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(The FASEB Journal. 2006;20:2027-2035.)
© 2006 FASEB

Inhibition of endothelial cell proliferation and angiogenesis by orlistat, a fatty acid synthase inhibitor

Cecille D. Browne1, Elizabeth J. Hindmarsh1,2 and Jeffrey W. Smith3

Cancer Research Center, Burnham Institute for Medical Research, La Jolla, California, USA

3Correspondence: Cancer Research Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Rd., La Jolla, CA 92037, USA. E-mail: jsmith{at}burnham.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Orlistat, an antiobesity drug, is cytostatic and cytotoxic to tumor cells (1). The antitumor activity of orlistat can be attributed to its ability to inhibit the thioesterase domain of fatty acid synthase (FAS). The objective of the present study was to test the effect of orlistat on endothelial cell proliferation and angiogenesis. Orlistat inhibits endothelial cell FAS, blocks the synthesis of fatty acids, and prevents endothelial cell proliferation. More significantly, orlistat inhibits human neovascularization in an ex vivo assay, which suggests that it may be useful as an antiangiogenic drug. The mechanism of these effects can be traced to the fact that orlistat prevents the display of the vascular endothelial growth factor (VEGF) receptor (VEGFR2/KDR/Flk1) on the endothelial cell surface. Thus, orlistat is an antiangiogenic agent with a novel mechanism of action.—Browne, C. D., Hindmarsh, E. J., and Smith, J. W. Inhibition of endothelial cell proliferation and angiogenesis by orlistat, a fatty acid synthase inhibitor.


Key Words: VEGF • KDR • neovascularization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TUMOR ANGIOGENESIS IS initiated when vascular endothelial growth factor (VEGF) binds to its receptor, VEGFR2/KDR/Flk1 (hereafter referred to as KDR), and activates a proliferative signaling cascade through the MAPK pathway (2 , 3) . The importance of this pathway as a drug target has been validated by the recent success of new antiangiogenesis drugs. For example, AVASTIN is a monoclonal antibody (mAb) that binds to VEGF and prevents this growth factor from binding to its cellular receptor (KDR) (4 , 5) . AVASTIN has been approved for treating patients with metastatic colon cancer (6) . Another example is SUTENT (SU11248), a small molecule antagonist of the kinase domain of KDR (7) . This drug has shown efficacy in both gastrointestinal stromal tumors (8) and metastatic renal cell carcinoma (9) . Along with results from other VEGF and KDR antagonists, these findings provide clinical validation of the VEGF/KDR pathway as a drug target for halting tumor progression.

In a series of seemingly unrelated studies, we and others have begun to explore fatty acid synthase (FAS) as a drug target in tumor cells. FAS is the enzyme responsible for cellular synthesis of palmitate, the precursor of long-chain nonessential fatty acids (10 11 12 13) . FAS, which contains seven separate enzymatic pockets, is situated as a head-to-tail dimer with the ketoacyl synthase and malonyl/acetyl transferase domains of one monomer working together with the dehydratase, enoyl reductase, ketoacyl reductase, acyl carrier protein, and thioesterase domains on the adjacent monomer (10 11 12 13) . These enzymatic domains act sequentially to condense acetyl-coenzyme A with malonyl-coenzyme A to form a four-carbon intermediate. Six additional turns of the enzyme’s cycle convert this intermediate to palmitate, which is then liberated from FAS by the action of the thioesterase domain (14) .

Initially, FAS attracted interest as a drug target in oncology because it is up-regulated in most solid tumors, including those of the breast (15 16 17) , prostate (18 19 20) , and ovary (21 , 22) . Further validation of FAS as a target comes from a number of studies showing that a pharmacologic blockade of FAS is cytostatic and cytotoxic to tumor cells. A synthetic analog of cerulenin, c75, targets the condensing enzyme of FAS (23) and can suppress tumor cell proliferation and in some cases induce tumor cell apoptosis (23 24 25 26 27 28 29) . We recently showed that orlistat, an approved antiobesity drug, is an unanticipated inhibitor of the thioesterase domain of FAS. Like c75, orlistat is cytotoxic and cytostatic to tumor cells in vitro and can inhibit tumor growth in vivo (1) .

