS (0.05 M, pH 7.9)-0.1% T for 1 h at room temperature. After washing with TBS-0.1% T, membranes were probed with CYP1B1 specific primary Ab (1:500 in TBS-0.01% T, 0.5% dried skimmed milk) for 1 h at room temperature. Membranes were rinsed with TBS-0.1% T and incubated for 1 h with horseradish peroxidase-conjugated goat antirabbit secondary Ab (1:500 in TBS-0.01% T, 0.5% dried milk). After rinsing the membranes (3 x 5 min with TBS-0.1% T and 2 x 5 min with TBS), CYP1B1 was detected by ECL.
Separation of Protein Bound to [14C]DF 203-derived
Radioactivity.
Cells were grown in 150-mm tissue culture Petri dishes to 50–70%
confluency and treated with 0.1, 1, and 10 µM
[14C]DF 203. After 24 h incubation, medium
was removed, and cells were rinsed with cold PBS (pH 7.4). Protein was
solubilized by addition of lysis buffer containing 50 mM
HEPES buffer (pH 7.4), 50 mM NaCl, 1% Triton X-100, 10%
glycerol, 5 mM EGTA, 15 mM
MgCl2, 20 mM NaF, 50 mM
ß-glycerophosphate, 2 mM phenylmethylsulfonyl fluoride, 1
mM Na3VO4, 10
µg/ml aprotinin, and 10 µg/ml leupeptin. After centrifugation at
14,000 rpm for 15 min at 4°C, the supernatant was collected for
estimation of protein content, and Laemmli’s buffer was added. Samples
were boiled for 5 min, and 200 µg of protein were loaded onto 4–20%
gradient SDS-polyacrylamide slab gel. SDS-PAGE was carried out
according to the method of Laemmli (14)
. After
electrophoresis, the gel was fixed for 1 h with 40% TCA, 30%
methanol, and 10% acetic acid and then kept for 1 h in
autoradiography enhancer (EN3
HANCE; NEN Research
Products, Boston, MA). The gel was stored in 2% glycerol solution
overnight, dried for 2 h under vacuum at 60°C, and
autoradiographed by exposure to X-ray film (Kodak) at -70°C for 2–4
weeks in the dark.
Immunoprecipitation.
Cells were grown in 150-mm Petri dishes to 50–70% confluency before
treatment with 10 µM [14C]DF 203
for 24 h, and cell lysates were prepared as described for
autoradiography.
Each immunoprecipitation sample consisted of 50 µl of protein A-agarose beads, 5 µl of CYP1A1 (or 10 µl of CYP1B1) antiserum, 500 µl of lysis buffer, and cell lysate (500 µg or 1 mg of protein). Samples were incubated for 4 h at 4°C with gentle end-on-end rocking, and beads were pelleted by centrifugation at 14,000 rpm for 5 s. Supernatant was aspirated, and beads were washed three times with 750 µl of wash buffer [20 mM HEPES (pH 7.4), 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol, with 1 mM Na3VO4 added fresh before use]. Laemmli buffer (25 µl) was added to each of the washed beads, and samples were boiled for 5 min before SDS-PAGE on 8% precast polyacrylamide gels. Gels were prepared for autoradiography as described above. At the same time, a duplicate set of immunoprecipitated samples was prepared for immunoblotting with CYP1A1 antiserum with ECL detection.
In Vitro Covalent Binding to Recombinant CYP1A1.
