Human ACAT inhibitory effects of shikonin derivatives from Lithospermum erythrorhizon

Article abstract of DOI:10.1016/j.bmcl.2006.11.024

Three naphthoquinones were isolated by bioassay-guided fractionation from the CHCl₃ extracts of roots of Lithospermum erythrorhizon. They were identified as acetylshikonin (1), isobutyrylshikonin (2), and β-hydroxyisovalerylshikonin (3) on the basis of their spectroscopic analyses. The compounds 1-3 were tested for their inhibitory activities against human ACAT-1 (hACAT-1) or human ACAT-2 (hACAT-2). Compound 2 preferentially inhibited hACAT-2 (IC₅₀ = 57.5 μM) than hACAT-1 (32% at 120 μM), whereas compounds 1 and 3 showed weak inhibitory activities in both hACAT-1 and -2. To develop more potent hACAT inhibitor, shikonin derivatives (5-11) were synthesized by semi-synthesis of shikonin (4), which was prepared by hydrolysis of 1-3. Among them, compounds 5 and 7 exhibited the strong inhibitory activities against hACAT-1 and -2. Furthermore, we demonstrated that compound 7 behaved as a potent ACAT inhibitor in not only in vitro assay system but also cell-based assay system.

Full text of DOI:10.1016/j.bmcl.2006.11.024

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1112–1116
Human ACAT inhibitory effects of shikonin derivatives from
Lithospermum erythrorhizon
a,c
a,b
c
a,
a,b,
*
*
Sojin An, Yong-Dae Park, Young-Ki Paik, Tae-Sook Jeong and Woo Song Lee
a
National Research Laboratory of Lipid Metabolism and Atherosclerosis, KRIBB, Daejeon 305-806, Republic of Korea
b
Molecular Science, University of Science and Technology, Daejeon 305-806, Republic of Korea
c
Department of Biochemistry, Yonsei University, Seoul 131-749, Republic of Korea
Received 26 May 2006; revised 19 October 2006; accepted 7 November 2006
Available online 10 November 2006
3
Abstract—Three naphthoquinones were isolated by bioassay-guided fractionation from the CHCl extracts of roots of Lithosper-
mum erythrorhizon. They were identified as acetylshikonin (1), isobutyrylshikonin (2), and b-hydroxyisovalerylshikonin (3) on
the basis of their spectroscopic analyses. The compounds 1–3 were tested for their inhibitory activities against human ACAT-1
(
(
hACAT-1) or human ACAT-2 (hACAT-2). Compound 2 preferentially inhibited hACAT-2 (IC50 = 57.5 lM) than hACAT-1
32% at 120 lM), whereas compounds 1 and 3 showed weak inhibitory activities in both hACAT-1 and -2. To develop more potent
hACAT inhibitor, shikonin derivatives (5–11) were synthesized by semi-synthesis of shikonin (4), which was prepared by hydrolysis
of 1–3. Among them, compounds 5 and 7 exhibited the strong inhibitory activities against hACAT-1 and -2. Furthermore, we dem-
onstrated that compound 7 behaved as a potent ACAT inhibitor in not only in vitro assay system but also cell-based assay system.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Acyl-CoA:
Cholesterol
acyltransferase
(ACAT,
inhibitors with different preferences on ACAT-1 and -2
may exert more favorable effects on atherosclerosis.
E.C.2.3.1.26) is the primary enzyme responsible for the
intracellular esterification of free cholesterol with fatty
acyl-CoA to produce cholesterol esters in various tis-
During search for new ACAT inhibitors from natural
7
1
sues. Accumulation of cholesteryl ester leads to the for-
mation of foam cells and a hallmark of atherosclerosis
lesions. Two isoenzymes, ACAT-1 and -2, have been
sources, we found that the CHCl extracts of Lithosper-
3
mum erythrorhizon roots at 100 lg/mL inhibited hA-
CAT-1 and -2 activities in 87% and 62%, respectively.