Here we show for the first time, that orlistat has antiproliferative effects on endothelial cells. We find that orlistat’s antiproliferative effects on endothelial cells can be traced to its ability to prevent the cell surface expression of KDR, a mechanism of action that is distinct from other antiangiogenic agents. Our findings indicate that further development of orlistat as an antiangiogenic drug is warranted. Such drugs could provide important therapeutic options, different pharmacologic profiles, and perhaps improved efficacy over currently approved antiangiogenic agents.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Orlistat (XENICAL, Roche Pharmaceuticals, Nutley, NJ) was extracted by solubilizing a tablet in ethanol (1 ml). The extract was centrifuged at 14,000 rpm for 5 min to remove insoluble excipients. Aliquots of orlistat (250 mM) were stored at –80°C until used.

Cell culture
Human umbilical vein endothelial cells (HUVEC; Clonetics, Cambrex Bioproducts, NJ) were maintained in Clonetics EGM-MV (normal growth medium) at 37°C in 5% CO2. Cells were maintained in 150 mm tissue culture plates and used in assays between cell passages 3 and 8.

Probing FAS activity with activity-based profiling
HUVECs were washed twice, harvested into ice-cold PBS, and collected by centrifugation at 1000 rpm for 5 min. Cell pellets were stored at –80°C until needed. Pellets were thawed on ice, resuspended in 50 mM Tris-HCl, 150 mM NaCl (pH 8.0), and lysed by sonication. Cell fractions were separated by ultracentrifugation at 64,000 rpm for 1 h at 4°C. Soluble fractions were measured spectrophotometrically for protein content at 280 nm.

Serine hydrolase activity profiles were determined using fluorophosphonate (FP)-TAMRA; polyethylene glycol (PEG)-6-carboxytetramethylrhodamine (provided by ActivX Biosciences, La Jolla, CA) (30) . Soluble protein fractions (25–60 µg) were incubated with orlistat (0–10 µM) or vehicle control for 30 min at room temperature. Fractions were then treated with 5 µM FP-TAMRA, for 1 h at room temperature. Reactions were stopped by addition of Laemmli buffer followed by boiling for 5 min. Nonspecific binding of the probe was assessed by using a protein sample preheated to 95°C for 10 min prior to labeling with FP-TAMRA. Labeled protein samples were resolved by SDS-PAGE, and fluorescent bands were visualized by scanning the gel with a Hitachi flatbed fluorescent scanner at 585 nm.

Effect of orlistat on the incorporation of acetate and malonyl-coenzyme A into cellular fatty acids
Inhibition of FAS activity in whole cells was measured by the incorporation of [14C] acetate into fatty acids. HUVECs (1x105 cells/well in 24-well plates) were cultured in EGM-MV for 24 h and subsequently serum-starved for 16 h in EBM containing VEGF (40 ng/ml; Chemicon International, Temecula, CA). Serum-starved cells were treated with orlistat for 4 h. [14C] acetate (50 µCi; Amersham Biosciences, Piscataway, NJ) was added to each well and incubated for 3 h at 37°C. Fatty acids were isolated by an initial hypotonic lysis of cells in 50 mM Tris, pH 7.5, followed by extraction in chloroform:water (1:1) for 30 min at room temperature. Lipid phases were dried under N2 and extracted with butanol:water (1:1). The butanol phase was evaporated under N2; radiolabeled lipids were counted by scintillation.

FAS inhibition in cell lysates was measured by fatty acid incorporation of [14C] malonyl coenzyme A (CoA) (24) . HUVECs were grown for 24 h in 150 mm dishes in EGM-MV. Cells were washed and harvested as before, centrifuged at 2000 rpm for 5 min and stored at –80°C. Cell pellets were lysed in hypotonic buffer (pH 7.5; 20 mM Tris, 1 mM DTT, 1 mM EDTA). Lysates were homogenized, sonicated, and spun at 14,000 rpm for 15 min at 4°C. Lysates (80 µl volumes) were treated with orlistat or vehicle for 1 h at room temperature. Treated lysates were added to a reaction mixture (1 ml; 500 µM NADPH, 167 µM acetyl CoA, 100 mM KCl) containing [14C] malonyl CoA (0.4 µCi, Amersham Biosciences) and incubated at 37°C for 25 min. Subsequently, unlabeled malonyl CoA (100 µM) was added, and mixtures were reincubated for 30 min. Lipids were extracted, dried, and counted by scintillation as before.

