Cancer Therapy: Preclinical Novel Inhibitors of Fatty Acid Synthase with Anticancer Activity

Purpose: Fatty acid synthase (FASN) is overexpressed in human breast carcinoma. Auth Biom Cièn Facu M.D d'On Bioq Barce Rece Gran RD06 T. Pu Colom 10, T dad A Span de la from Clin The natural polyphenol (-)-epigallocatechin-3-gallate blocks in vitro FASN activity and leads to apoptosis in breast cancer cells without any effects on carnitine palmitoyltransferase-1 (CPT-1) activity, and in vivo, does not decrease body weight. We synthesized a panel of new polyphenolic compounds and tested their effects on breast cancer models. Experimental Design: We evaluated the in vitro effects of the compounds on breast cancer cell growth (SK-Br3, MCF-7, and MDA-MB-231), apoptosis [as assessed by cleavage of poly(ADP-ribose) polymerase], cell signaling (HER2, ERK1/2, and AKT), and fatty acid metabolism enzymes (FASN and CPT-1). In vivo, we have evaluated their antitumor activity and their effect on body weight in a mice model of BT474 breast cancer cells. Results: Two compounds potently inhibited FASN activity and showed high cytotoxicity. Moreover, the compounds induced apoptosis and caused a marked decrease in the active forms of HER2, AKT, and ERK1/2 proteins. Interestingly, the compounds did not stimulate CPT-1 activity in vitro. We show evidence that one of the FASN inhibitors blocked the growth of BT474 breast cancer xenografts and did not induce weight loss in vivo. Conclusions: The synthesized polyphenolic compounds represent a novel class of FASN inhibitors, with in vitro and in vivo anticancer activity, that do not exhibit cross-activation of β-oxidation and do not induce weight loss in animals. One of the compounds blocked the growth of breast cancer xenografts. These FASN inhibitors may represent new agents for breast cancer treatment. (Clin Cancer Res 2009;15 (24):7608–15) Fatty acid synthase (E.C.2.3.1.85; FASN) is a lipogenic enzyme which catalyzes the de novo synthesis of long-chain fatty acids from acetyl-CoA, malonyl-CoA, and NADPH precursors (1). FASN expression is generally low or undetectable in human tissues other than the liver and adipose tissue, and nonmalignant cells preferentially use circulating dietary fatty acids for ors' Affiliations: Institut Català d'Oncologia and Institut d'Investigació èdica de Girona, Bioquímica i Biologia Molecular, Facultat de cies, Universitat de Girona, Girona, Spain; Química Orgánica I, ltad de Ciencias Químicas, Universidad Complutense de Madrid, . Anderson Cancer Center España, Madrid, Spain; Institut Català cologia and Institut d'Investigació Biomèdica de Bellvitge, and uímica i Biologia Molecular, Institut de Biomedicina, Universitat de lona, Barcelona, Spain ived 4/6/09; revised 9/22/09; accepted 9/27/09; publishedonline 12/15/09. t support: Instituto de Salud Carlos III (FIS PI04/1417, FIS PI082031, and -0020-0028; R. Colomer; ISCIII-RETIC RD06; D. Haro; and FIS PI082031; ig), the Susan G. Komen Breast Cancer Foundation (PDF-0504073, R. er), the Ministerio de Ciencia e Innovación (MICCN, CIT-090000-2008. Puig; MEC, SAF-2007-67008-CO2-01, M.L. López-Rodríguez), Comuniutónoma de Madrid (S-SAL-249-2006, M.L. López-Rodríguez), and the ish Society of Medical Oncology (SEOM08, I. Juez and T. Puig). Juan Cierva (JCI-2005-001616001; T. Puig) and Ramón y Cajal programs the Ministerio de Educación y Ciencia (RyC-07-039-04-02; S. Ortega7608 Cancer Res 2009;15(24) December 15, 2009 Cancer Rese on Sep clincancerres.aacrjournals.org Downloaded from the synthesis of new structural lipids. In contrast, high levels of FASN expression have been observed in breast cancer (2) and other human carcinomas (reviewed in ref. 3). Importantly, several reports have shown that FASN expression levels correlate with tumor progression, aggressiveness, and metastasis, and are found elevated in the serum of patients with cancer Gutiérrez), and Instituto de Salud Carlos III (CD07/00257). The MEC awarded C. Turrado with an FPU predoctoral grant. T. Puig and A. Urruticoechea received the support of a Claudia Elias award of the Fundació Institut Català d'Oncologia in 2007. 