Each incubation consisted of 100 mM Tris-HCl (pH 7.4), 2
mM MgCl2, 1 mM NADPH, and
10 µM [14C]DF 203. In control
samples, NADPH was omitted, whereas in competition assays, 100
µM 6-OH DF 203 or 1 mM GSH was added. The
final volume of each incubation was 200 µl. Reactions were started by
addition of 50 µg of CYP1A1 microsomes to preincubated mixtures and
terminated after 30 min at 37°C by the addition of 50 µl of cold
50% TCA solution (final concentration of 10% TCA) to precipitate the
proteins. Protein pellets were washed once with cold 10% TCA and then
exhaustively with 60°C methanol until radioactivity in the
supernatant fell to background level. Protein pellets were solubilized
with 1 N NaOH at 80°C and determined for protein content
and radioactivity.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Maximum activity occurred after 24 h exposure to 1
µM DF 203, levels of 6-hydroxylase activity
decreased with DF 203 concentrations >10 µM or
prolonged drug exposure. In MCF-7 cells, induction was observed as
early as 2 h after DF 203 treatment (Fig. 2B)
. In
addition, DF 203 6-hydroxylase activity was efficiently induced after
treatment of MCF-7 cells (24 h) with 3-MC (1
µM) or ß-NF (10 µM;
Fig. 2C
). However, 6-hydroxylation was not detected in
either vehicle-treated MCF-7 cells or unresponsive MDA-MB-435
homogenates, regardless of treatment conditions.
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-NF (15
, 16)
, furafylline
(17)
, coumarin (18)
, 7-ethoxycoumarin
(19)
, sulfaphenazole (20)
, quinidine
(21)
, DEDC (22)
, and TAO (23)
.
-NF strongly inhibited DF 203 6-hydroxylation, whereas furafylline
was a weak inhibitor (Fig. 3)
, implicating an oxidative role for CYP1A1 and/or CYP1B1. Human
CYP1A1-selective polyclonal goat antirat CYP1A1 serum inhibited the
6-hydroxylase activity induced in homogenates of MCF-7 cells treated
with 1 µM DF 203 for 24 h (Fig. 4)
. At the highest antisera concentration (0.3 ml/mg protein),
6-hydroxylation of DF 203 was inhibited by 90%. These data suggest
CYP1A1 as the major isoform that catalyzes 6-hydroxylation of DF 203.
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-NF
(Fig. 5A)
. This protective effect against growth inhibition was
similarly observed with 6-OH DF 203 (Fig. 5B)
. Furafylline
had no effect on DF 203-induced growth inhibition.
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and to a lesser extent by CYP1A2, CYP1B1, CYP2C9-ARG, and
CYP2D6. Other unidentified oxidation products of DF 203 were also
produced. The Kms for 6-hydroxylation
by CYP1A1, CYP1B1, and CYP2D6 were estimated by Lineweaver-Burk plots
(Fig. 6B)
: 4.2 µM (CYP1A1), 4.5
µM (CYP1B1), and 1 mM
(CYP2D6). Thus, CYP1A1 and CYP1B1 possess greater affinity for DF 203,
more readily catalyzing 6-hydroxylation under experimental
conditions.
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. The concentration-dependent profile of EROD activity is similar to
that of DF 203 6-hydroxylation, with maximum activity observed after 1
µM drug exposure.
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. After removal of DF 203 and recovery of microsomes by
ultracentrifugation, microsomes expressing human CYP1A1 showed little
recovery of EROD activity. In contrast, EROD activity was significantly
recovered in microsomes expressing human CYP1B1. Thus, DF 203
irreversibly inhibited CYP1A1 but not CYP1B1. In addition, 6-OH 203
inhibited EROD activity of CYP1A1 but not CYP1B1 microsomes (Fig. 9)
.
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. This was absent from insensitive MDA-MB-435 cells. This covalently
bound, Mr 50,000 protein was
immunoprecipitated by CYP1A1 (but not CYP1B1) antiserum, providing
evidence that it is the CYP1A1 enzyme
(Mr 52,000). An in vitro
covalent binding assay using [14C]DF 203 and
recombinant CYP1A1 microsomes showed NADPH-dependent binding of
radioactivity to CYP1A1 protein (Fig. 11)
. Coincubation with the inactive metabolite 6-OH 203 (100
µM) or GSH (1 mM) reduced
covalent binding by 74 and 33%, respectively.