Subsequent bioactivity-guided fractionation of the
2
identified. Under normal physiological conditions,
ACAT-1 is present in many tissues, including macro-
phages, and ACAT-2 is present in intestinal epithelial
CHCl extracts led to the isolation of three shikonin
3
8
derivatives that were identified as acetylshikonin (1),
isobutyrylshikonin (2), and b-hydroxyisovalerylshikonin
(3) on the basis of their spectroscopic analyses (Fig. 1).
3
cells and hepatocytes. Inhibition of ACAT was expect-
9
ed to reduce plasma lipid levels by inhibiting intestinal
cholesterol absorption and to prevent progression of
atherosclerotic lesions by inhibiting the accumulation
Although shikonin derivatives isolated from the extracts
of L. erythrorhizon roots have been reported to confer
many medicinal properties such as antibacterial, wound
4
of cholesteryl esters in macrophage. However, the
5
known ACAT inhibitors have no beneficial effects on
6
the regression of coronary atherosclerosis. Nissen
et al. have drawn a bold conclusion that nonselective
ACAT inhibition is not effective for treatment of
OH
OH
O
O
1
2
R = CH3
6
atherosclerosis and is probably harmful. Thus, ACAT
R = CH(CH3)2
O
R
3 R = CH C(CH ) OH
2 3 2
Keywords: ACAT inhibitor; Shikonin derivatives.
*
O
Corresponding authors. Tel.: +82 42 860 4558; fax: +82 42 861 2675
T.S.J.); tel.: +82 42 860 4278; fax: +82 42 861 2675 (W.S.L.); e-mail
addresses: tsjeong@kribb.re.kr; wslee@kribb.re.kr
(
Figure 1. Structures of isolated shikonin derivatives (1–3) from the
roots of L. erythrorhizon.
0
960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.024

S. An et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1112–1116
1113
healing, antiinflammatory, antithrombotic, and antitu-
hACAT-2 was 57.5 lM, while hACAT-1 was inhibited
only 32% at 120 lM. b-Hydroxyisovalerylshikonin (3)
having 3-hydroxy-3-methylbutanoyl was less potent in
both of enzymes. Shikonin (4) inhibited hACAT-1 with
2-fold greater potency (IC50 = 70.4 lM) compared to
hACAT-2 (IC50 = 138.2 lM). Interestingly, compound
5 involving n-propanoyl exhibited higher activity against
both hACAT-1 and -2 compared with the corresponding
analogue 1 having acetyl moiety, whereas n-butanoyl-
substituted analogue 6 showed an attenuated potency.
The compound 7 substituted with 3-methylbutanoyl
showed potent inhibitory activity with IC50 values of
13.8 and 25.1 lM, respectively. These results clearly
demonstrated that simple extension of carbon chain
without any polarity change is enough to significantly
enhance activity. Substitution of n-pentanoyl, 8, at this
position, however, led to decrease in potency. Keeping
the three or four acyl chain lengths indicated that per-
haps specific lipophilic interaction between enzymes
1
0
mor effects, their ACAT inhibitory activities have
not been reported. In this study, we wish to describe
the hACAT-1 and -2 inhibitory activities of the isolated
and semi-synthetic shikonin derivatives (1–11).
0
To investigate the best pharmacophore at 1 -OH of
shikonin (4) against hACAT inhibitory activity, shiko-
nin (4) was prepared by hydrolysis of compounds 1–3
with 1 N NaOH. Then, acylshikonins (5–11) were syn-
0
thesized by a selective acylation at 1 -OH of shikonin
0
(
4) in the presence of N-(3-dimethylaminopropyl)-N -
ethyl carbodiimide (EDC) and 4-(dimethylamino) pyri-
dine (DMAP) as shown in Scheme 1.
Three active compounds were isolated from CHCl ex-
3
tracts and identified to be acetylshikonin (1), isobutyr-
ylshikonin (2), and b-hydroxyisovalerylshikonin (3).
The biological activities of shikonin derivatives 1–11
were assessed against expressed hACAT-1 or -2 from
0
and acyl chains at 1 -OH of shikonin (4) was possible.