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: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). T. Puig and C. Turrado contributed equally to this work. Requests for reprints: Teresa Puig, Institut d'Investigació Biomèdica de Girona, Parc Científic i Tecnològic, Universitat de Girona, c/ Pic Peguera 11, E-17003, Girona, Spain. Phone: 34-972-940282; Fax: 34-972-485422; E-mail: [email protected]; María Luz López-Rodríguez, E-mail: mluzlr@quim. ucm.es; and Ramon Colomer, E-mail: [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-09-0856 www.aacrjournals.org arch. tember 10, 2017. © 2009 American Association for Translational Relevance The enzyme fatty acid synthase (FASN) has been shown to be a key therapeutic target in cancer given its importance in tumor biology. The present study evaluated novel anticancer compounds that inhibited FASN activity and did not have effects on fatty acid oxidation or caused weight loss in experimental animals. These compounds also represent promising agents in preliminary in vivo studies. Here, we open a new therapeutic perspective providing preliminary in vitro and in vivo proof of evidence of a new generation of synthetic FASN inhibitors with low toxicity profile. Novel Fatty Acid Synthase Inhibitors for Breast Cancer Treatment (4–6). The widespread expression of FASN in human cancer and its association with poor prognosis suggest that fatty acid synthesis provides an advantage for tumor growth, and could be a promising target for antitumor drug development (7). Pharmacologic inhibition of FASN has been used to study the loss of FASN function in tumor cells. Among the characterized inhibitors of FASN, cerulenin, C75, and (-)-epigallocatechin-3-gallate (EGCG) deserve special attention (Fig. 1). Cerulenin, a natural antibiotic product of the fungus Cephalosporium ceruleans (8), inhibits FASN activity leading to apoptotic cancer cell death in vitro (9, 10). C75, a synthetic analogue of cerulenin, is also a potent FASN inhibitor (11), which is cytotoxic for various tumor cell lines in vitro (12, 13), and shows significant in vivo antitumor activity against human cancer cell xenografts (14, 15). The use of C75 in vivo is limited by anorexia and body weight loss, which we and others have associated with the stimulation of carnitine palmitoyltransferase-1 (CPT-1; refs. 16–18), the enzyme responsible for the regulation of mitochondrial fatty acid oxidation. Recent studies have reported that EGCG, the main polyphenolic catechin of green tea, and other naturally occurring flavonoids (such as luteolin, quercetin, and kaempferol) inhibit FASN, induce apoptosis of several tumor cell lines in vitro (18–24), and reduce the size of mammary tumors in animal models (25). We have recently reported a simultaneous comparison of the cellular, molecular, and functional effects on the key fatty acid metabolism enzymes FASN and CPT-1 of C75 versus EGCG, in breast cancer cells (18). We showed that EGCG achieves all cellular, functional, and molecular antitumor effects of known FASN inhibitors but, unlike C75, it did not increase CPT-1 activity (18). However, the in vivo effect of EGCG might be limited by its IC50 value, as well as its relative instability under the slightly neutral or alkaline physiologic conditions (24), which may be illustrated by the limited activity of green tea in prostate cancer clinical trials (25, 26). We now report the synthesis and biological evaluation of a new series of polyphenolic derivatives related to EGCG, which has resulted in the identification of two potent FASN inhibitors with high antitumor activity in vitro. Their antitumor effects occurred without stimulating CPT-1 activity, and most importantly, without inducing weight loss in vivo. Interestingly, one of the novel compounds blocked tumor growth in an in vivo breast cancer xenograft model. Therefore, these novel compounds might represent suitable new agents for the treatment of cancer. 