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3 µM,
72 h; Fig. 12
), ß-NF (10 µM), or 3-MC (1
µM; results not shown). Expression of CYP1A1 in
sensitive cell lines was strictly dependent upon induction by drug
treatment. CYP1A1 protein could be detected in MCF-7 cell lysates after
4 h exposure to low (100 nM) concentrations
of DF 203 (results not shown).
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. Induction in both of these cell lines
occurred within 24 h of drug exposure. DMSO solvent (0.5% v/v)
did not alter expression of either enzyme (results not shown). Neither CYP1A1 nor CYP1B1 were expressed constitutively or induced in cell lines unresponsive to 2-(4-aminophenyl)benzothiazoles (breast MDA-MB-435, HBL 100, and MCF-7/ADR; prostate PC 3 and DU 145).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sensitive breast cancer cell lines metabolized DF 203 to its inactive
6-hydroxy derivative (6)
. Data herein report DF 203 to be
efficiently oxidized by microsomal CYP1A1 and CYP1B1 expressed in human
B-lymphoblastoid cells and to a lesser extent by CYP1A2 and CYP2D6.
Moreover, induction of CYP1A1 and modulation of CYP1B1 expression
occurred specifically in breast cell lines sensitive to growth
inhibition by 2-(4-aminophenyl)benzothiazoles, irrespective of ER
status. Further experimental evidence implicates CYP1A1 as the major
isoform catalyzing 6-hydroxylation of DF 203, following its induction
in sensitive breast cell lines. Neither CYP1A1 expression, EROD, nor DF
203 6-hydroxylase activities were detected constitutively; in contrast,
CYP1B1 protein was constitutively expressed in sensitive breast cell
lines. Profiles of 6-hydroxylation and EROD activity in MCF-7 cells
were biphasic and inverse to growth inhibition and induction of
6-hydroxylase activity by DF 203 was inhibited by CYP1A1-selective goat
antirat CYP1A1 serum and -NF. Coincubation of MCF-7 cells with DF
203 and -NF abolished DF 203-induced growth inhibition. Thus, a
mechanistic link between CYP1A1 catalytic activity and DF 203-induced
growth inhibition may be inferred.
CYP-catalyzed metabolism of DF 203 appears to produce both mitogenic
and inhibitory metabolites, such as in the case of estradiol
(25)
. Indeed, 6-OH 203 not only protected cells from
growth inhibition by DF 203 but evoked a mitogenic response in MCF-7
cells cultured under conditions suboptimal for growth (phenol red-free
medium supplemented with 5% charcoal-stripped
FCS).5
In addition, in culture medium of sensitive MCF-7 cells, the
3'-hydroxymethyl metabolite IH 224 (Fig. 1)
was identified as a minor
oxidation product of DF 203 (IC50, <0.01
µM; Ref. 6
).
In MCF-7 and T-47D cell lysates, radioactivity derived from [14C]DF 203 covalently bound to a Mr 50,000 protein, which was immunoprecipitated with specific CYP1A1 (but not CYP1B1) Ab. Moreover, NADPH-dependent covalent binding between [14C]DF 203 and recombinant CYP1A1 was detected. 6-OH 203 reversibly antagonized binding of [14C]DF 203 to CYP1A1 but was readily removed during the experimental procedure, suggesting no covalent binding between 6-OH 203 and CYP1A1. In addition, 6-OH 203 significantly inhibited CYP1A1 activity but had a negligible effect on CYP1B1 activity. As covalent binding of [14C]DF 203 to recombinant CYP1A1 was reduced by GSH, we speculate that DF 203 is metabolized by CYP1A1 to a reactive intermediate that covalently binds and then inactivates the enzyme. Indeed, DF 203 irreversibly inhibited the activity of CYP1A1 but not CYP1B1 microsomes. A multidisciplinary effort is under way to isolate and identify the chemical species covalently bound to CYP1A1 and assess its biological activity. Our observations are consistent with the hypothesis that CYP1A1-dependent metabolism of DF 203 is crucial for execution of its antitumor activity. However, covalent labeling of CYP1A1 by CYP1A1-dependent metabolism of DF 203 may be partially protective, and biotransformation to 6-OH 203 may underlie proliferation associated with the unique biphasic dose response, as a consequence of its mitogenic activity and inhibition of both DF 203-derived covalent binding to CYP1A1 and CYP1A1 activity.
