[CANCER RESEARCH 64, 2070–2075, March 15, 2004]
Orlistat Is a Novel Inhibitor of Fatty Acid Synthase with Antitumor Activity
Steven J. Kridel,1 Fumiko Axelrod,1 Natasha Rozenkrantz,2 and Jeffrey W. Smith1
1Cancer Research Center, The Burnham Institute, and 2Activx Biosciences, La Jolla, California
ABSTRACT
One of the fundamental principles of pharmacology is that most drugs
have side effects. Although considerable attention is paid to detrimental
side effects, drugs can also have beneficial side effects. Given the time and
expense of drug development, it would be particularly exciting if a sys-
tematic method could be applied to reveal all of the activities, including
the unappreciated actions, of a potential drug. The present study takes the
first step along this path. An activity-based proteomics strategy was used
to simultaneously identify targets and screen for their inhibitors in pros-
tate cancer. Orlistat, a Food and Drug Administration-approved drug
used for treating obesity, was included in this screen. Surprisingly, we find
a new molecular target and a potential new application for Orlistat.
Orlistat is a novel inhibitor of the thioesterase domain of fatty acid
synthase, an enzyme strongly linked to tumor progression. By virtue of its
ability to inhibit fatty acid synthase, Orlistat halts tumor cell proliferation,
induces tumor cell apoptosis, and inhibits the growth of PC-3 tumors in
nude mice.
INTRODUCTION
Most drugs have side effects. These range in magnitude from
simple nuisances to life-threatening complications. Although focus is
placed on negative side effects, beneficial side effects are also ob-
served. Unfortunately, the unanticipated effects of a drug are often
revealed in the later stages of development or even after the drug has
been approved for use. Given the time and expense of drug develop-
ment, it would be particularly exciting if all activities of a compound
could be revealed at the outset of its development. With such infor-
mation, care could be taken to minimize detrimental side effects, and
testing of the drug could be expanded to other indications should its
activity profile warrant.
The ability to perform all of the encompassing screens of the
activity of a drug may be on the horizon. In principle, one could
predict the effects of a drug by knowing all of its targets. Recent
emphasis on global profiling strategies, including gene expression
profiling and proteomics, drives this type of thinking (1). Yet these
profiling technologies measure abundance, not function, and they fall
short of making it possible to screen drugs against a plethora of
targets. Recent work in the area of chemical biology points the way
toward direct profiling of protein activity, offering a possible solution
to this hurdle. Two groups have created chemical probes that react at
the active site of multiple enzymes of a given class. Liu et al. (2)
synthesized a probe containing fluorophosphonate as the warhead and
biotin as the reporter, and then used this probe to reveal the serine
hydrolase activity profile in biological samples. Greenbaum et al. (3)
showed that the cysteine proteinases profile could be visualized with
probes containing reactive epoxides. Because activity-based probes
bind at an active site of the enzyme, a direct measure of the level of
active enzyme can be obtained. Consequently, it becomes possible to
use straightforward competition assays to screen for inhibitors of all
of the enzymes within a family.
Here we apply the activity-based screening strategy to identify
serine hydrolases in prostate cancer cells. The activity-based nature of
the screen also allows us to identify inhibitors of these enzymes. Of
particular interest is fatty acid synthase (FAS), which is up-regulated
in the prostate cancer (PCa) cells compared with normal prostate
epithelial cells, and has been implicated in the progression of various
types of tumors (4–9). Interestingly, Orlistat is a novel and rather
selective inhibitor of FAS in tumor cells. This drug inhibits the
thioesterase function of the enzyme, interferes with cellular fatty acid
synthesis, and can halt tumor cell proliferation and induce tumor cell
apoptosis. Orlistat also inhibits the growth of PC-3 prostate tumors in
vivo. Altogether the study reaffirms the significance of FAS in tumor
progression and underscores the fact that this enzyme is a valid
oncology target. The study also indicates that compounds with reac-
tive
-lactones, such as Orlistat, should be evaluated as potential
antitumor agents.
MATERIALS AND METHODS
Activity Profiling of Serine Hydrolases. LNCaP, DU-145, and PC-3 cell
lines (American Type Culture Collection) were maintained in RPMI 1640
(Irvine Scientific) supplemented with 10% fetal bovine serum at 37°C in 5%
CO2. The PrEC cell line (Clonetics) was maintained in defined medium
supplied by Clonetics. Each cell line was maintained in 150-mm tissue culture
dishes. To generate protein lysates, cells were washed with ice-cold PBS and
harvested by scraping with a cell lifter into cold PBS. Cells were collected by
centrifugation, resuspended in 50 mM Tris-Cl (pH 8.0), and then lysed by
sonication as described previously (2, 10). Soluble and insoluble cell fractions
were separated by ultracentrifugation for 1 h at 64,000 rpm at 4°C. Protein
concentrations were determined by BCA assay (Pierce).