Cell proliferation assays
HUVECs were plated at 2 x 103 cells/well in 96-well plates and cultured for 24 h in EGM-MV (100 µl/well). Where noted, cells were washed twice with PBS and incubated in serum-starvation medium (EBM with 0.2% FBS) for 24 h. Medium was replaced with EGM-MV or with starvation medium containing VEGF (40 ng/ml) for 24 h. Cells were incubated in the presence of orlistat (10 µM). Bromodeoxyuridine (BrdU) (100 µM) was added, and its uptake was measured using colorimetric Cell Proliferation ELISA (Roche).

Western blotting and immune-precipitation
HUVECs were seeded in 150 mm TC dishes at 1–2 x 106 cells per plate in EGM-MV. After 48 h, subconfluent monolayers were starved with 0.2% EBM basal medium containing 15 µM orlistat and incubated for an additional 18–20 h. Cells were then stimulated for 2–40 min with VEGF (50 ng/ml). Monolayers were washed three times with ice-cold PBS and lysed in 50 mM Tris-HCl, pH 7.5 (1% Triton X-100, 150 mM NaCl, 1.0 mM EGTA, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, and 1 mM sodium orthovanadate). Cell lysates were spun at 14,000 rpm for 15 min at 4°C. Clarified lysates were immune-precipitated with anti-KDR (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Immune-precipitates and lysates were immune-blotted with antibodies to KDR (Santa Cruz; Abcam, Cambridge, MA) and phosphorylated KDR (Santa Cruz). FAS immune-precipitation and immune-blotting were performed using antibodies against the FAS N-terminal region (Pharmingen, BD Biosciences, San Jose, CA).

Angiogenesis assays
This procedure is a modification of the placental vessel neovascularization assay developed by Brown et al. (31) . Placentas were obtained within 48 h of delivery, having been stored at 4°C under sterile conditions. Superficial vessels, ~1–2 mm in diameter and 2–5 cm in length, were excised from the apical placenta surface and dissected into 1–2 mm fragments (but for each assay a consistent length was used). Medium 199 (M199; 3 mg/ml fibrinogen) was added to 48-well tissue culture plates. One vessel fragment was placed in the center of each well. Thrombin (0.25 U; Amersham) was added to induce clot formation. Gels were overlaid with M199 supplemented with 20% FBS, 0.1% {epsilon}-aminocaproic acid, 2 mM L-glutamine, and 1% penicillin/streptomycin/fungizone, plus orlistat (25 or 50 µM) or an equivalent volume of ethanol (vehicle control). Fumagillin (200 nM), a known inhibitor of angiogenesis (32) , was also overlaid in designated wells as a positive control. Vessels were cultured for 14–21 d, with daily medium replacement. Vessel fragments were stained with Phalloidin-AlexaFluor 594 (Molecular Probes, Eugene, OR) and quantified at 645 nm using the Cytofluor Fluorescence Plate Reader (Applied Biosystems, Foster City, CA). In pilot studies we found that, prior to the growth of explants, vessels embedded in fibrin typically yielded a baseline of fluorescence between 1000 and 2000 relative fluorescence units. Experiments on the growth of vessel explants were considered to be valid only if fumagillin could block the increase in fluorescence to baseline levels.

Vessel fragments were fixed in Carnoy solution and sectioned for immunostaining. Endogenous peroxidase activity was inhibited by incubation in 3% H2O2 in PBS for 10 min, and slides were trypsinized to enhance subsequent immunostaining (Digest-All 2, Zymed Laboratories, South San Francisco, CA). To demonstrate neovascularization, slides were blocked (Avidin/Biotin Blocking Kit, Vector Laboratories, Burlingame, CA), incubated overnight at 4°C with primary CD31 MAb (33.5 µg/ml; DakoCytomation, Carpinteria, CA), with biotinylated secondary antimouse IgG (10 µg/ml; Jackson IR USA, West Grove, PA) for 45 min at room temperature, and with Vectastain avidin-biotin complex (ABC) peroxidase-conjugated biotin-avidin complex (Vector) for 30 min. For FAS visualization, slides were incubated overnight at 4°C with rabbit anti-human FAS (2 µg/ml; IBL-America, Minneapolis, MN) and then with secondary anti-rabbit HRP conjugate (DakoCytomation) for 45 min at room temperature. Peroxidase activity was visualized with diaminobenzadine tetrahydrochloride substrate [3,3'-diaminobenzidine (DAB); Vector] for 10 min; slides were counterstained with hematoxylin.

