Membrane disruption, but not metabolic rewiring, is the key mechanism of anticancer-action of FASN-inhibitors: a multi-omics analysis in ovarian cancer.

Fatty-acid(FA)-synthase(FASN) is a druggable lipogenic oncoprotein whose blockade causes metabolic disruption. Whether drug-induced metabolic perturbation is essential for anticancer drug-action, or is just a secondary-maybe even a defence response-is still unclear. To address this, SKOV3 and OVCAR3 ovarian cancer(OC) cell lines with clear cell and serous histology, two main OC subtypes, were exposed to FASN-inhibitor G28UCM. Growth-inhibition was compared with treatment-induced cell-metabolomes, lipidomes, proteomes and kinomes. SKOV3 and OVCAR3 were equally sensitive to low-dose G28UCM, but SKOV3 was more resistant than OVCAR3 to higher concentrations. Metabolite levels generally decreased upon treatment, but individual acylcarnitines, glycerophospholipids, sphingolipids, amino-acids, biogenic amines, and monosaccharides reacted differently. Drug-induced effects on central-carbon-metabolism and oxidative-phosphorylation (OXPHOS) were essentially different in the two cell lines, since drug-naïve SKOV3 are known to prefer glycolysis, while OVCAR3 favour OXPHOS. Moreover, drug-dependent increase of desaturases and polyunsaturated-fatty-acids (PUFAs) were more pronounced in SKOV3 and appear to correlate with G28UCM-tolerance. In contrast, expression and phosphorylation of proteins that control apoptosis, FA synthesis and membrane-related processes (beta-oxidation, membrane-maintenance, transport, translation, signalling and stress-response) were concordantly affected. Overall, membrane-disruption and second-messenger-silencing were crucial for anticancer drug-action, while metabolic-rewiring was only secondary and may support high-dose-FASN-inhibitor-tolerance. These findings may guide future anti-metabolic cancer intervention.

miR-130b is a potent stimulator of hepatic very-low-density lipoprotein assembly and secretion via marked induction of microsomal triglyceride transfer protein.

miR-130b is a microRNA whose expression is particularly elevated within adipose tissue and in the circulation in diabetic states. Hepatic miR-130b expression has been linked to hepatocellular carcinoma and changes in lipid metabolism. Here, we investigated the role of miR-130b in hepatic lipid homeostasis and lipoprotein export. We observed that overexpression of miR-130b-3p or -5p in HepG2 cells markedly enhanced the secretion of very-low-density lipoprotein (VLDL) particles, enhanced the secretion of [3H]glycerol metabolically labeled triglyceride (TG), and significantly increased the number or the average size of lipid droplets (LDs), respectively. Overexpression of miR-130b also altered the expression of key genes involved in lipid metabolism and in particular markedly increased both mRNA and protein expression levels of microsomal triglyceride transfer protein (MTP). Conversely, the miR-130b inhibitor decreased mRNA levels of MTP and fatty acid synthase (FAS) in HepG2 cells. However, dual-luciferase reporter assays indicated that MTP is not a direct target of miR-130b-3p. miR-130b overexpression did not alter de novo synthesized TG or the stability and secretion of apolipoprotein B 100. Interestingly, knockdown of phosphatase and tensin homolog (PTEN) blocked the upregulation of MTP mRNA induced by miR-130b. Finally, miR-130b-induced stimulation of VLDL secretion was also observed in a second hepatocyte cell culture model, immortalized human hepatocytes, confirming the effects observed in HepG2 cells. Overall, these data suggest a potential role for miR-130b in promoting hepatic VLDL assembly and secretion mediated by marked stimulation of MTP expression and TG mobilization. Thus miR-130b overexpression corrects the defect in VLDL production in HepG2 cells.

Fatty Acid Inhibition Sensitizes Androgen-Dependent and -Independent Prostate Cancer to Radiotherapy via FASN/NF-κB Pathway.

Elevated fatty acid synthase (FASN) has been reported in both androgen-dependent and -independent prostate cancers. Conventional treatment for prostate cancer is radiotherapy (RT); however, the following radiation-induced radioresistance often causes treatment failure. Upstream proteins of FASN such as Akt and NF-κB are found increased in the radioresistant prostate cancer cells. Nevertheless, whether inhibition of FASN could improve RT outcomes and reverse radiosensitivity of prostate cancer cells is still unknown. Here, we hypothesised that orlistat, a FASN inhibitor, could improve RT outcomes in prostate cancer. Orlistat treatment significantly reduced the S phase population in both androgen-dependent and -independent prostate cancer cells. Combination of orlistat and RT significantly decreased NF-κB activity and related downstream proteins in both prostate cancer cells. Combination effect of orlistat and RT was further investigated in both LNCaP and PC3 tumour-bearing mice. Combination treatment showed the best tumour inhibition compared to that of orlistat alone or RT alone. These results suggest that prostate cancer treated by conventional RT could be improved by orlistat via inhibition of FASN.

Triclosan Is an Aminoglycoside Adjuvant for Eradication of Pseudomonas aeruginosa Biofilms.

One of the most important clinical obstacles in cystic fibrosis (CF) treatment is antibiotic treatment failure due to biofilms produced by Pseudomonas aeruginosa The ability of this pathogen to survive eradication by tobramycin and pathoadapt into a hyperbiofilm state leading to chronic infections is key to its success. Retrospective studies have demonstrated that preventing this pathoadaptation by improving eradication is essential to extend the lives of CF patients. To identify adjuvants that enhance tobramycin eradication of P. aeruginosa, we performed a high-throughput screen of 6,080 compounds from four drug-repurposing libraries. We identified that the Food and Drug Administration (FDA)-approved compound triclosan, in combination with tobramycin, resulted in a 100-fold reduction of viable cells within biofilms at 6 h, but neither compound alone had significant antimicrobial activity against biofilms. This synergistic treatment significantly accelerated the killing of biofilms compared to that with tobramycin treatment alone, and the combination was effective against 6/7 CF clinical isolates compared to tobramycin treatment alone, including a tobramycin-resistant strain. Further, triclosan and tobramycin killed persister cells, causing a 100-fold reduction by 8 h and complete eradication by 24 h. