Identification and pharmacological characterization of cholesterol-5,6- epoxide hydrolase as a target for tamoxifen and AEBS ligands

Article abstract of DOI:10.1073/pnas.1002922107

The microsomal antiestrogen binding site (AEBS) is a high-affinity target for the antitumor drug tamoxifen and its cognate ligands that mediate breast cancer cell differentiation and apoptosis. The AEBS, a hetero-oligomeric complex composed of 3β-hydroxysterol-Δ⁸-Δ⁷- isomerase (D8D7I) and 3β-hydroxysterol-Δ⁷-reductase (DHCR7), binds different structural classes of ligands, including ring B oxysterols. These oxysterols are inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), a microsomal epoxide hydrolase that has yet to be molecularly identified. We hypothesized that the AEBS and ChEH might be related entities. We show that the substrates of ChEH, cholestan-5α,6α-epoxy-3β-ol (α-CE) and cholestan-5β,6β-epoxy-3β-ol (β-CE), and its product, cholestane-3β,5α,6β-triol (CT), are competitive ligands of tamoxifen binding to the AEBS. Conversely, we show that each AEBS ligandis aninhibitor of ChEH activity, and that there is a positive correlation between these ligands' affinity for the AEBS and their potency to inhibit ChEH (r² = 0.95; n = 39; P < 0.0001). The single expression of D8D7I or DHCR7 in COS-7 cells slightly increased ChEH activity (1.8- and 2.6-fold), whereas their coexpression fully reconstituted ChEH, suggesting that the formation of a dimer is required for ChEH activity. Similarly, the single knockdown of D8D7I or DHCR7 using siRNA partially inhibited ChEH in MCF-7 cells, whereas the knockdown of both D8D7I and DHCR7 abolished ChEH activity by 92%. Taken together, our findings strongly suggest that the AEBS carries out ChEH activity and establish that ChEH is a new target for drugs of clinical interest, polyunsaturated fatty acids and ring B oxysterols.

Full text of DOI:10.1073/pnas.1002922107

Correction
PHARMACOLOGY
Correction for “Identification and pharmacological character-
ization of cholesterol-5,6-epoxide hydrolase as a target for ta-
moxifen and AEBS ligands,” by Philippe de Medina, Michael R.
Paillasse, Gregory Segala, Marc Poirot, and Sandrine Silvente-
Acad Sci USA (107:13520–13525; first published July 6, 2010;
10.1073/pnas.1002922107).
The authors note that, due to a printer’s error, the online
version of Table 1 was formatted incorrectly. The table appears
Poirot, which appeared in issue 30, July 27, 2010, of Proc Natl correctly in the print version and is shown below.
3
Table 1. Inhibition of [ H]Tam binding to the AEBS and catalytic activity of ChEH by drugs
Compound
K
i
AEBS, nM
K
i
ChEH, nM
Selective AEBS ligands
PBPE
PCPE
Tesmilifene
MBPE
MCPE
PCOPE
MCOPE
MCOCH2PE
SERMs
1
2
3
4
5
6
7
8
9 ± 1
10 ± 1
56 ± 2
18 ± 1
48 ± 2
64 ± 4
102 ± 16
850 ± 12
27 ± 6
35 ± 8
62 ± 3
27 ± 6
57 ± 8
203 ± 11
241 ± 7
902 ± 13
Tamoxifen
9
10
11
12
13
14
2.5 ± 0.2
11 ± 1
6 ± 1
2.4 ± 0.3
1.5 ± 0.2
38 ± 1
34 ± 8
145 ± 4
36 ± 4
18 ± 6
9 ± 2
4
OH-Tamoxifen
Raloxifene
Nitromiphene
Clomiphene
RU 39,411
155 ± 8
σ receptor ligands
BD-1008
Haloperidol
SR-31747A
Ibogaine
AC-915
15
16
17
18
19
20
21
22
83 ± 1
99 ± 9
18,067 ± 14
6 ± 2
2,150 ± 11
3,527 ± 9
2,325 ± 8
733 ± 9
5,322 ± 9
1.2 ± 0.1
920 ± 12
1,120 ± 8
640 ± 5
Rimcazole
Amiodarone
Trifluoroperazine
Cholesterol biosynthesis inhibitors
Ro 48–8071
U-18666A
AY-9944
Triparanol
Terbinafine
SKF-525A
432 ± 22
14 ± 2
135 ± 7
23
24
25
26
27
28
110 ± 4
84 ± 2
358 ± 12
17 ± 2
3,720 ± 16
897 ± 10
89 ± 5
90 ± 5
649 ± 6
39 ± 3
9,105 ± 33
1,904 ± 11
Ring B oxysterols
6
7
7
7
7
6
-Ketocholestanol
-Ketocholestanol
-Ketocholesterol
α-Hydroxycholesterol
β-Hydroxycholesterol
-Keto-5α-hydroxycholestanol
29
30
31
32
33
34
35
1,122 ± 30
580 ± 12
2,251 ± 21
864 ± 22
1,223 ± 31
2,252 ± 32
4,471 ± 22
5,320 ± 22
7,131 ± 28
4,212 ± 32
6,151 ± 22
6,941 ± 21
8,522 ± 12
9,744 ± 11
Cholestane-3β,5α,6β-triol
Fatty acids
Oleic acid
α-Linolenic acid
ARA
36
37
38
39
48,144 ± 19
38,232 ± 41
26,171 ± 17
18,284 ± 19
54,235 ± 38
36,341 ± 42
24,094 ± 18
12,111 ± 16
DHA
3
Rat liver microsomes were incubated with a single concentration of 2.5 nM [ H]Tam and increasing concentrations of inhibitors ranging from 0.1 nM to
1
,000 μM under the conditions described in SI Appendix, SI Materials and Methods. IC50 values were determined using the iterative curve-fitting program
3
3
GraphPad Prism version 4 (GraphPad Software). For the AEBS, the apparent K
of 2 nM. The K
i
was expressed as K
i
= [IC50]/(1 + ([[ H]Tam])/K
d
), using 2.5 nM [ H]Tam and a K
d
14
i
values of drugs for ChEH inhibition were determined using 150 μg of rat liver microsomal protein and 10 and 20 μM of [ C]α-CE with
increasing concentrations of inhibitors ranging from 0.01 to 1000 μM, under the conditions described in SI Appendix, SI Materials and Methods. K
i
was
measured as the projection on the x-axis of the intersection of the lines obtained from 1/V versus [inhibitor] plots for ChEH. Values are the average of three
experiments ± SEM, each carried out in duplicate.
www.pnas.org/cgi/doi/10.1073/pnas.1010447107
www.pnas.org
PNAS
|
August 17, 2010
|
vol. 107
|
no. 33
|
14937

Identification and pharmacological characterization of
cholesterol-5,6-epoxide hydrolase as a target for
tamoxifen and AEBS ligands
a,b
a,b
a,c,d
a,c,d,1
a,c,d
Philippe de Medina , Michael R. Paillasse , Gregory Segala
, Marc Poirot
, and Sandrine Silvente-Poirot
a
Institut National de la Santé et de la Recherche Médicale, U563, Équipe: Métabolisme, Oncogenése et Différenciation Cellulaire, 31052 Toulouse, France;
Affichem, 31400 Toulouse, France; Institut Claudius Regaud, 31052 Toulouse, France; and Université Toulouse III Paul Sabatier, 31062 Toulouse, France
b
c
d
Edited by David J. Mangelsdorf, University of Texas Southwestern Medical Center, Dallas, TX, and approved June 9, 2010 (received for review March 5, 2010)
The microsomal antiestrogen binding site (AEBS) is a high-affinity
target for the antitumor drug tamoxifen and its cognate ligands that
mediate breast cancer cell differentiation and apoptosis. The AEBS,
3β-ol-7-one (7-ketocholestanol), and 5α-cholestan-3β-ol-6-one (6-
ketocholestanol) (8).
In previous work, we established that the coexpression of 3β-
8
7
8
7
a hetero-oligomeric complex composed of 3β-hydroxysterol-Δ -Δ -
hydroxysterol-Δ -Δ -isomerase (D8D7I) and 3β-hydroxysterol-
7
7
isomerase (D8D7I) and 3β-hydroxysterol-Δ -reductase (DHCR7),
Δ -reductase (DHCR7) in mammalian cells is necessary and suf-
3
binds different structural classes of ligands, including ring B oxyster-
ols. These oxysterols are inhibitors of cholesterol-5,6-epoxide hydro-
lase (ChEH), a microsomal epoxide hydrolase that has yet to be
molecularly identified. We hypothesized that the AEBS and ChEH
might be related entities. We show that the substrates of ChEH,
cholestan-5α,6α-epoxy-3β-ol (α-CE) and cholestan-5β,6β-epoxy-3β-
ol (β-CE), and its product, cholestane-3β,5α,6β-triol (CT), are compet-
itive ligands of tamoxifen binding to the AEBS. Conversely, we show
thateachAEBSligandisaninhibitorofChEHactivity, and that there is
a positive correlation between these ligands’ affinity for the AEBS
ficient to reconstitute the high-affinity binding site for [ H]Tam
(
i.e., the AEBS) (5). D8D7I and DHCR7 are two enzymes in-
volved in specific catalytic steps in the postlanosterol biosynthesis
of cholesterol. Consistent with the fact that the AEBS binds σ-
receptor ligands (7), D8D7I (an AEBS subunit) carries a binding
site for σ-receptor ligands (10). AEBS expression is ubiquitous,
and AEBS is highly expressed in proliferative cells, such as tumor
cells, and in cholesterogenic tissues, such as the liver and brain, in
accordance with its relationship with cholesterol metabolism
(5, 10, 11). We recently established that AEBS ligands induce
breast cancer cell differentiation and apoptosis through a mecha-
nism involving the production of sterol autoxidation products (3, 4,
2
and their potency to inhibit ChEH (r = 0.95; n = 39; P < 0.0001). The
single expression of D8D7I or DHCR7 in COS-7 cells slightly increased
ChEH activity (1.8- and 2.6-fold), whereas their coexpression fully
reconstituted ChEH, suggesting that the formation of a dimer is re-
quired for ChEH activity. Similarly, the single knockdown of D8D7I or
DHCR7 using siRNA partially inhibited ChEH in MCF-7 cells, whereas
the knockdown of both D8D7I and DHCR7 abolished ChEH activity by
1
2). Cholestan-5α,6α-epoxy-3β-ol (α-CE), cholestan-5β,6β-epoxy-
3β-ol (β-CE), and 7-ketocholesterol are among the major autoxi-
dation products of cholesterol (13). CEs (α-CE and β-CE) are the
only known substrates of cholesterol epoxide hydrolase (ChEH),
and 7-ketocholesterol is an inhibitor of ChEH (14, 15). ChEH (EC
3.3.2.11) catalyzes the hydrolysis of α-CE and β-CE into a unique
geminal trans-diol, cholestane-3β,5α,6β-triol (CT) (16) (SI Ap-
pendix, Fig. S1).
9
2%. Taken together, our findings strongly suggest that the AEBS
carries out ChEH activity and establish that ChEH is a new target for
drugs of clinical interest, polyunsaturated fatty acids and ring
B oxysterols.
We have observed that the AEBS and ChEH share similar
characteristics and pharmacological properties. Indeed, ring B
oxysterols, such as 6-ketocholestanol, 7-ketocholestanol, and 7-
ketocholesterol, inhibit ChEH (14) and bind to the AEBS (8) with
the same order of potency. In addition, ChEH is inhibited by an
breast cancer chemoprevention
| cholesterol metabolism | oxysterol
|
|
docosahexaenoic acid
amoxifen (Tam) is one of the most commonly used drugs
T
worldwide for hormonal treatment and chemoprevention of autoxidation product of 7-dehydrocholesterol (17), and 7-dehy-
estrogen receptor (ER)-positive breast cancers (1). In this applica- drocholesterol is the substrate of DHCR7, an AEBS subunit (5).
tion, Tam activity is mediated through the modulation of gene ex-
Moreover, as with the AEBS, ChEH is located in the endoplasmic
pression under the control of ERs. Tam’s pharmacology is complex, reticulum of cells and is found in most mammalian tissues, with
however; it exerts nongenomic effects through other targets at the liver being the richest source of both the AEBS and ChEH
therapeutic doses (2). After the ER, the microsomal antiestrogen (18, 19). ChEH is the last member of the epoxide hydrolase family
binding site (AEBS) is the target of highest affinity for Tam. The
with an unidentified coding gene. The results from these studies,
AEBS has no affinity for estrogens, but binds selective ER along with the relationship that we established between sterol
modulators (SERMs); these ligands contain a hydrophobic core autoxidation products and the functions of the AEBS (3, 4, 12),
that mimics the steroid backbone of estrogens, grafted to a dia- suggested to us a pharmacological and structural link between the
lkylaminoalkyl side chain. Thus, in addition to Tam, SERMs, such
as raloxifene, 4-OH-Tam, and RU-39411, are ligands of the AEBS
AEBS and ChEH, which we investigated in the present study. Our
results indicate that the enzymes that form the AEBS are involved
in the catalytic activity of ChEH.
(
3–5). The AEBS selectively binds diphenylmethane derivatives of
Tam, including (4-benzyl-phenoxy)-ethyl-N-pyrrolidine (PBPE)
and tesmilifene (6) and it also binds σ-receptor ligands and inhib-
itors of cholesterol biosynthesis, such as triparanol and AY-9944
Author contributions: P.d.M., M.R.P., G.S., and M.P. performed research; P.d.M., M.P., and
S.S.-P. analyzed data; M.P. and S.S.-P. designed research; and M.P. and S.S.-P. wrote the
paper.