1
1
Hi5 cells and confirmed by the positive control with
oleic acid anilide which inhibited hACAT-1 and -2 with
To investigate the lipophilicity, compound 9 was exam-
ined for inhibitory potency. It was equipotent to 8 in
hACAT-2, whereas compound 9 having more lipophilic
group led to 3-fold increase in potency in hACAT-1.
Compound 10 having linoleoyl as the long acyl residue
resulted in a significant loss in potency. In addition, 11
introduced with benzoyl group showed a slight increase
in potency in hACAT-1, but the potency was increased
1
1
IC50 values of 0.14 and 0.17 lM, respectively. The bio-
logical data for shikonin derivatives (1–11) have been
shown in Table 1. Acetylshikonin (1) inhibited hA-
CAT-1 and -2 with IC50 values of 128.9 and 112.2 lM,
respectively. Isobutyrylshikonin (2) dominantly inhibit-
ed hACAT-2 not hACAT-1; the IC50 value for
OH
OH
O
O
OH
OH
O
O
i
ii
1-3
OH
OCOR
5-11
4
Scheme 1. Reagents: (i) 1 N NaOH (1.2 equiv), MeOH; (ii) EDC (1.1 equiv), DMAP (0.2 equiv), RCOOH (1.1 equiv), CH
2 2
Cl .
Table 1. ACAT inhibitory activities of shikonin derivatives (1–11) in in vitro assay system
OH
OH
O
O
OR
a
Compound
R
Yield (%)
IC50 (lM)
hACAT-1
128.9
hACAT-2
1
2
3
4
5
6
7
8
9
Acetyl
—
—
—
71
67
59
57
62
53
70
28
112.2
57.5
169.8
138.2
28.7
49.6
25.1
84.5
71.4
b
Isobutanoyl
3-Hydroxy-3-methylbutanoyl
H
32%
186.9
70.4
22.4
77.6
13.8
94.8
30.7
n-Propanoyl
n-Butanoyl
3-Methylbutanoyl
n-Pentanoyl
4-Pentenoyl
Linoleoyl
b
b
1
0
1
5%
53.7
4%
56.2
1
Benzoyl
a
In vitro ACAT inhibitory activity was measured using expressed human ACAT-1 and -2. Data were shown as mean values of two independent
experiments performed in duplicate.
b
Percentage at 120 lM.

1114
S. An et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1112–1116
approximately 2-fold in hACAT-2 with IC50 of 56.2 lM
as compared to shikonin (4). To investigate that these
compounds function as ACAT inhibitor in physiological
condition, we established stable cell line expressing hA-
CAT-1 or -2 from AC-29 cells, ACAT-deleted CHO
100
8
0
TM
12
cells, using Flp-In system. The expression of hA-
CAT-1 and -2 was confirmed by immunoblot analysis
60
(
Fig. 2) and fluorescence microscopy using NBD choles-
4
2
0
0
0
terol (Fig. 3). Among compounds 1–11, the most potent
ACAT inhibitor, compound 7, was tested in cell–base-
fluorescent ACAT assay system.
ure 4, compound 7 inhibited significantly the enzymatic
activities of both hACAT-1 and -2 in cell-based assay
system with even lower IC50 values (IC50 = 0.6 lM for
hACAT-1 and IC50 = 0.8 lM for hACAT-2) than that
of in vitro assay system. However, compound 7 exhibit-
ed no selectivity against hACAT isoforms in both assay
systems, showing similar fold difference of IC50 value
hACAT1
hACAT2
1
3,14
As shown in Fig-
1 -
0
2
10-1
100
Compound 7 (μM)
101
102
Figure 4. hACAT inhibitory activity of compound 7 in cell-based
fluorescent assay system.
A
B
C
between hACAT-1 and -2 (1.8-fold for in vitro assay
and 1.3-fold for cell-based assay).
anti hACAT-1
According to these results, it was found that the inhibi-
tory potency was strongly dependent on the size and
length of acyl group. The chain length of acyl group
for the maximal inhibition of hACAT was found to be
three or four carbon atoms. These data suggest that
there may be a size limitation of acyl moiety for the
effective interaction of acylshikonins with hACAT and
human microsomal ACAT-1 and -2 enzymes have prov-
en to be very sensitive to the property of compounds.