Activation of the CYP1A1 gene is mediated by the AhR
(26
, 27)
; in addition, CYP1B1 is regulated by AhR
(28)
. After ligand binding, AhR heterodimerizes with its
protein partner, arylhydrocarbon receptor nuclear translocator
(29
, 30)
. This heterodimer complex binds xenobiotic
responsive elements located within the 5'-flanking region of responsive
genes to stimulate the synthesis of their protein products (31
, 32)
. Most breast cancer cell lines express AhR and the
arylhydrocarbon receptor nuclear translocator.
2,3,7,8-Tetrachlorodibenzo-p-dioxin is a strong inducer of
CYP1B1 in both ER+ and ER- breast cancer cell lines (33
, 34)
, whereas CYP1A1 induction by
2,3,7,8-tetrachlorodibenzo-p-dioxin appears dependent on ER
status (34, 35, 36)
. This suggests differences in the
regulatory controls of CYP1A1 and CYP1B1. Indeed, the profile of CYP1B1
modulation by DF 203 differed from that of CYP1A1; concentrations of DF
203 <300 nM down-regulated CYP1B1 expression in
both MCF-7 and MDA 468 cell lines. Induction of CYP1B1 occurred at
concentrations of DF 203, which elicited the second proliferative phase
of the in vitro dose-response curve (2)
,
suggesting a possible correlation between these two observations. That
activity of CYP1B1 was neither irreversibly inhibited by DF 203 (Fig. 8)
nor inhibited by 6-OH 203 (Fig. 9)
corroborates this deduction.
Human mammary tumors express CYP1A1 and CYP1B1, both of which
contribute significantly to the bioactivation of carcinogenic
polycyclic aromatic hydrocarbons (37, 38, 39, 40)
. Known inducers
of CYP1A1, such as 3-MC, induced DF 203 6-hydroxylase activity in MCF-7
lysates as efficiently as DF 203 (Fig. 2C)
. However, 3-MC
and B[a]P displayed very poor profiles of growth
inhibition in the National Cancer Institute in vitro drug
screen and, more importantly, did not COMPARE with DF 203.
Significantly, regulation of CYP1A1 and CYP1B1 by
2-(4-aminophenyl)benzothiazoles is cell line specific and may determine
drug sensitivity. Induction and inactivation of CYP1A1 by DF 203 has
potentially important clinical implications. Because CYP1A1 metabolizes
many drugs and xenobiotics and can be induced in human liver,
manipulation of CYP1A1 levels can potentially influence drug
interactions and toxicities. Polymorphisms exist within the
CYP gene family, e.g., individuals (
5% of the
population) possessing a CYP2D6 defect metabolize drugs more slowly,
causing accumulation of toxic drugs (41)
. Thus,
pharmacogenomic assessment will provide essential information to reduce
adverse drug effects and optimize therapeutic efficacy in patients with
favorable genetic profiles.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 To whom requests for reprints should be
addressed, at Cancer Research Laboratories, School of Pharmaceutical
Sciences, University of Nottingham, University Park, Nottingham NG7
2RD, United Kingdom.
3 M. C. Bibby and J. A. Double, personal
communication.