(5, 7). All of these classes of synthetic AEBS ligands have a pro-
tonable dialkylaminoalkyl chain in common that is necessary
for high-affinity binding to the AEBS (6). In the quest for natural
AEBS ligands, several unsaturated fatty acids and ring B oxysterols
have been identified (8, 9). Oxysterols that bind to the AEBS are
cholesterol-oxidized on ring B of the cholesterol backbone and in-
clude 5-cholesten-3β-ol-7-one (7-ketocholesterol), 5α-cholestan-
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: marc_poirot@hotmail.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1002922107/-/DCSupplemental.
1
3520–13525
|
PNAS
|
July 27, 2010
|
vol. 107
|
no. 30
www.pnas.org/cgi/doi/10.1073/pnas.1002922107

Results
in the presence of known inhibitors (6-ketocholestanol and 7-
ketocholestanol) (14), as well as in the presence of prototypical
AEBS ligands (Tam and PBPE). The conversion of α-CE or β-CE
to CT was inhibited by 10 μM 6-ketocholestanol or 7-ketocho-
lestanol and by 1 μM Tam or PBPE (SI Appendix, Fig. S2C).
Taken together, these results indicate that Tam and PBPE inhibit
ChEH activity.
α-CE, β-CE, and CT Are Competitive Ligands of the AEBS. We began
by investigating whether the substrates of ChEH (α-CE and
β-CE) and CT, the product of ChEH activity, are ligands of the
AEBS. We performed assays on rat liver microsomes, the richest
source of the AEBS (6). Competitive binding assays showed that
increasing concentrations of unlabeled Tam, α-CE, β-CE, or CT
3
inhibited the binding of 2.5 nM [ H]Tam to the AEBS in
a concentration-dependent manner (Fig. 1A). The IC50 was 5.9 ±
Tam and PBPE Are Competitive Inhibitors of ChEH. We next carried
out experiments to determine the modality of ChEH inhibition
by Tam and PBPE. Tam inhibited ChEH in a concentration-
dependent manner, as shown by a double-reciprocal plot (Line-
0
.5 nM for Tam, 153 ± 0.5 nM for α-CE, 394 ± 1 nM for β-CE,
and 16.3 ± 0.6 μM for CT. To study the modality of the inhibition
by α-CE, β-CE, and CT, we performed Scatchard analyses of
14
3
weaver-Burk) of the inhibition of [ C]α-CE hydration by Tam in
Fig. 2A. The x-intercept (i.e., the 1/K value) decreased, whereas
[
H]Tam binding in the absence or presence of two concen-
m
trations of α-CE (Fig. 1B), β-CE (Fig. 1C), and CT (Fig. 1D). For
the y-intercept (i.e., the Vmax value) was not affected; these changes
are characteristic of competitive inhibition. This was confirmed
through a Dixon plot of the inverse of the velocity as a function of
increasing Tam concentration in the presence of two concen-
each experiment, a diminished slope of the lines indicated de-
3
creasing [ H]Tam affinity, whereas the Bmax remained un-
changed. These data demonstrate that the substrates (α-CE and
β-CE) and the product (CT) of ChEH are competitive ligands of
14
trations of [ C]α-CE (SI Appendix, Fig. S3A). The x-intercept gave
a K value of 34 ± 8 nM for Tam. The same experiments carried out
with PBPE, a selective AEBS ligand, showed competitive inhibition
i
the AEBS, with apparent K values of 66.5 ± 0.2 nM for α-CE,
i
1
71.3 ± 1.4 nM for β-CE, and 7.1 ± 0.6 μM for CT.
i
of ChEH, with a K value of 27 ± 6 nM (Fig. 2B and SI Appendix,
The Prototypical AEBS Ligands Tam and PBPE Are Inhibitors of ChEH.
To test the effects of AEBS ligands on ChEH, we first measured
ChEH activity by incubating rat liver microsomes with 20 μM
Fig. S3B). These results indicate that Tam and PBPE are potent
competitive inhibitors of ChEH activity.
1
4
[
C]α-CE and separated it from CT by TLC. The migration of CE
Synthetic AEBS Ligands Are Inhibitors of ChEH. We tested the potency
of AEBS ligands of different pharmacological classes to inhibit
ChEH. All of the selective AEBS ligands (compounds 1–8; SI Ap-
pendix, Fig. S4A) inhibited ChEH activity (Table 1). The most potent
inhibitors were PBPE, 1-{2-[4-(2-phenylpropan-2-yl) phenoxy]ethyl}
pyrrolidine (PCPE), tesmilifene, 4-[2-(4-benzylphenoxy)ethyl]mor-
pholine (MBPE), and 4-{2-[4-(2-phenylpropan-2-yl)phenoxy]ethyl}
morpholine (MCPE) (5), with potencies in the nanomolar range,
whereas 1-[4-(2-morpholino ethoxy)phenyl]-2-phenylethanone (MC-
and CT was validated by reference to commercially available
standards. As shown in the TLC autoradiogram (SI Appendix, Fig.
S2A, Left), the conversion of α-CE to CT was time-dependent.
Steady-state kinetics were apparent for the first 10 min of in-
cubation (SI Appendix, Fig. S2A, Right). Consequently, all sub-
sequent experiments were performed using a 9-min incubation
period. The Km was 7.4 ± 0.5 μM, and the Vmax was 0.62 ± 0.01
nmol CT/mg protein/min (SI Appendix, Fig. S2B), consistent with
data reported in the literature (14, 15). We then assessed ChEH
1
4
14
OCH2PE) was the least potent, with a K value in the micromolar
i
activity, measured using [ C]α-CE or [ C]β-CE as the substrate,
range for ChEH (Table 1). In the subsequent series, we found that
SERMs (compounds 9–14; SI Appendix, Fig. S4B) inhibited ChEH
3
Fig. 1. Competition for [ H]Tam binding to the microsomal AEBS by Tam,
α-CE, β-CE, and CT. (A) Competition assays with increasing concentrations of
unlabeled Tam (▼), α-CE (●), β-CE (▲), and CT ( ) were performed on rat
▪
3
3
liver microsomes using 2.5 nM [ H]Tam. (B) Scatchard plots of [ H]Tam binding
to the microsomal AEBS in the absence (▼) or presence of 100 nM α-CE (○) or
3
2
00 nM α-CE (●). (C) Scatchard plots of [ H]Tam binding to the microsomal
AEBS in the absence (▼) or presence of 200 nM β-CE (△) or 500 nM β-CE (▲).
3
(
D) Scatchard plots of [ H]Tam binding to the microsomal AEBS in the absence
(
▼) or presence of 5 μM CT (□) or 10 μM CT ( ). The lines intercept on the x-
▪
Fig. 2. Inhibition of ChEH by Tam, PBPE, and oleic acid. The relationship be-
tween the conversion rates of α-CE to CT and inhibitor concentrations is shown
using 10 and 20 μM Tam and PBPE with rat liver microsomal ChEH. Shown are
axis, indicating that α-CE, β-CE, and CT are competitive ligands of the AEBS
with respect to Tam binding. Measurements were made in triplicate for at
least three separate experiments. Data are presented as the mean ± SEM.