Previous studies have demonstrated that the selective
inhibition of ACAT-1 and -2 provides evidence for
Anti β-actin
anti hACAT-2
Anti β-actin
uniqueness in structure and function of these two en-
11,15
Figure 2. Human ACAT-1 or -2 expression in Flp-In AC29 cells. The
expression of hACAT-1 or 2 was identified in cell lysates isolated from
Flp-In AC29 cells (A) and Flp-In AC29 cells stably expressing
hACAT-1 (B) or hACAT-2 (C) using immunoblot analysis. The
intensity of anti-b-actin band was used as an internal control to
compensate loading error.
zymes.
ACAT catalytic domains, which includes His-460 for
Chang et al. proposed that the putative
1
6a
16b
hACAT-1
and His-434 for ACAT-2,
are located
within a hydrophobic membrane, enabling the enzyme
to produce cholesteryl ester in the same membrane. In
Figure 3. Fluorescence microscopy after incubation of cells with NBD-cholesterol. Flp-In AC29 cells (A) and Flp-In AC29 cells stably transfected
with hACAT-1 (B) or hACAT-2 (C) were incubated with 5 lg/mL NBD-cholesterol for 3 h and fixed with 2% paraformaldehyde. The fluorescence
images were viewed with green channel filters (488 nm excitation, 540 nm emission), and their corresponding optical images were shown below each
fluorescence image. The results shown are representative of two separate experiments. Magnification 400·.

S. An et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1112–1116
1115
this context, different inhibitory potency of shikonin
derivatives against hACAT-1 and -2 may have resulted
from the degree of lipophilicity provided by acyl chain
and, providing optimal lipophilicity, three to four car-
bon length of acyl chain could increase a chance to inter-
act with catalytic site of hACAT-1 and -2 located in the
4. (a) Bocan, T. M.; Mueller, S. B.; Hendorf, P. D.; Brown,
E. Q.; Mazur, M. J.; Black, A. E. Atherosclerosis 1993, 99,
1
75; (b) Kusunoki, J.; Hansoty, D. K.; Aragane, K.;
Fallon, J. T.; Badimon, J. J.; Fisher, E. A. Circulation
001, 103, 2604; (c) Delsing, D. J.; Offerman, E. H.;
2
Duyvenvoorde, W. V.; Boom, H.; Wit, E. C.; Gijbels, M.
J.; Laarse, A.; Jukema, J. W.; Havekes, L. M.; Princen, H.
M. Circulation 2001, 103, 1778.
1
7
hydrophobic membrane. Recently, An et al. revealed
that His-386, another histidine residue imbedded in a
hydrophobic membrane, has the critical role for cataly-
sis of hACAT-1, whereas His-360 and His-399, cytoplas-
mic histidine residues, are essential for catalysis of
hACAT-2 suggesting catalysis in the plane of membrane
is limited in ACAT-1 and that catalysis of ACAT-2
takes place in the cytoplasmic side as well as hydropho-
bic membrane. We discovered that compound 2 contain-
ing isopropyl moiety preferentially inhibited hACAT-2
than hACAT-1, whereas compound 5, equivalent com-
pound with compound 2 except containing n-propyl
moiety instead of isopropyl moiety, considerably inhib-
ited both hACAT-1 and -2. Thus, we could speculate
that this isopropyl moiety of shikonin derivatives might
specifically interact with catalytic site of hACAT-2
located in the cytoplasmic side.
5
. For recent reviews of ACAT inhibitor, see (a) Giovannoni,
M. P.; Dal Piaz, V.; Vergelli, C.; Barlocco, D. Mini-Rev.
Med. Chem. 2003, 3, 576; (b) Sliskovic, D. R.; Picard, J.
A.; Krause, B. R. Prog. Med. Chem. 2002, 39, 121.