Flow cytometry
HUVECs were grown in EGM-MV for 48 h and starved an additional 24 h in 0.2% EBM basal medium containing vehicle control or 15 µM orlistat. Monolayers were released from the culture plate with PBS containing EDTA, aliquoted at ~1 million cells per tube, and collected by microcentrifugation at 1000 rpm for 5 min. Prior to addition of primary antibody (Ab), nonspecific binding sites on cells were blocked with PBS containing 1% BSA. Cells were then incubated with primary Ab or an irrelevant isotype control (25 µg/ml) for 40 min on ice. Cells were spun at 2000 rpm for 2 min at 4°C and then resuspended in 100 µl PBS-BSA. Cell suspensions were incubated for 20 min at 4°C with 20 µg/ml of FITC-conjugated goat anti-mouse Ab (Santa Cruz). Following addition of PBS-BSA (100 µl) containing propidium iodide (10 µg/ml; Molecular Probes), samples were analyzed using BD FACSort 3-color Analyzer and BD CellQuest software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Orlistat blocks the FAS thioesterase, fatty acid synthesis, and proliferation in HUVECs
The ability of orlistat to inhibit the FAS thioesterase in HUVECs was measured by activity-based profiling of the active site of the FAS thioesterase (1) . HUVEC lysates were incubated with a concentration range orlistat (0–10 µM) and with FP-PEG-TAMRA, a probe that reacts with the serine nucleophile of the active site ofthe thioesterase (33) , and therefore competes with orlistat for binding (1) . The FAS thioesterase was covalently tagged with the activity-based probe (Fig. 1 A, lane 1) and orlistat competed for the labeling in a dose-dependent manner (Fig. 1A ). In the HUVEC lysates, half-maximal inhibition occurred at ~130 nM orlistat, as determined by densitometry scanning. The TAMRA-labeled protein at ~230 kDa (Fig. 1A ) has been identified as FAS in other cells lines using mass spectrometry (1) . In this study we confirmed its identity in HUVECs by immune-precipitation with anti-FAS Ab (Fig. 1B ), as we have done previously with tumor cells (1) .


Figure 1
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  		Figure 1. Orlistat inhibits the thioesterase domain of fatty acid synthase and inhibits the metabolic function of FAS in human umbilical vein endothelial cells. A) The effect of orlistat on the FAS thioesterase was probed in HUVEC lysates that were treated with orlistat (0–10 µM) or vehicle control for 30 min. The treated lysates were exposed to the activity-based probe, FP-TAMRA (5 µM), resolved using SDS-PAGE and visualized by scanning with a Hitachi fluorescent gel scanner. B) Lysates treated with orlistat or control and exposed to FP-TAMRA (5 µM) were immune-precipitated with antibodies against FAS and resolved using SDS-PAGE. Bands were visualized by scanning with a Hitachi fluorescent gel scanner and the gel was subsequently transferred onto nitrocellulose paper and immunoblotted with antibodies against FAS. C) The effect of orlistat on the incorporation of [14C] malonyl CoA was measured in lysates of HUVECs. The lysates, containing FAS, were treated with orlistat or vehicle for 1 h. The lysates were subsequently incubated with [14C] malonyl CoA for 25 min at 37°C to allow the malonyl CoA to incorporate into palmitate. Fatty acids were extracted from each sample and quantified by scintillation counting. Triplicate wells were assigned for each condition and statistically significant differences were observed between control treated lysate and each of orlistat-treated lysate (P<0.2 for 0.25 µM and P<0.05 for 0.5 µM). D) The effect of orlistat on the incorporation of acetate into fatty acids was probed in live HUVECs. The cells (1x105 cells/well in 24-well plates) were cultured in EGM-MV for 24 h and subsequently serum-starved for 16 h in EBM containing VEGF (40 ng/ml; Chemicon International, Temecula, CA). Serum-starved cells were treated with 15 µM orlistat for 4 h. [14C] acetate was added to each well and incubated for 3 h at 37°C. After incubation with [14C] acetate, fatty acids were extracted and quantified by scintillation counting. Six wells were designated as control + VEGF; three wells were designated as orlistat + VEGF. Statistically significant differences were observed between these two conditions (P<0.05). Three wells were grown without VEGF as growth control. E) The effect of orlistat on the proliferation of HUVECs was determined by measuring the incorporation of BrdU following treatment of cells with the drug. HUVECs were seeded in 96-well plates at a density of 2000 cells/well and cultured in endothelial growth media for 24 h at 37°C. Cells were subsequently cultured in media containing EBM and 2% FCS for 24 h. Culture media was replaced with EBM containing 2% FCS and VEGF (20 ng/ml) to stimulate proliferation. At this time cells were treated with orlistat (0–40 µM) and BrdU (100 µM) was added to track DNA synthesis. After a 24-h incubation the incorporation of BrdU uptake was measured using colorimetric Cell Proliferation ELISA (Roche) as described in Materials and Methods. Triplicate wells were used for each condition. SE in three repetitions of this experiment was typically 10%. F) The effect of orlistat on endothelial cell death was also measured across a concentration range of the drug. HUVECs were grown to 60% confluency (proliferating •) or 100% confluency (contact-inhibited {blacksquare}) and then exposed to orlistat for 48 h. Cell death was measured with the Roche Cell Death ELISA kit. Each point is the average of triplicate data points. SE was less than 12% for each point.