7609 www.aacrjournals.org Cancer Re on Se clincancerres.aacrjournals.org Downloaded from Materials and Methods Synthesis of polyphenolic compounds I(1-8). Target compounds 1-8 were synthesized via standard chemical procedures starting from conveniently protected gallic acid 17 (27), as described in Fig. 2 and in the Supplementary Data. Spectroscopic data of all synthesized compounds were consistent with the proposed structures. For series 1-8 and 9-16, we include the data of compounds 4, 6, 12, and 14. The rest of the compounds are described in the Supplementary Data. 1,3-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)benzene (12) was prepared following the general procedure, using gallic acid and resorcinol as starting materials. Chromatography: dichloromethane/hexane 9:1; yield: 70%; mp 194–196°C; IR ν 1734, 1593, 1500, 1128; H-NMR (CDCl3) δ 5.19 (s, 12H), 7.13–7.45 (m, 34H), 7.55 (s, 4H); CNMR (CDCl3) δ 71.3, 75.2, 109.7, 115.9, 119.3, 124.2, 127.6, 128.0, 128.1, 128.2, 128.5, 128.6, 136.5, 137.3, 143.2, 151.9, 152.7, 164.4. 1,3-Bis[(3,4,5-trihydroxybenzoyl)oxy]benzene (4) was prepared following the general procedure, starting from intermediate 12. Yield: 60%; mp 194–195°C; IR ν 3370, 1718, 1618, 1200; H-NMR (CD3OD) δ 7.06–7.14 (m, 3H), 7.20 (s, 4H), 7.48 (t, J = 8.4, 1H); C-NMR (CD3OD) δ 110.7, 117.1, 120.3, 130.8, 140.8, 146.8, 153.3, 166.7; ESI-MS 412.8 (M H). 2,3-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)naphthalene (14) was prepared following the above described general procedure, using gallic acid and naphthalene-2,3-diol as starting materials. Chromatography: dichloromethane/hexane 6:4; yield: 58%; mp 130–131°C; IR ν 1740, 1589, 1500, 1194; H-NMR (CDCl3) δ 4.83 (s, 8H), 4.91 (s, 4H), 7.05– 7.28 (m, 30H), 7.38 (s, 4H), 7.44–7.49 (m, 2H), 7.78–7.83 (m, 4H); CNMR (CDCl3) δ 71.2, 75.1, 109.5, 121.2, 123.7, 126.5, 127.6, 127.9, 128.1, 128.2, 128.3, 128.5, 131.7, 136.3, 137.4, 141.4, 143.3, 152.7, 164.2. 2,3-Bis[(3,4,5-trihydroxybenzoyl)oxy]naphthalene (6) was prepared following the general procedure, starting from intermediate 14. Yield: 60%; mp 182–183°C; IR ν 3308, 1743, 1618, 1194; H-NMR (CD3OD) δ 7.12 (s, 4H), 7.51–7.56 (m, 2H), 7.79 (s, 2H), 7.89–7.92 (m, 2H); C-NMR (CD3OD) δ 109.3, 118.4, 120.6, 125.9, 126.9, 131.7, 139.2, 141.9, 145.1, 165.0; ESI-MS 487.0 (M + Na). Cell lines and cell culture. Cells were routinely incubated at 37°C with 5% CO2. BT474, MCF-7, and MDA-MB-231 breast cancer cells were obtained from the American Type Culture Collection, and were routinely grown in DMEM (Life Technologies) containing 10% fetal bovine serum (Bio Whittaker), 1% L-glutamine, 1% sodium pyruvate, 50 units/mL penicillin, and 50 μg/mL streptomycin. SK-Br3 breast cancer cells were obtained from Eucellbank, and were passaged in McCoy's 5A medium containing 10% fetal bovine serum, 1% L-glutamine, 1% sodium pyruvate, 50 units/mL of penicillin, and 50 μg/mL of streptomycin. Cytotoxicity assay. EGCG and 3,4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma. Drug sensitivity was determined using a standard colorimetric MTT assay. Briefly, cells were plated out at a density of 7 × 10 cells/100 μL/well in 96-well microtiter plates, and allowed an overnight period for attachment. Then the medium was removed and cells were incubated for 48 h with fresh medium containing different concentrations of EGCG or the novel polyphenolic compound 1-8. Following treatment, drug-free medium (100 μL/well) and 10 μL of a 5mg/mLMTT solution were added and cells were incubated for 3 h at 37°C. After careful removal of the