14
double reciprocal plots of Tam (A), PBPE (B), and oleic acid (C) versus [ C]α-CE.
de Medina et al.
PNAS
|
July 27, 2010
|
vol. 107
|
no. 30
|
13521

3
Table 1. Inhibition of [ H]Tam binding to the AEBS and catalytic
Table S1). Of the σ-receptor ligands (SI Appendix, Fig. S5A), com-
pounds 15–22 were both ligands of the AEBS and inhibitors of ChEH
with the following order of potency: SR-31747A > BD-1008 > tri-
fluoroperazine > amiodarone > ibogaine ≥ rimcazole > AC-915 >
haloperidol (Table 1). SR-31747A had the highest affinity for the
AEBS and was the most potent ChEH inhibitor of the compounds
activity of ChEH by drugs
Compound
K
i
AEBS, nM
K
i
ChEH, nM
Selective AEBS ligands
PBPE
PCPE
Tesmilifene
MBPE
MCPE
PCOPE
MCOPE
MCOCH2PE
SERMs
1
2
3
4
5
6
7
8
9 ± 1
10 ± 1
56 ± 2
18 ± 1
48 ± 2
64 ± 4
102 ± 16
850 ± 12
27 ± 6
35 ± 8
62 ± 3
27 ± 6
57 ± 8
203 ± 11
241 ± 7
902 ± 13
tested, with a K value of 1.2 ± 0.1 nM for the inhibition of Tam
i
binding to the AEBS and a K value of 6 ± 2 nM for ChEH inhibition.
i
Other σ-receptor ligands (compounds S6–S10; SI Appendix, Fig. S7),
including ditolyl guanidine (DTG), (+)-pentazocine, (+)-3PPP,
PRE-084, and progesterone, failed to bind to the AEBS and inhibit
ChEH, even at concentrations up to 1,000 μM (SI Appendix, Table
S1). In the last series of synthetic compounds, inhibitors of cholesterol
biosynthesis already reported to be AEBS ligands (5) (compounds
Tamoxifen
9
10
11
12
13
14
2.5 ± 0.2
11 ± 1
6 ± 1
2.4 ± 0.3
1.5 ± 0.2
38 ± 1
34 ± 8
145 ± 4
36 ± 4
18 ± 6
9 ± 2
2
3–28; SI Appendix, Fig. S5A), including U-18666A, triparanol, AY-
944, and SKF-525A, and newly identified AEBS ligands, such as
4
OH-Tamoxifen
9
Raloxifene
Nitromiphene
Clomiphene
RU 39,411
terbinafine and Ro 48–8071, were inhibitors of ChEH (Table 1).
Together, these results establish that every tested drug that bound to
the AEBS was an inhibitor of ChEH.
155 ± 8
σ receptor ligands
BD-1008
Haloperidol
SR-31747A
Ibogaine
AC-915
Unesterified Ring B Oxysterols Are Inhibitors of ChEH. We next
evaluated a set of oxysterols (SI Appendix, SI Appendix,Figs. S6 and
S8). Ring B oxysterols (compounds 29–34; SI Appendix, Fig. S6)
inhibited ChEH according to the following order of potency: 7-
ketocholestanol > 6-ketocholestanol > 7-ketocholesterol > 7α-
hydroxycholesterol > 7β-hydroxycholesterol > 6-keto-5α-hydrox-
ycholestanol > CT (Table 1). In contrast, side-chain oxysterols
(compounds S13–S16; SI Appendix, Fig. S8) did not inhibit ChEH
activity or bind to the AEBS (SI Appendix, Table S1). Ring B oxy-
sterols were previously shown to be competitive inhibitors of ChEH
15
16
17
18
19
20
21
22
83 ± 1
99 ± 9
18,067 ± 14
6 ± 2
2,150 ± 11
3,527 ± 9
2,325 ± 8
733 ± 9
5,322 ± 9
1.2 ± 0.1
920 ± 12
1,120 ± 8
640 ± 5
Rimcazole
Amiodarone
Trifluoroperazine
Cholesterol biosynthesis inhibitors
Ro 48–8071
U-18666A
AY-9944
Triparanol
Terbinafine
SKF-525A
Ring B oxysterols
432 ± 22
14 ± 2
135 ± 7
23
24
25
26
27
28
110 ± 4
84 ± 2
358 ± 12
17 ± 2
3,720 ± 16
897 ± 10
89 ± 5
90 ± 5
649 ± 6
39 ± 3
9,105 ± 33
1,904 ± 11
(14) as well as of Tam binding to the AEBS (8). In addition, the
sulfate ester α-CE (S17) and the stearic acid ester of CE (S18) had no
affinity for the AEBS and were not inhibitors of ChEH (SI Appendix,
Table S1). Thus, unlike α-CE, esterified forms of α-CE are not
substrates of ChEH. Our data indicate that unesterified ring B oxy-
sterols are both inhibitors of ChEH and ligands of the AEBS,
whereas side-chainoxysterols and esterified ring B oxysterols are not.
6
7
7
7
7
6
-Ketocholestanol
-Ketocholestanol
-Ketocholesterol
α-Hydroxycholesterol
β-Hydroxycholesterol
-Keto-5α-hydroxycholestanol
29
30
31
32
33
34
35
1,122 ± 30
580 ± 12
2,251 ± 21
864 ± 22
Unsaturated Fatty Acids That Are AEBS Ligands Are Inhibitors of
ChEH. Because oleic acid is a noncompetitive ligand of the AEBS
(20), we next studied whether oleic acid can inhibit ChEH activity,
and analyzed the modality of its inhibition. Using Lineweaver-
Burk analysis (Fig. 2C) and Dixon analysis (SI Appendix, Fig.
S3C), we found that oleic acid is a noncompetitive inhibitor of
1,223 ± 31
2,252 ± 32
4,471 ± 22
5,320 ± 22
7,131 ± 28
4,212 ± 32
6,151 ± 22
6,941 ± 21
8,522 ± 12
9,744 ± 11
Cholestane-3β,5α,6β-triol
Fatty acids
Oleic acid
α-Linolenic acid
ARA
DHA
i
ChEH, with a K value of 54 μM (Table 1). We extended this study
36
37
38
39
48,144 ± 19
38,232 ± 41
26,171 ± 17
18,284 ± 19
54,235 ± 38
36,341 ± 42
24,094 ± 18
12,111 ± 16
by testing other fatty acids [compounds 36–39 (SI Appendix, Fig.
S6) and S19–S21 (SI Appendix, Fig. S8)]. Unsaturated fatty acids,
such as docosahexaenoic acid (DHA), α-linoleic acid, and ara-
chidonic acid (ARA), are inhibitors of ChEH activity, whereas the
saturated fatty acids stearic acid and palmitic acid and the methyl
ester of oleic acid are not (SI Appendix, Table S1). These data
indicate that unsaturated fatty acids are inhibitors of ChEH, and
that oleic acid is a noncompetitive inhibitor.