. Nissen, S. E.; Tuzcu, E. M.; Brewer, H. B.; Sipahi, I.;
Nicholls, S. J.; Ganz, P.; Schoenhagen, P.; Waters, D. D.;
Pepine, C. J.; Crowe, T. D.; Davidson, M. H.; Deanfield,
J. E.; Wisniewski, L. M.; Hanyok, J. J.; Kassalow, L. M.
N. Engl. J. Med. 2006, 354, 1253.
6
7
. Lee, W. S.; Lee, D. W.; Baek, Y. I.; An, S. J.; Cho, K. H.;
Choi, Y. K.; Kim, H. J.; Park, H. Y.; Bae, K. H.; Jeong, T.
S. Bioorg. Med. Chem. Lett. 2004, 14, 3109.
8. The dried roots of L. erythrorhizon (500 g) were cut into
small pieces and extracted with CHCl3 (2 L) at room
temperature for 3 days. After filtration, the solution was
evaporated to remove CHCl
the inhibitory activity against hACAT-1 and -2 (87% and
2% inhibition at 100 lg/mL), and was chromatographed
3
. The extract (11 g) showed
6
on a silica gel (230–400 mesh, Merck, Ø50 · 150 mm),
eluting with n-hexane/EtOAc (10:1, 5:1, 1:1, and EtOAc),
to yield eight fractions. Each fraction was monitored by
in vitro ACAT activity assay. Purification of fraction 2
was carried out on Sephadex LH-20 column eluting with
MeOH to yield five subfractions. Subfraction 1 was
further purified by silica gel (230–400 mesh, Merck,
Ø50 · 150 mm) chromatography, eluted with n-hexane/
EtOAc (10:1) to yield 1 (50 mg). Active fraction 1 was
separated by RP C18 (40–63 mesh, Merck, Ø30 · 100 mm)
In summary, we have discovered a new class of hACAT
inhibitors. Shikonin derivatives (1–3) were isolated by
bioassay-guided fractionation from the CHCl extracts
3
of L. erythrorhizon roots. The compounds 5–11 were
modified using semi-synthesis of shikonin (4). Among
them, compound 7 exhibited the most potent hACAT-
1
and -2 inhibitory activities with IC50 values of 13.8
and 25.1 lM, respectively. Notably, we confirmed the
ACAT inhibition capacity of compound 7 in cell-based
assay system, newly established in our laboratory via
construction of stable cell line expressing hACAT-1 or
2
column chromatography, eluted with MeOH/H O (9:1) to
yield 2 (30 mg). Fraction 4 was purified by chromatogra-
phy on silica gel (230–400 mesh, Merck, Ø50 · 150 mm),
eluted with n-hexane/EtOAc (5:1) to yield 3 (70 mg).
-
2. Further structure–activity relationship (SAR) studies
are being pursued to find more potent and selective
inhibitors. Although the negative effects of nonselective
ACAT inhibitors have been reported, hACAT-1 or -2
selective inhibitor may prove to have clinical benefit to
reduce atherosclerosis via directly reducing the size of
9
. Physical and spectroscopic data: compound 1: C18
HREI-MS m/z: 330.1103 (calcd for C18H O [M] :
18 6
H O ;
+
18 6
1
330.1104); H NMR (CDCl , 300 MHz): d 12.57 (1H, s),
3
12.41 (1H, s), 7.18 (2H, s), 6.98 (1H, s), 6.02 (1H, dd,
J = 6.9, 4.2 Hz), 5.12 (1H, t, J = 7.2 Hz), 2.62 (1H, m), 2.46
1
1H, m), 2.14 (3H, s), 1.69 (3H, s), 1.57 (3H, s); C NMR
3
1
8
(
the lipid-rich core in the atherosclerotic plaques or
1
9
(CDCl , 300 MHz): d 178.2, 176.7, 169.7, 167.5, 166.9,
the absorption of cholesterol in intestine, respectively.
Thus, it is important to continue the search for ACAT
isotype-selective inhibitors.