supernatants, the MTT-formazan crystals formed by metabolically viable cells were dissolved in DMSO (100 μL/well) and absorbance was measured at 570 nm in a multi-well plate reader (Model Anthos Labtec 2010 1.7). FASN activity assay. EDTA, dithiotreitol, acetyl-CoA, malonyl-CoA, and NADPH were purchased from Sigma. After 6, 12, or 24 h of exposure to drug, cells were harvested by treatment with trypsin-EDTA solution, pelleted by centrifugation, washed twice, and resuspended in cold PBS. Cells were sonicated for 30 min at 4°C (PSelecta Ultrasons) and centrifuged for 15 min at 4°C to obtain particle-free supernatants. A supernatant sample was taken to measure protein content by a Clin Cancer Res 2009;15(24) December 15, 2009 search. ptember 10, 2017. © 2009 American Association for Cancer Therapy: Preclinical Lowry-based Bio-Rad assay (Bio-Rad Laboratories) and the adequate volume was diluted to a concentration of 1 μg/μL. One-hundred and twenty microliters of this particle-free supernatant were preincubated for 15 min at 37°C for temperature equilibration. The sample was then added to 150 μL of the reaction buffer followed by 30 μL of 500 μmol/L malonyl-CoA (FASN substrate), and the final volume of 0.3 mL of reaction mixture [200 mmol/L potassium phosphate buffer (pH 7.0), 1 mmol/L EDTA, 1 mmol/L DTT, 30 μmol/L acetyl-CoA, 0.24 mmol/L NADPH, and 50 μmol/L malonyl-CoA] was assayed for 10 min to determine FASN-dependent oxidation of NADPH. Before the addition of malonyl-CoA, the background rate of NADPH oxidation in the presence of acetyl-CoA was monitored at 340 nm for 3 min. FASN activity was expressed in nmol NADPH oxidized × min × mg protein. Immunoblot analysis of FASN, p185, phosphorylated p185, ERK1/2, phosphorylated ERK1/2, AKT, phosphorylated AKT, and poly(ADP-ribose) polymerase. The primary antibody for FASN immunoblotting was a mouse IgG1 FASN monoclonal antibody from BD Biosciences PharMingen. Monoclonal anti–β-actin mouse antibody 7610 Clin Cancer Res 2009;15(24) December 15, 2009 Cancer Rese on Sep clincancerres.aacrjournals.org Downloaded from (clone AC-15) was from Sigma. Rabbit polyclonal antibodies against poly(ADP-ribose) polymerase (PARP), AKT, phosphorylated AKT, and mouse monoclonal antibodies against p185 (clone Ab-3) and phosphorylated p185 were from Cell Signaling Technology. Following treatment of SK-Br3 cells with EGCG or novel polyphenolic compounds at corresponding concentrations and time intervals, cells were harvested by treatment with trypsin-EDTA, washed twice with PBS, and stored at -80°C. Cells were lysed in lysis buffer [1 mmol/L EDTA, 150 mmol/L NaCl, 100 μg/mL phenylmethylsulfonyl fluoride, 50 mmol/L Tris-HCl (pH 7.5)] and kept at 4°C while they were routinely mixed every 2 min on the vortex for 30 min. A sample was taken for measurement of protein content by the Lowry-based BioRad assay. Equal amounts of protein were heated in SDS sample buffer (Laemmli) for 5 min at 95°C, separated on a 3% to 8% SDS-polyacrylamide gel (FASN, p185, phosphorylated p185) or 4% to 12% SDS-polyacrylamide gel (AKT, phosphorylated AKT, ERK1/2, phosphorylated ERK1/2, and PARP), and transferred onto nitrocellulose membranes. Membranes were incubated for 1 h at room temperature in blocking buffer (2.5% powdered skim milk in tris-buffered solution with 0.05% Tween 20; TBS-T [10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, and 0.05% Tween 20]) to prevent nonspecific antibody binding, and incubated with the corresponding primary antibody diluted in blocking buffer overnight at 4°C. After three 5-min washes in TBS-T, blots were incubated for 1 h with corresponding peroxidase-conjugated secondary antibody and developed using a commercial kit (West Pico chemiluminescent substrate). Blots were reprobed with an antibody against β-actin as control of protein loading and transfer. Measurement of CPT-1 activity. CPT-1 activity