4 The abbreviations used are: CYP, cytochrome
P450; NF, naphthoflavone; DEDC, diethyldithiocarbamate; TAO,
triacetyloleandomycin; MTT,
3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide; ECL,
enhanced chemiluminescence; Ab, antibody; HPLC, high-performance liquid
chromatography; EROD,
7-ethoxyresorufin-O-deethylase; GSH, glutathione;
AhR, arylhydrocarbon receptor; ER, estrogen receptor; 3-MC,
3-methylcholanthrene; B[a]P,
benzo[a]pyrene; TCA, trichloroacetic acid.
Received 6/15/99. Accepted 8/ 2/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 E. Brantley, V. Trapani, M. C. Alley, C. D. Hose, T. D. Bradshaw, M. F. G. Stevens, E. A. Sausville, and S. F. Stinson FLUORINATED 2-(4-AMINO-3-METHYLPHENYL)BENZOTHIAZOLES INDUCE CYP1A1 EXPRESSION, BECOME METABOLIZED, AND BIND TO MACROMOLECULES IN SENSITIVE HUMAN CANCER CELLS Drug Metab. Dispos., December 1, 2004; 32(12): 1392 - 1401. [Abstract] [Full Text] [PDF]
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 C. O. Leong, M. Suggitt, D. J. Swaine, M. C. Bibby, M. F.G. Stevens, and T. D. Bradshaw In vitro, in vivo, and in silico analyses of the antitumor activity of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazoles Mol. Cancer Ther., December 1, 2004; 3(12): 1565 - 1575. [Abstract] [Full Text] [PDF]
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A. Levine-Fridman, L. Chen, and C. J. Elferink Cytochrome P4501A1 Promotes G1 Phase Cell Cycle Progression by Controlling Aryl Hydrocarbon Receptor Activity Mol. Pharmacol., February 1, 2004; 65(2): 461 - 469. [Abstract] [Full Text] [PDF]
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C. D. Hose, M. Hollingshead, E. A. Sausville, and A. Monks Induction of CYP1A1 in tumor cells by the antitumor agent 2-[4-amino-3-methylphenyl]-5-fluoro-benzothiazole: A potential surrogate marker for patient sensitivity Mol. Cancer Ther., December 1, 2003; 2(12): 1265 - 1272. [Abstract] [Full Text] [PDF]
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A. Monks, E. Harris, C. Hose, J. Connelly, and E. A. Sausville Genotoxic Profiling of MCF-7 Breast Cancer Cell Line Elucidates Gene Expression Modifications Underlying Toxicity of the Anticancer Drug 2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole Mol. Pharmacol., March 1, 2003; 63(3): 766 - 772. [Abstract] [Full Text] [PDF]
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M. J. Kuffel, J. C. Schroeder, L. J. Pobst, S. Naylor, J. M. Reid, S. H. Kaufmann, and M. M. Ames Activation of the Antitumor Agent Aminoflavone (NSC 686288) Is Mediated by Induction of Tumor Cell Cytochrome P450 1A1/1A2 Mol. Pharmacol., July 1, 2002; 62(1): 143 - 153. [Abstract] [Full Text] [PDF]
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T. D. Bradshaw, M. C. Bibby, J. A. Double, I. Fichtner, P. A. Cooper, M. C. Alley, S. Donohue, S. F. Stinson, J. E. Tomaszewjski, E. A. Sausville, et al. Preclinical Evaluation of Amino Acid Prodrugs of Novel Antitumor 2-(4-Amino-3-Methylphenyl)Benzothiazoles Mol. Cancer Ther., February 1, 2002; 1(4): 239 - 246. [Abstract] [Full Text] [PDF]
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A. I. Loaiza-Perez, V. Trapani, C. Hose, S. S. Singh, J. B. Trepel, M. F. G. Stevens, T. D. Bradshaw, and E. A. Sausville Aryl Hydrocarbon Receptor Mediates Sensitivity of MCF-7 Breast Cancer Cells to Antitumor Agent 2-(4-Amino-3-methylphenyl) Benzothiazole Mol. Pharmacol., January 1, 2002; 61(1): 13 - 19. [Abstract] [Full Text] [PDF]
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