3
1
3
48.2, 136.1, 132.8, 132.7, 131.4, 117.6, 111.8, 111.5, 69.5,
2.8, 25.7, 20.9, 17.9. Compound 2: C20 ; HREI-MS
22 6
H O
+
1
m/z: 358.1416 (calcd for C20
22 6
H O [M] : 358.1418), H
NMR (CDCl , 300 MHz): d 12.58 (1H, s), 12.42 (1H, s),
3
7
5
.18 (2H, s), 6.97 (1H, s), 6.02 (1H, dd, J = 7.5, 4.2 Hz),
.12 (1H, t, J = 7.2 Hz), 2.63 (2H, m), 2.48 (1H, m), 1.69
Acknowledgment
1
3
(
3H, s), 1.58 (3H, s), 1.21 (6H, dd, J = 6.9, 3.0 Hz);
C
This work was supported by grants from National Re-
search Laboratory Program of the Ministry of Science
and Technology and KRIBB Research Initiative Pro-
gram, Korea.
NMR (CDCl , 300 MHz): d 178.3, 176.8, 175.8, 167.4,
3
166.8, 148.6, 135.9, 132.8, 132.7, 131.3, 117.8, 111.8, 111.6,
69.0, 34.0, 32.9, 25.7, 18.9, 18.8, 17.9. Compound 3:
C H O ; HREI-MS m/z: 388.1522 (calcd for C H O
21 24
+
7
21 24
7
1
[
M] : 388.1521); H NMR (CDCl , 300 MHz): d 12.56
3
(
1H, s), 12.37 (1H, s), 7.15 (2H, s), 7.01 (1H, s), 6.07 (1H,
dd, J = 7.5, 4.2 Hz), 5.10 (1H, t, J = 7.2 Hz), 3.14 (1H, s),
.62 (1H, m), 2.57 (2H, s), 2.48 (1H, m), 1.67 (3H, s), 1.57
References and notes
2
1
3
(
3
3H, s), 1.29 (6H, d, J = 4.2 Hz); C NMR (CDCl ,
1
2
3
. Largis, E. E.; Wang, V. H.; DeVries, V. G.; Schaffer, S. A.
J. Lipid Res. 1989, 30, 681.
. Chang, T. Y.; Chang, C. C.; Cheng, D. Annu. Rev.
Biochem. 1997, 66, 613.
. Chang, T. Y.; Chang, C. C.; Lin, S.; Yu, C.; Li, B. L.;
Miyazaki, A. Curr. Opin. Lipidol 2001, 12, 289.
3
1
3
00 MHz): d 176.9, 175.4, 171.6, 168.5, 167.9, 147.4, 136.3,
33.2, 133.0, 131.3, 117.6, 111.7, 111.5, 69.7, 69.0, 46.4,
2.8, 29.2, 29.0, 25.7, 17.9.
1
0. Papageorgiou, V. P.; Assimopoulou, A. N.; Couladouros,
E. A.; David, H.; Nicolaou, K. C. Angew. Chem. Int. Ed.
1
999, 38, 270.

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S. An et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1112–1116
1
1. Cho, K. H.; An, S.; Lee, W. S.; Paik, Y. K.; Kim, Y. K.;
Jeong, T. S. Biochem. Biophys. Res. Commun. 2003, 309,
64.
2. Generation of cell lines expressing hACAT-1 or hACAT-2:
we generated stable cell line expressing human ACAT-1 or
used to determine background fluorescence attributable to
free NBD-cholesterol. After incubation, the medium was
removed, and the cells were washed two times with cold
balanced salt solution (BSS, Invitrogen). The fluorescent
intensities of 96-well culture plates were read from the top
using VICTOR3 fluorescent plate reader (Perkin-Elmer)
equipped with 485 nm excitation and 535 nm emission
filters. After measurement, cellular protein was digested
through incubation with 25 lL of 0.4 N NaOH for 2 h.
The fluorescent intensity was normalized by cellular
protein content and calculated by subtracting the back-
ground fluorescent intensity from the total fluorescent
intensity.