The effect of orlistat on the function of FAS in HUVECs was tested by measuring the incorporation of metabolic precursors into palmitate, the end product of FAS (1) . Two types of studies were done. First, we measured the incorporation of [14C] malonyl CoA into palmitate in lysates of HUVECs. The activity of FAS was measured in cell lysates because whole cells are not permeable to malonyl CoA. In this case, where the cell membrane was not a barrier to orlistat, the IC50 for inhibition of the incorporation of [14C] malonyl CoA into palmitate fell between 250 and 500 nM (Fig. 1C ), a value that is close to the IC50 of orlistat for inhibition of activity-based labeling of FAS (Fig. 1A ).

In the second type of assay, we measured the effects of orlistat on the incorporation of [14C] acetate into palmitate in live HUVECs. Unlike malonyl CoA, acetate can traverse the plasma membrane. In live cells orlistat had minimal effect on the incorporation of acetate into fatty acids, unless cells were stimulated with VEGF. VEGF stimulated the incorporation of [14C] acetate into fatty acids, suggesting that an increase in fatty acid synthesis may be a consequence of VEGF signaling. Importantly, 15 µM of orlistat completely ablates the VEGF-induced increase in the incorporation of acetate into palmitate in whole cells (Fig. 1D ).

The effect of orlistat on endothelial cell proliferation was measured by assessing the incorporation of BrdU in response to VEGF. Proliferation of HUVECs was challenged with a concentration range of orlistat, with the IC50 equaling ~1–3 µM (Fig. 1E ). We also tested the effects of orlistat on apoptosis in HUVECs. In this instance, we compared the effects of orlistat on cell death in proliferating vs. contact-inhibited HUVECs. Cells were treated with a dose range of orlistat, and cell death was measured 48 h following treatment (Fig. 1F ). The level of cell death was normalized to values obtained with untreated cultures of HUVECs. While orlistat elicits extensive cell death in proliferating HUVECs, the drug is without effect on nonproliferating HUVECs. Importantly, cell death is not observed until 48 h after treatment with orlistat, and the effects of orlistat on other cell processes were made well before this time. Therefore, the effects of orlistat on fatty acid synthesis and proliferation are not due to a toxic effect of the drug.

Orlistat inhibits neovascularization in a model of human angiogenesis
The antiproliferative effect of orlistat on HUVECs led us to test its effects on neovascularization in an ex vivo model of explanted human blood vessels (31) . Human placental vessels were embedded within fibrin clots, and the sprouting of new vessels from the explants was monitored. Vessel explants were cultured in the presence or absence of orlistat or fumagillin, a compound known to inhibit angiogenesis (32) . When visualized by bright-field microscopy, the degree of new vessel sprouts was lower in inhibitor-treated vs. control vessels (Fig. 2 A, upper panel). To quantify the degree of vessel spouting, the vessels were stained with fluorescent phalloidin, which binds to actin within the newly sprouted vessels (Fig. 2A , lower panel). We found that staining of explants immediately after embedding them in fibrin, and prior to the growth of explants, yielded far less staining with fluorescent phalloidin, with the relative fluorescence measures being closer to 500–2000 RFU rather than the values of 2500–8000, obtained when new vessels had grown.