Activity profiling was performed with fluorophosphonate (FP)-polyethylene
glycol (PEG)-6-carboxytetramethylrhodamine (TAMRA) using methods de-
scribed previously (2, 10). Briefly, soluble fractions (40 l; 1 mg/ml) were
treated with 2 M FP-PEG-TAMRA for 1 h at ambient temperature. Reactions
were stopped by the addition of Laemmli buffer and boiling. Nonspecific
reaction of the probe was determined with a duplicate sample boiled for 10 min
before labeling with FP-PEG-TAMRA. The labeled samples were resolved by
10% SDS-PAGE and visualized by scanning with a Hitachi flatbed scanner at
605 nm.
Serine hydrolase activity in whole cells was measured with a membrane-
permeable probe, FP-BODIPY. After addition of Orlistat, the probe was added
to cells (final concentration of 2 M), and the reaction was allowed to proceed
to completion (1 h). Cells were lysed by the addition of Laemmli sample buffer
and boiled; samples were resolved on SDS-PAGE and visualized by scanning
with a Hitachi flatbed scanner at 605 nm.
Inhibition of Serine Hydrolase Activity by
-Lactones. Ebelactone A
and B stocks were made in DMSO. Orlistat (Roche) was solubilized from pills
in absolute ethanol. Cell lysates were generated at 1 mg/ml as described above.
Samples (40 g) were incubated with inhibitors for 20 min, and FP-PEG-
TAMRA was added and reacted for an additional 30 min.
Identification of Labeled Serine Hydrolases. To identify serine hydro-
lases, a fluorophosphonate probe linked to biotin was used (2, 10). Cell lysates
were preadsorbed to avidin-agarose to reduce nonspecific binding of proteins
during the purification. Cell lysates were labeled with FP-PEG-biotin (5 M)
for 1 h at room temperature. Protein was separated from unincorporated
FP-PEG-biotin by gel filtration on Nap 25 columns. SDS was added to the
eluate to a concentration of 0.5%, and the sample was denatured by boiling.
Samples were diluted with 50 mM Tris (pH 7.5) and 150 mM NaCl, and
Received 11/20/03; revised 12/23/03; accepted 1/20/04.
Grant support: Grants CA69306 and CA82713 from the NIH, Grant 1701-1-0031
from the Department of Defense Prostate Cancer Program, Cancer Center Grant CA
30199 from the National Cancer Institute, and support from Activx Biosciences.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Note: S. Kridel and F. Axelrod contributed equally to this work.
Requests for reprints: Jeffrey Smith, Cancer Research Center, The Burnham Institute,
10901 North Torrey Pines Road, La Jolla, CA 92037. E-mail: jsmith@burnham.org.
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incubated with avidin-agarose for 1 h at room temperature. The agarose beads
were washed eight times with 50 mM Tris (pH 7.5) and 150 mM NaCl
containing 1% Tween 20. Labeled protein was eluted with Laemmli buffer
containing 1% SDS. Protein was resolved by 10% SDS-PAGE and detected by
silver staining. Specific bands were extracted and subjected to in-gel digestion
by trypsin and peptide mass fingerprinting with matrix-assisted desorption
ionization-time of flight as described previously (11, 12).
Expression of the Recombinant Thioesterase Domain of Fatty Acid
Synthase. The portion of the FAS gene (gi:21618359) encoding the thioes-
terase domain was amplified by PCR using the following primers: 5
ATG
ACG CCC AAG GAG GAT GGT CTG GCC CAG CAG (corresponds to
nucleotides 6727–6756) and 3
GCC CTC CCG CAC GCT CAC GCG TGG
CT (corresponds to nucleotides 7625–7650). The recombinant thioesterase
domain was cloned into pTrcHis (Invitrogen) and expressed in Escheria coli.
The recombinant protein corresponds to residues 2202 through 2509 of FAS.
The thioesterase was purified by Ni-affinity chromatography, and analyzed for
activity and inhibition by Orlistat, using methods described above.