Rat liver microsomes were incubated with a single concentration of 2.5
3
nM [ H]Tam and increasing concentrations of inhibitors ranging from 0.1 nM
to 1,000 μM under the conditions described in SI Appendix, SI Materials and
Methods. IC50 values were determined using the iterative curve-fitting pro-
gram GraphPad Prism version 4 (GraphPad Software). For the AEBS, the
3
apparent K
[
i
was expressed as K
H]Tam and a K of 2 nM. The K
determined using 150 μg of rat liver microsomal protein and 10 and 20 μM of
i
= [IC50]/(1 + ([[ H]Tam])/K
i
values of drugs for ChEH inhibition were
d
), using 2.5 nM
Ligands’ Affinity for the AEBS Positively Correlates with Their
3
d
Inhibition of ChEH. Plotting the pK
[-log(K
)] of compounds that
values for their
i
i
bound to the AEBS as a function of their pK
i
1
4
[
C]α-CE with increasing concentrations of inhibitors ranging from 0.01 to
000 μM, under the conditions described in SI Appendix, SI Materials and
was measured as the projection on the x-axis of the intersection
inhibitory potency of ChEH activity yielded a positive linear
correlation between both parameters, with an r value of 0.95 (n =
1
2
Methods. K
i
3
9; P < 0.0001) (Fig. 3). This demonstrates a clear correlation
of the lines obtained from 1/V versus [inhibitor] plots for ChEH. Values are
the average of three experiments ± SEM, each carried out in duplicate.
between the affinity for the AEBS and ChEH inhibition for the
different classes of molecules.
in the following order of potency: clomiphene >nitromiphene >Tam
D8D7I and DHCR7 Coexpression Allows the Reconstitution of ChEH.
We previously reported that the coexpression of D8D7I and
DHCR7 is necessary for reconstitution of the AEBS in mamma-
lian COS-7 cells (5). We evaluated whether these two enzymes
were involved in ChEH activity. As shown in Fig. 4A, the basal
≥
raloxifene > 4OH-tamoxifen ≥ RU 39,411 (Table 1). ER ligands
with no affinity for the AEBS, such as 17β-estradiol, diethylstilbestrol,
ICI-164,384, RU-56668, and ICI-182,780 (SI Appendix, Fig. S7), did
not inhibit the ChEH at concentrations up to 10 μM (SI Appendix,
1
3522
|
www.pnas.org/cgi/doi/10.1073/pnas.1002922107
de Medina et al.

activity of ChEH was low in COS-7 cells transfected with the
vector control (mock). The single expression of D8D7I (D8) or
DHCR7 (D7) in COS-7 cells induced a slight increase in ChEH
activity (1.8- and 2.6-fold, respectively) compared with the vector
control. In contrast, the coexpression of D8D7I and DHCR7
proteins) (Fig. 4H). Transfection of the cells with DHCR7 siRNA,
but not with scrambled siRNA, decreased DHCR7 expression at
the mRNA level (73%) (Fig. 4D) and protein level (64%) (Fig. 4E).
Knockdown of DHCR7 increased the Km value of ChEH by 66%
(Km = 17.22 μM) with no significant changes in the V
max
value
(
(
D8 + D7) in COS-7 cells potentiated ChEH activity by 8.5-fold
(Fig. 4G), and reduced the affinity of Tam for the AEBS with no
changes in the Bmax value (K = 7.2 ± 0.6 nM, B = 1,445 ± 23
Fig. 4A). A Vmax value of 0.46 ± 0.04 nmol CT/mg protein/min
d
max
and a Km value of 4.47 ± 0.05 μM was measured in the D8D7I
fmol/mg proteins) (Fig. 4H). Finally, transfection of the cells with
D8D7I siRNA and DHCR7 siRNA produced a comparable de-
crease in D8D7I and DHCR7 expression as in the single knock-
down experiments, associated with a drastic reduction (92%) in
and DHCR7 coexpression experiments, compared with a Vmax
0
=
.05 ± 0.002 nmol CT/mg protein/min and Km = 7.01 ± 0.05 μM
for the vector-only transfected cells (Fig. 4B). These data indicate
that coexpression of D8D7I and DHCR7 led to the reconstitution
of robust ChEH activity in mammalian cells, indicating that
ChEH activity requires both enzymes. We tested the inhibition of
ChEH by various AEBS ligands on COS-7 cell lysates that coex-
pressed D8D7I and DHCR7 (Fig. 4C). The order of potency of
AEBS ligands for inhibiting the reconstituted ChEH was as fol-
ChEH activity (Fig. 4F) that was more than additive (V = 0.023 ±
m
0.06 nmol CT/mg protein/min, with no change in the Km value
(5.75 ± 0.07 μM) (Fig. 4G). A similar effect on the AEBS was ob-
served, with a 93% decrease in the B
max
value (103.5 ± 29 fmol/mg
proteins) with no changes in the K value (Fig. 4H). These decreases
d
are greater than expected based on the reduction in protein ex-
lows: clomiphene (K
i
= 32.3 ± 0.5 nM) > PBPE (K
i
= 43.3 ± 0.4
i
pression and strongly suggest that D8D7I and DHCR7 cooperate
in the ChEH activity. These data further demonstrate that D8D7I
and DHCR7 are involved in the ChEH activity.
nM) > Tam (K = 57.3 ± 0.6 nM) > 4OH-tamoxifen (K = 295.8 ±
i
0
.8 nM) > 7-ketocholestanol (K
i
= 711.6 ± 0.9 nM). 17β-Estra-
diol, which does not bind to the AEBS (SI Appendix, Table S1),
did not inhibit the reconstituted ChEH. These data establish that
the pharmacological profile obtained with the ChEH is similar to
that of the AEBS (5).
A
α-CE
B
C
Knockdown of D8D7I and DHCR7 Abolishes ChEH Activity. To confirm
that D8D7I and DHCR7 both contribute to ChEH, we conducted
knockdown experiments using siRNA against D8D7I and DHCR7
in a human breast adenocarcinoma cell line, MCF-7 cells. The
siRNA specificities were evaluated at the mRNA and at the pro-
tein levels for the expression of D8D7I and DHCR7. The impact
of knockdowns on the kinetic parameters of ChEH and on the
CT
3
binding parameters of [ H]Tam to the AEBS was measured. MCF-
D
ED8
7
d
cells expressed the AEBS (K = 5.2 ± 1.4 nM, Bmax = 1,553 ± 25
28kd
0kd
fmol/mg proteins) (4), and ChEH activity was found, with a Vmax
value of 0.38 ± 0.07 nmol CT/mg protein/min and a K value of
.91 ± 0.06 μM (Fig. 4 G and H). Transfection of the cells with
4
D7
m
37kd
5
Actin
D8D7I siRNA, but not with scrambled siRNA, led to decreased
D8D7I expression at the mRNA level (72%) (Fig. 4D) and protein
level (60%) (Fig. 4E). Interestingly, it also reduced ChEH activity
by 47% (Fig. 4F), with Vmax = 0.18 ± 0.09 nmol CT/mg protein/
min, K
m
= 3.87 ± 0.07 μM (Fig. 4G), and a 42% decrease in the
F
α-CE
G
H
amount of AEBS (K = 6.1 ± 0.4 nM, B
= 897 ± 18 fmol/mg
d
max
CT
Fig. 4. Expression and knockdown of D8D7I and DHCR7 in mammalian cells:
Impact on ChEH and AEBS activities. (A) ChEH activity of microsomal extracts
from COS-7 cells transfected with control vector (mock), D8, D7, and D8 + D7.