Figure 2
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  		Figure 2. Inhibition of neovascularization in human placental vessels by orlistat. A) A model of sprouting of placental vessels implanted in fibrin was used to determine if orlistat treatment blocked neovascularization. Vessels were cultured for 14–21 d in medium containing vehicle (left panel), orlistat (50 µM), or fumagillin (200 nM). Vessels were visualized by staining actin within the vessels with phalloidin. B) The effect of orlistat on neovascularization was measured in vessels from five separate donors. Two experiments were performed with explants from Donor #20. Placental explants were embedded in fibrin as described in Materials and Methods and then treated with vehicle, fumagillin as a positive control, or with orlistat. The growth of explants was measured by staining vessel fragments with Phalloidin-AlexaFluor 594 and subsequent quantification at 645 nm as described in Materials and Methods. Each data point (•) represents the extent of neovascularization from a single explant. The statistical significance of each treatment was measured by comparing the control group to the treated groups with the Student’s t test. P values are noted above each data set in parentheses. C) The expression of FAS was assessed in cross sections of excised human placental blood vessels by immunohistochemistry. Vessels were stained with an anti-CD31 (upper right) or anti-FAS Ab (lower right), or with irrelevant isotype control (left panels).

The effect of orlistat on the growth of new vessels was measured with explants obtained from five donors (resulting in six repetitions). In each case fumagillin was used as a positive control, which decreased the labeling fluorescent phalloidin and indicated the inhibition of new vessel growth. In all cases, the inhibitory effect of fumagillin was statistically significant with P values < 0.01. In 30–40% of the placentas that we obtained, vessel growth was absent or was not inhibited by fumagillin. Results from these placentas are not included in our analysis. The growth of new vessels was also challenged with multiple concentrations of orlistat (Fig. 2B ). For each condition, five to seven explants were studied. The effects of orlistat on vessel growth were generally dose-dependent and were statistically significant (P values for the Student’s t test are shown above each data set, Fig. 2B ). The concentrations of fumagillin and orlistat required to elicit effects were substantially higher than reports in other types of angiogenesis assays (1 , 32) , almost certainly reflecting poor penetration of these drugs into the solid fibrin clot. Altogether, the results in Fig. 2B strongly suggest that orlistat can block angiogenesis in human vessels.

To confirm the expression of FAS in placental vessel explants, cross sections of vessel fragments were stained with antibodies against FAS and CD31, an endothelial marker. Positive staining of CD31 (Fig. 2C , upper right panel) highlights the position of a vessel within surrounding placental tissue. Immunostaining with anti-FAS Ab showed FAS in the endothelium and in the surrounding placental vessel cells (lower right panel). Specific staining was absent with irrelevant isotype control antibodies (upper and lower left panels).

Orlistat inhibits KDR function in HUVECs
To examine the mechanism by which orlistat prevents endothelial cell proliferation, we measured the phosphorylation of KDR in response to VEGF. Endothelial cells were stimulated with VEGF, lysed, and probed with a general Ab against phosphotyrosine and, subsequently, with Ab against KDR. Orlistat blocked the VEGF-stimulated phosphorylation of a band with the mass equivalent to KDR (Fig. 3 A, top panel) but was without effect on the total levels of KDR in cells (Fig. 3A , lower panel). To determine if the band phosphorylated in response to VEGF is KDR, a similar blot was probed with antibodies specific for phosphorylated KDR (Fig. 3B ). As shown in this figure, orlistat blocked the VEGF-stimulated phosphorylation of KDR in a dose-dependent manner, with an apparent cellular IC50 of ~1 µM (Fig. 3B ). These findings were confirmed by immunoblotting an immune-precipitation of KDR, followed by immunoblotting with Ab specific for phosphorylated KDR (Fig. 3C ). The effects of orlistat were time-dependent; an ~18 h treatment with the drug was required to block the phosphorylation of KDR in HUVECs (Fig. 3D ).


Figure 3
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  		Figure 3. Orlistat inhibits the activity of KDR in human endothelial cells. A) HUVECs were serum-starved with EBM plus 0.2% FBS with or without orlistat (15 µM) for 18 h. Cells were stimulated with VEGF (50 ng/ml) for 1, 2, and 5 min. Lysates were immunoblotted with antiphosphotyrosine (upper panel) and with anti-KDR (middle panel). B) The effect of orlistat on KDR phosphorylation was examined in HUVECs treated with orlistat (0.5–8 µM) for 18 h. Cells were then stimulated for 5 min with VEGF (50 ng/ml, and lysed as described in Materials and Methods). Cell lysates were immunoblotted with an antiphospho-KDR Ab. C) To confirm the observations in (A), the effect of orlistat on KDR phosphorylation was examined as described for (A) and the cell lysates were immune-precipitated with anti-KDR Ab. These precipitates were transferred to nitrocellulose and then probed with antiphospho KDR Ab. D) The effect of orlistat on KDR phosphorylation was examined as a function of time. HUVECs were incubated with orlistat for either 11 or 16 h, and then stimulated with VEGF as described above. Cells were lysed, separated by SDS-PAGE, and transferred to nitrocellulose. The blots were probed with Ab against the phosphorylated form of KDR.