assay was done using palmitoyl-CoA lithium salt from Sigma, fatty acid–free bovine serum albumin from Roche, L-carnitine hydrochloride from Sigma, and L-[methyl-H]carnitine hydrochloride (82 Ci/mmol) from Amersham Biosciences. CPT-1 activity was assayed by the forward exchange method using L-[H]carnitine as we previously described (17, 18). Briefly, reactions (total volume of 0.5 mL) consisted of the standard enzyme assay mixture with 200 μmol/L of the corresponding FASN inhibitor, 2 mmol/L of L-[H]carnitine (∼5,000 dpm/nmol), 80 μmol/L of Fig. 1. Natural and synthetic FASN inhibitors. Fig. 2. General synthetic route for the synthesis of new polyphenolic compounds. www.aacrjournals.org arch. tember 10, 2017. © 2009 American Association for Novel Fatty Acid Synthase Inhibitors for Breast Cancer Treatment palmitoyl-CoA, 20 mmol/L of HEPES (pH 7.0), 1% fatty acid–free albumin, and 40 to 75 mmol/L of KCl. Reactions were initiated by the addition of isolated intact yeastmitochondria. The reactionwas linear up to 4 min and all incubations were done at 30°C for 3 min. Reactions were stopped by the addition of 6% perchloric acid and were then centrifuged at 2,500 rpm for 5 min. The resulting pellet was suspended in water and the product [H]palmitoylcarnitine was extracted with butanol at low pH. After centrifugation at 2,300 rpm for 3min, an aliquot of the butanol phase was transferred to a vial and counted by liquid scintillation. Animal short-term weight loss experiments. Male mice C57BL/6J (12 wk, 23-25 g) were purchased from Harlan Laboratories (France), fed ad libitum with a standard rodent chow and housed in a light/dark 12 h/12 h cycle at 22°C for 1 wk. C75 was from Alexis Biochemicals. Animals were randomized into five groups of four animals each: control, C75-treated, EGCG-treated, and compounds 7and 8-treated. All experiments were done in accordance with guidelines on animal care and use established by the University of Barcelona School of Pharmacy Institutional Ethic and Scientific Committee (Barcelona, Spain). Treatments were done as previously described (16, 28). Briefly, mice were fasted for 12 h during the dark cycle before treatment. Each group received a single i.p. injection (0.5 mL) of FASN inhibitor (30 mg/kg) or vehicle alone (DMSO), dissolved in RPMI 1640. After i.p. injections, mice were given free access to rodent chow for 24 h. At this time, the experiment finished and body weight was registered. Human breast tumor xenograft experiments. Female athymic nude BALB/c mice (4-5 wk old) were purchased from Harlan Laboratories (France) and housed in a pathogen-free facility. Tumor xenograft was established by s.c. injection of 10 × 10 BT474 cells mixed in Matrigel (BD Bioscience) into the female's flank. Tumors were allowed to increase 150 to 250 mm size, and the mice were randomized into groups of six animals each. Mice were treated by i.p injection daily with 40 mg/kg of compound 7 for 45 days. Tumors were measured daily with electronic calipers, and tumor volumes were calculated by the formula: π/6 × (v1 × v2 × v2), where v1 represents the largest tumor diameter, and v2 the smallest one. Weight from control and treatment groups was registered daily. At the end of the experiment, all mice were euthanized, and tumors were stored at -80°C. All experiments were conducted in accordance with guidelines on animal care and use established by Biomedical Research Institute of Bellvitge (IDIBELL) Institutional Animal Care and Scientific Committee. 7611 www.aacrjournals.org Cancer Re on Se clincancerres.aacrjournals.org Downloaded from Statistical analysis. Results were analyzed by Student's t test or by one-way ANOVA using a Tukey test as a post-test. P < 0.05 was considered statistically significant. Further details. For details concerning apoptosis, and malignant and nonmalignant cell proliferating assays, see Supplementary Data.