(
B) Michaelis-Menten plot of velocity versus α-CE in ChEH assays from COS-7
cells transfected with mock (●) or D8 + D7 ( ). (C) Inhibition of ChEH in
▪
microsomal extracts from COS-7 cells coexpressing human recombinant D8
and D7 with increasing concentrations of clomiphene (♢), PBPE (○), Tam (□),
4
OH-tamoxifen (△), 7-ketocholestanol (▽), or 17β-estradiol ( ). (D, E) Ex-
▪
pression of D8 and D7 in MCF-7 cells transfected with siSC scrambled, siD8,
siD7, or siD8 + siD7 at the mRNA level (D) and at the protein level (E). (F)
Representative TLC autoradiogram showing ChEH activity in MCF-7 cells
from three independent experiments. (G) Michaelis-Menten plot of velocity
versus α-CE in ChEH assays from MCF-7 cells transfected with control
Fig. 3. Correlation between affinity of AEBS ligands for the AEBS and their
potency to inhibit ChEH. Graph of the pK for 39 compounds tested for the
inhibition of [ H]Tam binding as a function of pK on ChEH activity. The drug
numbers and the corresponding pK values [−log(K )] are listed in Table 1.
Here r is the correlation coefficient between pK values calculated for the
inhibition of Tam binding and ChEH activity. The r value of 0.95 and sig-
nificance of correlation (P < 0.0001) are given for all structural classes of
compounds (n = 39).
i
3
i
i
i
scrambled siRNA (siSC; ●), siD8 (○), siD7 (□), or siD8 + siD7 (
▪
). (I) Scatchard
plots of [ H]Tam binding to microsomal AEBS from MCF-7 cells transfected
with siSC (●), siD8 (○), siD7 (□), or siD8 + siD7 ( ). Measurements were
3
i
2
▪
made in triplicate for at least three separate experiments. Data are pre-
sented as mean ± SEM.
de Medina et al.
PNAS
|
July 27, 2010
|
vol. 107
|
no. 30
|
13523

Discussion
the mechanism of induction of breast cancer cell differentiation
and apoptosis by AEBS ligands that require sterol autoxidation
products. The therapeutic plasma concentration of Tam is 1–10 μM
The present study provides evidence that the AEBS and ChEH are
pharmacologically and molecularly related. We have shown that
substrates (CEs) and the product (CT) of ChEH are competitive
inhibitors of Tam binding to the AEBS, and have established that
ChEH is inhibited by all AEBS ligands tested, demonstrating that
inhibition of ChEH is a hallmark of AEBS ligands. We found
that different structural classes of AEBS ligands inhibit ChEH with
(
24), and that of tesmilifene is 5 μM (25), whereas their respective
K values for ChEH are 33 nM and 26 nM, indicating total in-
i
hibition of ChEH at therapeutic concentrations. The plasma
concentration of nonesterified DHA is 2–10 μM in humans re-
ceiving a diet supplemented with 1.5 g DHA/d (26), whereas the
3
DHA K value for the AEBS is 12.1 μM, indicating possible in-
the same modality as they inhibit [ H]Tam binding to the AEBS.
i
We established the pharmacological similarities by showing a pos- hibition of ChEH. Thus, the accumulation of CEs is likely to be
itive correlation between inhibition of ChEH activity and affinity of part of the pharmacologic action of these compounds and might
compounds for the AEBS. Importantly, compounds belonging to be involved in the anticancer and chemopreventive actions of
different pharmacological or biochemical classes that had no af- SERMs and DHA; this merits further investigation.
finity for the AEBS did not inhibit ChEH. With the series of syn-
CEs are autoxidation products of cholesterol, and their pro-
thetic compounds, the arylaminoalkyl structure was not sufficient duction can be blocked by lipophilic antioxidants such as vitamin
for ChEH inhibition; such compounds as (+)-3-PPP, (+)-pentaz- E (27). We recently established that AEBS ligands induce dif-
ocine, PRE-084, and MCH
with Tam for binding to the AEBS.
3
PE did not inhibit ChEH or compete
ferentiation and apoptosis in breast cancer cells through a mech-
anism involving the production of sterol autoxidation products,
As shown previously for reconstitution of the AEBS (5), tran- and that vitamin E inhibits these effects (3, 4, 12). Consistent with
sient coexpression of D8D7I and DHCR7 in COS-7 cells led to the these data, previous reports have shown a limited clinical outcome
reconstitution of ChEH. We confirmed the involvement of D8D7I with Tam treatment when cholesterol metabolism enzymes (28)
and DHCR7 in ChEH through knockdown experiments using or antioxidant enzymes (29) are overexpressed in breast tumors.
siRNA directed against D8D7I and DHCR7. These data show that These data, along with our present findings, suggest that the
both D8D7I and DHCR7 are required for the dual reconstitution modulation of CE metabolism resulting from ChEH inhibition by
of ChEH and the AEBS and provide more evidence of the mo-
lecular nature of the ChEH. The fact that our single knockdown
experiments showed that D8D7I and DHCR7 affect the kinetic
AEBS ligands might be involved in these effects. Further in-
vestigation is warranted. In particular, it will be interesting to
study the relationship that may exist between CE metabolism and
parameters of ChEH differently suggests that D8D7I carries the the sensitivity of and resistance to Tam of breast cancer cell lines
catalytic activity, and that DHCR7 cooperates in the binding of and to define how accumulation of CEs or the products of their
substrates to ChEH (Fig. 5). We find it interesting that three ep- transformation might be involved in the effects of AEBS ligands.
oxide hydrolases in three unrelated structural classes have such
The physiological function of ChEH has been proposed to be
high structural complexity. The leukotriene A4 hydrolase/amino- involved in the control of lipid metabolism (16) based on the bi-
peptidase (21) and the soluble epoxide hydrolase/phosphatase (22) ologicalpropertiesofCT, not fordetoxificationasinitiallyproposed
are both bifunctional enzymes. Our data suggest even greater
(14). As opposed to toxic aliphatic and aromatic epoxides, which
complexity with ChEH in being bifunctional and composed of two can spontaneously alkylate proteins and nucleic acids in association
independent gene products that unite in the microsomes to create withtheircytotoxicityorcarcinogenicity, CEsarestable(30)andare
a functional protein.
less toxic than CT (13, 31) and nontumorigenic substances (32).