Orlistat inhibits the cell-surface display of KDR in HUVECs and its association with G{alpha}q11
The effects of orlistat on the cell surface distribution of KDR were examined by flow cytometry (Fig. 4 ). Orlistat reduced expression of KDR on the cell surface (left panel), but had no effect on other membrane markers like CD31 (middle panel) or the {alpha}vß3 integrin (right panel). Therefore, orlistat elicits a rather selective clearance of KDR from the cell surface.


Figure 4
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  		Figure 4. Orlistat prevents the display of KDR on the cell surface.

Flow cytometry was used to measure the effects of orlistat on the cell surface expression of KDR. HUVECs were treated with orlistat, harvested from dishes, and then stained with antibodies against KDR, CD31, and {alpha}vß3 as described in Materials and Methods. FITC-conjugated secondary Ab was used to detect cell surface staining for each protein. Staining was performed in the absence of fixation. Staining of control HUVECs is shown in red, and staining of orlistat-treated cells is shown with blue shading.

To further probe the effect of orlistat on KDR function at the cell surface, we examined its association with G{alpha}q11, a member of the heterotrimeric G protein family that is required for KDR signaling. In fact, the physical association between KDR and G{alpha}q11 is required for VEGF-induced proliferation (34) . Coimmune-precipitation experiments were conducted to determine if orlistat disrupts the interaction of G{alpha}q11 with KDR. HUVECs were stimulated with VEGF, which induces the association of KDR and G{alpha}q11, and then lysed at various times after treatment. The lysates were immune-precipitated with Ab against G{alpha}q11, separated on SDS gels, and transferred to nitrocellulose. Western blots were performed to detect the presence of KDR in the immune-precipitates. In the absence of VEGF we observed a basal level of association between KDR and G{alpha}q11. In the minutes following stimulation with VEGF, the levels of KDR associated with G{alpha}q11 increased (Fig. 5 , left panel). In contrast, the VEGF-stimulated increase in this association was less profound and delayed in orlistat-treated cells (Fig. 5 , right panel). The same blot was probed with antibodies against G{alpha}q11, showing that the amount of this protein that was immune-precipitated is essentially equivalent in all samples (Fig. 5 , lower panel). These observations lend additional support to the idea that orlistat disrupts the proper display and subsequent protein associations at the cell surface.


Figure 5
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  		Figure 5. Orlistat disrupts the association between KDR and G{alpha}q11.

The association between KDR and G{alpha}q11 was examined by coimmune-precipitation studies. HUVECs were temporarily starved by culturing them in EBM containing 0.2% FBS with or without orlistat (15 µM) for 18 h. Then, cells were stimulated with VEGF (50 ng/ml) and lysed at various times (0–5 min). HUVEC lysates were immune-precipitated with anti-G{alpha}q11 Ab, and the lysates were transferred to nitrocellulose membranes. The blots were probed with antibodies against KDR or G{alpha}q11.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we find that orlistat, a FAS inhibitor, is an antiangiogenic agent. Orlistat: 1) blocks the FAS thioesterase and inhibits fatty acid synthesis in endothelial cells; 2) prevents endothelial cell proliferation and neovascularization; and 3) blocks VEGF-induced activation of KDR by clearing the receptor from the cell surface. The current drugs that antagonize this signaling pathway block either the binding of VEGF to KDR (AVASTIN, Genentech), or block the tyrosine kinase activity of KDR (e.g., SUTENT, Pfizer). Therefore, orlistat defines a new class of antiangiogenic agent that prevents the cell surface expression of KDR.

Orlistat blocks VEGF-induced proliferation of HUVECs and induces apoptosis in proliferating HUVECs at concentrations in the low micromolar range. Because orlistat blocked fatty acid biosynthesis in whole cells in the same concentration range, the simplest interpretation of these findings is that the antiproliferative and proapoptotic effects of orlistat result from its inhibition of FAS. This conclusion is also supported by our observation that an siRNA knock down of FAS in HUVECs also blocks VEGF-stimulated proliferation (data not shown). While similar concentrations of orlistat (~1–5 µM) halt proliferation and induce apoptosis, the two effects are observed at different times. A halt to proliferation is observed within 24 h after treatment with drug, whereas the apoptotic effects are not observed until 48 h after treatment. These observations are consistent with our prior finding in tumor cells showing that orlistat-induced apoptosis also follows a halt in cell proliferation (1) . In that report we also showed that orlistat fails to elicit apoptosis in normal human fibroblasts. Altogether, these findings argue against a general toxicity of orlistat and suggest that its effects are the result of interception of specific cellular pathways.