Detection of Fatty Acid Synthase by Western Blot. PC-3 cells (5  104)
treated with Orlistat were boiled in Laemmli buffer, resolved by SDS-PAGE,
and transferred to nitrocellulose. The membrane was blocked with nonfat milk
and probed with an anti-FAS monoclonal antibody (PharMingen). Binding was
visualized with an horseradish peroxidase-conjugated rabbit antimouse IgG
(Bio-Rad) followed by chemiluminescent detection with the Western Lighting
Chemiluminescence Reagent (Perkin-Elmer).
Inhibition of Fatty Acid Synthesis by Orlistat. Cellular fatty acid syn-
thesis was measured by the incorporation of [14C]acetate (13, 14). Cells
(2.5  104 cells/well in 24-well plates) were washed twice with PBS and
incubated in defined serum-free medium containing 300 g/ml BSA and
insulin, transferrin, and selenium as supplements. Medium was added to the
cells in the presence or absence of Orlistat. Cells were incubated with Orlistat
for up to 2 h before the addition of 1 Ci of [14C]acetate. Cells were incubated
with [14C]acetate for 2 h, at which time medium was removed, and the cells
were washed with PBS/EDTA and trypsinized. Cell pellets were washed twice
more with PBS, and fatty acids were extracted with chloroform-methanol (1:1)
for 30 min. The extract was dried under N2 and extracted with water-saturated
butanol. Butanol was evaporated under N2, and labeled fatty acids were
detected by scintillation counting.
Effects of Orlistat on Cell Proliferation. PC-3 cells were exposed to
Orlistat along with different concentrations of palmitate for 48 h. Fresh
medium, along with Orlistat and palmitate, were added every 24 h. Prolifer-
ation was assessed by measuring bromodeoxyuridine labeling using the Cell
Proliferation ELISA (Roche).
Effects of Orlistat on Cell Death. Cells were plated in 96-well tissue
culture plates in complete medium. After 24 h, the cells were exposed to
Orlistat for an additional 24 h. Apoptosis was measured with the Cell Death
Detection ELISA (Roche), which was performed according to the manufac-
turer’s protocol. As an independent assessment of apoptosis, the amount of
cleaved poly(ADP-ribose) polymerase was measured in cells after treatment
with Orlistat. Cells were cultured with the Orlistat (25 M) or ethanol for 72 h,
or with Staurosporine (1 M) for 5 h. At each time point, total cell extracts
were generated by addition of 1 SDS sample buffer. Samples were subjected
to Western analysis using antibodies against the cleaved form of poly(ADP-
ribose) polymerase (Cell Signaling). Western blotting was performed accord-
ing to protocols established by the manufacturer of the anti-poly(ADP-ribose)
polymerase antibody
PC-3 Xenograft Tumor Model. The effect of Orlistat on growth of PC-3
tumors in nude mice was assessed with a staged model. PC-3 cells (1  106)
were injected into the flank of male athymic nude mice 4–5 weeks of age.
Tumors were allowed to grow until they reached a size of 100 mm3, at which
time Orlistat administration was initiated. Orlistat was administered in 30 l of
vehicle containing 33% ethanol and 66% PEG 400. Animals received 240
mg/kg/day of Orlistat. Tumor size was measured with calipers twice weekly,
and volume was calculated with the formula volume  /6  XY2 (15).
RESULTS AND DISCUSSION
An activity-based proteomics screen was used to identify serine
hydrolases in PCa cells and to screen for their inhibitors. Serine
hydrolases were revealed with an activity-based probe composed of aFig. 1. Structures of
lactones.
Fig. 2. Activity profiling of normal and neoplastic prostate epithelial cells. Lysates
were generated from primary cultures of normal prostatic epithelial cells (PrECs) and
from three prostate tumor cell lines (LNCaP, DU-145, and PC-3). Lysates were incubated
with fluorophosphonate-polyethylene glycol-6-carboxytetramethylrhodamine (FP-PEG-
TAMRA) for 1 h at room temperature. Nonspecific labeling with the activity probe was
measured in samples denatured by boiling (lanes marked ). Samples were resolved by
10% SDS-PAGE and visualized at 605 nm using a Hitachi flatbed gel scanner (lanes 1–8).
The effect of three
-lactones on the activity labeling of serine hydrolases from prostate
cancer cells was assessed in a similar manner. Before incubation with FP-PEG-TAMRA,
lysates were preincubated with ebelactone A (lane 10), ebelactone B (lane 11), or Orlistat
(lane 12). After labeling with FP-PEG-TAMRA, the reactions were halted and enzyme
activity visualized as described above.
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ORLISTAT’S ANTITUMOR ACTIVITY