8
1
-
2 (Accession No. BC028940 and AF059203 for ACAT-1
and ACAT-2, respectively) from AC-29 cells, ACAT-
TM
deleted CHO cells, using Flp-In system (Invitrogen)
according to the manufacturer’s suggestion. AC-29 cells
1
5
were kindly gifted by Dr. Rudel, L. L. . Briefly, Flp-In
AC-29 cells containing five copies of integrated FRT
sequence were generated by transfection of pFRT/lacZeo
into AC-29 cells. FRT site-integrated clones were selected
by 100 lg/mL zeocin (Invitrogen) for 1 week. Several
colonies were picked and maintained in F-12 medium
supplemented with 10% fetal bovine serum (FBS) and
14. Methyl-b-cyclodextrin (MbCD)–NBD-cholesterol com-
2
0
plex preparation: 100 lM of MbCD was dissolved in
10 mL F-12 medium, and 6 lM NBD-cholesterol, and
10 lg/mL BSA were added to this solution with vigorous
vortex. The dispersion was sonicated at room temperature
for 5 min using a Fisher model 60 sonic dismembrator at
setting 5. The solution was filtered with 0.45 lm mem-
brane filter and kept in a glass tube under argon at 4 ꢁC
for up to a week.
100 lg/mL zeocin. The presence and number of FRT-site
were verified by b-galactosidase assay and Southern blot
analysis, respectively. To construct stable cell lines express-
ing hACAT-1 or hACAT-2, Flp-In AC-29 cells were plated
on 60-mm dishes and transfected with 1.8 lg of the Flp-In
recombinase-encoding pOG44 vector and either 0.2 lg of
pcDNA5/FRT-hACAT-1 or pcDNA5/FRT-hACAT-2.
After 2 days, we proceeded to select the single colony with
15. Lada, A. T.; Davis, M.; Kent, C.; Chapman, J.; Tomoda,
ꢀ
H.; Omura, S.; Rudel, L. L. J. Lipid Res. 2004, 45, 378.
5
00 lg/mL hygromycin (Invitrogen) for 2 weeks. Colonies
16. (a) Guo, Z.-Y.; Lin, S.; Heinen, J. A.; Chang, C. C. Y.;
Chang, T.-Y. J. Biol. Chem 2005, 280, 37814; (b) Lin, S.;
Lu, X.; Chang, C. C. Y.; Chang, T.-Y. Mol. Biol. Cell
2003, 14, 2447.
17. An, S.; Cho, K. H.; Lee, W. S.; Lee, J. O.; Paik, Y. K.;
Jeong, T. S. FEBS Lett. 2006, 580, 2741.
18. Ikenoya, M.; Yoshinaka, Y.; Kobayashi, H.; Kawamine,
K.; Shibuya, K.; Sato, F.; Sawanobori, K.; Watanabe, T.;
Miyazaki, A. Atherosclerosis. 2006, July 1; (Epub. ahead
of print).
19. Rudel, L. L.; Lee, R. G.; Cockman, T. L. Curr. Opin.
Lipidol. 2001, 12, 121.
were picked up, expanded, and assayed for expression of
hACAT-1 or -2. Established cell lines were maintained in
F-12/10% FBS containing 100 lg/mL hygromycin.
1
5
1
3. Cell-based fluorescent ACAT assay: a total of 30,000
cells per well were plated on 96-well culture plates and
allowed to recover overnight. Assays were done with cells
at least 80% confluent. Cells were incubated in Ham’s F-12
medium supplemented with 1% Eagle’s vitamins and 10%
heat-inactivated FBS containing 5 lg/mL NBD-cholester-
ol as methyl-b-cyclodextrin complex with or without
ACAT inhibitor for 9 h. NBD-cholesterol was added
from a 5-mg/mL stock solution in methanol, and meth-
anol concentrations in the medium did not exceed 0.1%.
Flp-In AC-29 cells incubated with NBD-cholesterol were
20. Li, Y.; Ge, M.; Ciani, L.; Kuriakose, G.; Westover, E. J.;
Dura, M.; Covey, D. F.; Freed, J. H.; Maxfield, F. R.;
Lytton, J.; Tabas, I. J. Biol. Chem. 2004, 279, 37030.