In endothelial cells, one of these pathways involves signaling by VEGF through its cell surface receptor KDR. In the presence of orlistat, very little KDR is detected on the HUVEC surface by flow cytometry, even though the surface display of other common cell surface proteins is not affected. The fact that orlistat disrupts the VEGF-induced association between G{alpha}q11 and KDR is also consistent with the idea that the drug prevents the proper display of KDR on the cell. It was previously reported that the G protein G{alpha}q11 physically associates with KDR in response to VEGF and that this association is necessary for VEGF-mediated proliferation of endothelial cells (34) . Interestingly, we find that orlistat diminishes the level of association between G{alpha}q11 and KDR, and substantially delays the increase in this association that results from treatment with VEGF. Our findings differ slightly from previous analysis of the association of KDR with G{alpha}q11. We observed a basal association between the two proteins in the absence of VEGF stimulation. This type of ligand-independent interaction was not observed in previous reports (34) . Our findings on the links between KDR and G{alpha}q11 are qualitatively similar to prior reports (34) , but differ in that we observed association at slightly different times after VEGF stimulation. Although there is no explanation for these distinctions, we suspect they may relate to differences in protocol. Moreover, the differences that are observed have no bearing on the interpretation of the present study.

The observation that orlistat disrupts the cell surface expression of KDR generally parallels ideas put forth by others showing that a pharmacologic blockade of FAS interferes with cellular localization of the human growth factor (HER2) receptor, p185HER2, in tumor cells (35) . Consequently, it has been suggested that FAS inhibition disrupts targeting of lipid domain-associated p185HER2 to the cell membrane (36) . Altogether then, these observations suggest that orlistat disrupts the cellular trafficking, or cellular localization, of growth factor receptors. It is tempting to speculate that such misregulation is linked to the reduction in palmitate synthesis. While a precise mechanism for improper trafficking will require further study, it may relate to the inability of orlistat-treated cells to assemble lipid rafts, or the disruption of protein palmitoylation, which targets many signaling proteins to the inner leaflet of the plasma membrane.

The work presented here shows, for the first time, a requirement for FAS in VEGF-stimulated endothelial cell proliferation and angiogenesis. Moreover, orlistat, an inhibitor of FAS, is identified as a potential antiangiogenic agent. The drug blocks VEGF-stimulated proliferation of endothelial cells by a unique mechanism of action; the down-regulation of KDR from the cell surface. Orlistat can block neovascularization in explants of human placental vessels, an ex vivo model of human angiogenesis. In the original report characterizing the growth of vessel explants from placental vessles (31) , the authors showed a clear contribution of both the VEGF and basic fibroblast growth factor (bFGF) pathways. Consequently, the inhibitory effects of orlistat in this assay may extend beyond the drugs ability to down-regulate the display of KDR on the cell surface. Therefore, we cannot exclude the possibility that orlistat has similar effects of the bFGF pathway. Nevertheless, our findings provide an alternative therapeutic route for antiangiogenic therapy. In addition, the fact that orlistat kills proliferating HUVECs, but not contact-inhibited HUVECs, suggests that the drug could selectively hit the pathogenic formation of new vessels in vivo.


   ACKNOWLEDGMENTS
 
We thank the staff of Scripps Encinitas Birth Pavilion for providing placenta. This work was supported by grants from the Department of Defense Breast Cancer Center of Excellence, DAMD17–02-0693, and from the National Institutes of Health (U54 RR020843–02, P01 CA82713–06, R 01 CA106582–02). E. J. H. was supported by a fellowship (6FB-0037) from the California Breast Cancer Research Program.


   FOOTNOTES
 
1 These authors contributed equally to this work.

2 Present address: Immunology and Genetics Division, John Curtin School of Medical Research, The Australian National University, Canberra ACT 0200, Australia. E-mail: liesa.hindmarsh{at}anu.edu.au

Received for publication November 15, 2005. Accepted for publication May 25, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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