CT’s greater toxicity and the mutagenic nature of its oxidation
products (33, 34) suggest that the inhibition of ChEH and the in-
hibition of CT production protects cells against cytotoxic insults.
Several other lines of evidence point to the existence of a dynamic
metabolism centered on CEs. α-CE is the only epimer of CE found
in the adrenal cortex, where it is produced by an as-yet-unidentified
cytochrome p450 (35). α-CE can be esterified by Sult2B1b (36) to
give a 3β-sulfated product that antagonizes liver X-receptor sig-
Here we have shown that anticancer drugs of clinical interest
are inhibitors of ChEH at pharmacologically and therapeutically
active concentrations. In vitro studies on breast cancer cells have
previously used 1- to 40-μM concentrations of selective AEBS
ligands, SERMS, or σ-receptor ligands (3, 4, 7) and 10–100 μM
polyunsaturated fatty acids (23). The present study has demon-
strated the existence of ChEH activity in MCF-7 cells, and thus
ChEH inhibition and CE accumulation are likely to play a role in
A : DHCR7 and D8D7I (AEBS) carry out the ChEH activity
CT production
CE
CT
CE
D8D7I
D8D7I
D8D7I
DHCR7
DHCR7
DHCR7
Fig. 5. Functional relationship between
B : TAM and AEBS ligands bind to the AEBS and inhibit the ChEH activity
the AEBS and ChEH. (A) The subunits of the
AEBS that bind Tam (D8D7I and DHCR7)
carry out the ChEH activity. (B) Ligands of
the AEBS, such as cationic SERMs (Tam and
raloxifene), diphenylmethane compounds
CE
CT
CE
CE
CE
Inhibition of
CT production
CE accumulation
(
tesmilifene andPBPE), ring B oxysterols (7-
ketocholesterol and 7-hydroxycholesterol)
and polyunsaturated fatty acids (DHA), are
inhibitors of ChEH, leadingto theblockage
of CT production and CE accumulation.
Tam
or
AEBS ligands
D8D7I
D8D7I
DHCR7
DHCR7
1
3524
|
www.pnas.org/cgi/doi/10.1073/pnas.1002922107
de Medina et al.

naling (37, 38), or it can be transformed by glutathione transferase
into 3β,5α-dihydroxycholestan-6β-yl-S-glutathione (39, 40). In ad-
dition, we have reported that the aminolysis of α-CE by biogenic
amines under catalytic conditions is possible and generates pow-
erful cell-differentiating alkylaminooxysterols (30). In contrast,
β-CE is nonreactive even under catalytic conditions (30) and has
been reported to accumulate in breast fluids (41) and in the plasma
of endometrial cancer patients (42). Thus, it would be interesting to
study whether β-CE can deregulate CE metabolism at the level of
the epoxidation step or of CE metabolism, including ChEH, leading
to the appearance of toxic CT.
ChEH Activity Assays. Rat liver microsomes were prepared as described pre-
viously (43), and ChEH activity was assayed as described previously (14).
14
Drugs and [ C]α-CE were dissolved in acetonitrile for the biological tests.
14
The concentration of [ C]α-CE in the test tubes was 10 or 20 μM for the
Dixon analyses and 5, 10, 20, 30, or 40 μM for the Lineweaver-Burk analyses.
m
The maximal velocity (Vmax) and Michaelis constant (K ) were determined by
nonlinear regression analysis using GraphPad Prism version 4.01 for Win-
dows (GraphPad Software). One-way ANOVA with Dunnett’s multiple-
comparison posttest was performed with vector-only cells as the control
using GraphPad Prism 4.01.
ACKNOWLEDGMENTS. We thank Dr. Michel Record for his critical reading of
the manuscript. This work was funded by Institut National de la Santé et de
la Recherche Médicale Conseil Regional Midi-Pyrénées, the Institut National
du Cancer through the ResisTH network, the Ministère Français de la
Recherche et de l’Enseignement Supérieur through the GenHomme project
and a predoctoral fellowship (G.S.), Affichem, and European Commission FP6
Integrated Project EuroHear (LSHG CT-2004-512063A). S.S.-P. is in charge of
research at the Centre National de la Recherche Scientifique.
In conclusion, these data shed light on the molecular nature
and potential functions of ChEH and open up the existence of an
active metabolic pathway at the level of the CEs.
Materials and Methods
Descriptions of chemical synthesis, AEBS binding, cell culture, and trans-
fections are provided in SI Appendix, SI Materials and Methods.
1
2
3
. Jordan VC (2003) Tamoxifen: A most unlikely pioneering medicine. Nat Rev Drug
Discov 2:205–213.
23. Chamras H, Ardashian A, Heber D, Glaspy JA (2002) Fatty acid modulation of MCF-7
human breast cancer cell proliferation, apoptosis and differentiation. J Nutr Biochem
13:711–716.
. de Médina P, Favre G, Poirot M (2004) Multiple targeting by the antitumor drug
tamoxifen: A structure-activity study. Curr Med Chem Anticancer Agents 4:491–508.
. de Medina P, et al. (2009) Ligands of the antiestrogen-binding site induce active cell
death and autophagy in human breast cancer cells through the modulation of
cholesterol metabolism. Cell Death Differ 16:1372–1384.
. Payré B, et al. (2008) Microsomal antiestrogen-binding site ligands induce growth
control and differentiation of human breast cancer cells through the modulation of
cholesterol metabolism. Mol Cancer Ther 7:3707–3718.
24. Trump DL, et al. (1992) High-dose oral tamoxifen, a potential multidrug-resistance–reversal
agent: Phase I trial in combination with vinblastine. J Natl Cancer Inst 84:1811–1816.
25. Brandes LJ, Bracken SP, Ramsey EW (1995) N,N-diethyl-2-[4-(phenylmethyl)phenoxy]
ethanamine in combination with cyclophosphamide: An active, low-toxicity regimen
for metastatic hormonally unresponsive prostate cancer. J Clin Oncol 13:1398–1403.
26. Bougnoux P, Hajjaji N, Maheo K, Couet C, Chevalier S (2010) Fatty acids and breast
cancer: Sensitization to treatments and prevention of metastatic re-growth. Prog
Lipid Res 49:76–86.
4
5
6
7
. Kedjouar B, et al. (2004) Molecular characterization of the microsomal tamoxifen
binding site. J Biol Chem 279:34048–34061.
2
7. Bergstrom S, Samuesson B (1961) The autoxidation of cholesterol. Autoxidation and
Antioxidants, ed Lundberg WO (Interscience, New York), Vol I, pp 233–248.
8. Pitroda SP, Khodarev NN, Beckett MA, Kufe DW, Weichselbaum RR (2009) MUC1-
induced alterations in a lipid metabolic gene network predict response of human
breast cancers to tamoxifen treatment. Proc Natl Acad Sci USA 106:5837–5841.
9. Schiff R, et al. (2000) Oxidative stress and AP-1 activity in tamoxifen-resistant breast
tumors in vivo. J Natl Cancer Inst 92:1926–1934.
. Poirot M, et al. (2000) Synthesis, binding and structure-affinity studies of new ligands
for the microsomal anti-estrogen binding site (AEBS). Bioorg Med Chem 8:2007–2016.
. Kedjouar B, et al. (1999) Structural similitudes between cytotoxic antiestrogen-binding
site (AEBS) ligands and cytotoxic sigma-receptor ligands: Evidence for a relationship
between cytotoxicity and affinity for AEBS or sigma-2 receptor but not for sigma-1
receptor. Biochem Pharmacol 58:1927–1939.
. Hwang PL, Matin A (1989) Interactions of sterols with antiestrogen-binding sites:
Structural requirements for high-affinity binding. J Lipid Res 30:239–245.
. Hwang PL (1986) Unsaturated fatty acids as endogenous inhibitors of tamoxifen
binding to antioestrogen-binding sites. Biochem J 237:749–755.
2
2
3
0. de Medina P, Paillasse MR, Payré B, Silvente-Poirot S, Poirot M (2009) Synthesis of new
alkylaminooxysterols with potent cell differentiating activities: Identification of leads
8
9
for the treatment of cancer and neurodegenerative diseases.
765–7777.
J Med Chem 52:
7
3
3
3
1. Smith LL, Johnson BH (1989) Biological activities of oxysterols. Free Radic Biol Med 7:
285–332.
1
1
1
0. Moebius FF, et al. (1998) Pharmacological analysis of sterol delta8-delta7 isomerase
3
proteins with [ H]ifenprodil. Mol Pharmacol 54:591–598.
2. el-Bayoumy K, et al. (1996) Lack of tumorigenicity of cholesterol epoxides and
estrone-3,4-quinone in the rat mammary gland. Cancer Res 56:1970–1973.
3. Cheng YW, Kang JJ, Shih YL, Lo YL, Wang CF (2005) Cholesterol-3-beta, 5-alpha, 6-
beta-triol induced genotoxicity through reactive oxygen species formation. Food
Chem Toxicol 43:617–622.
1. Gray JM, Ziemian
L (1992) Antiestrogen binding sites in brain and pituitary of
ovariectomized rats. Brain Res 578:55–60.
2. de Medina P, Silvente-Poirot S, Poirot M (2009) Tamoxifen and AEBS ligands induced
apoptosis and autophagy in breast cancer cells through the stimulation of sterol
accumulation. Autophagy 5:1066–1067.
3
4. Black HS (1980) Analysis and physiologic significance of cholesterol epoxide in animal
tissues. Lipids 15:705–709.
1
1
1
1
1
3. Schroepfer GJ,, Jr. (2000) Oxysterols: Modulators of cholesterol metabolism and other
processes. Physiol Rev 80:361–554.
3
5. Watabe T, Sawahata
3
3
T
(1979) Biotransformation of cholesterol to cholestane-
beta,5alpha,6beta-triol via cholesterol alpha-epoxide (5alpha,6alpha-epoxycholestan-
beta-ol) in bovine adrenal cortex. J Biol Chem 254:3854–3860.
4. Sevanian A, McLeod LL (1986) Catalytic properties and inhibition of hepatic
cholesterol-epoxide hydrolase. J Biol Chem 261:54–59.
5. Nashed NT, Michaud DP, Levin W, Jerina DM (1985) Properties of liver microsomal
cholesterol 5,6-oxide hydrolase. Arch Biochem Biophys 241:149–162.
6. Newman JW, Morisseau C, Hammock BD (2005) Epoxide hydrolases: Their roles and
interactions with lipid metabolism. Prog Lipid Res 44:1–51.
7. Nashed NT, Michaud DP, Levin W, Jerina DM (1986) 7-Dehydrocholesterol 5,6 beta-
oxide as a mechanism-based inhibitor of microsomal cholesterol oxide hydrolase.
J Biol Chem 261:2510–2513.
3
3
6. Fuda H, Javitt NB, Mitamura K, Ikegawa S, Strott CA (2007) Oxysterols are substrates
for cholesterol sulfotransferase. J Lipid Res 48:1343–1352.
7. Song C, Hiipakka RA, Liao S (2001) Auto-oxidized cholesterol sulfates are antagonistic
ligands of liver X receptors: Implications for the development and treatment of
atherosclerosis. Steroids 66:473–479.
3
8. Argmann CA, et al. (2005) Regulation of macrophage cholesterol efflux through
hydroxymethylglutaryl-CoA reductase inhibition: A role for RhoA in ABCA1-mediated
cholesterol efflux. J Biol Chem 280:22212–22221.
1
8. Watts CK, Murphy LC, Sutherland RL (1984) Microsomal binding sites for nonsteroidal
anti-estrogens in MCF 7 human mammary carcinoma cells: Demonstration of high
affinity and narrow specificity for basic ether derivatives of triphenylethylene. J Biol
Chem 259:4223–4229.
3
9. Meyer DJ, Ketterer B (1982) 5 alpha,6 alpha-epoxy-cholestan-3 beta-ol (cholesterol
alpha-oxide): A specific substrate for rat liver glutathione transferase B. FEBS Lett 150:
499–502.
1
2
2
2
9. Aström A, et al. (1986) Subcellular and organ distribution of cholesterol epoxide
hydrolase in the rat. Biochim Biophys Acta 882:359–366.
0. Hwang PL (1987) Interaction of unsaturated fatty acids with antioestrogen-binding
sites. Biochem J 243:359–364.
1. Haeggström JZ (2004) Leukotriene A4 hydrolase/aminopeptidase, the gatekeeper of
chemotactic leukotriene B4 biosynthesis. J Biol Chem 279:50639–50642.
2. Newman JW, Morisseau C, Harris TR, Hammock BD (2003) The soluble epoxide
hydrolase encoded by EPXH2 is a bifunctional enzyme with novel lipid phosphate
phosphatase activity. Proc Natl Acad Sci USA 100:1558–1563.
40. Watabe T, Sawahata T, Horie J (1979) Evidence for the formation of a steroid S-
glutathione conjugate from an epoxysteroid precursor. Biochem Biophys Res Commun
87:469–475.
41. Wrensch MR, et al. (1989) Breast fluid cholesterol and cholesterol beta-epoxide
concentrations in women with benign breast disease. Cancer Res 49:2168–2174.
42. Küçük O, et al. (1994) Increased plasma level of cholesterol-5 beta,6 beta-epoxide in
endometrial cancer patients. Cancer Epidemiol Biomarkers Prev 3:571–574.
43. de Medina P, et al. (2004) Tamoxifen is a potent inhibitor of cholesterol esterification
and prevents the formation of foam cells. J Pharmacol Exp Ther 308:1165–1173.
de Medina et al.
PNAS
|
July 27, 2010
|
vol. 107
|
no. 30
|
13525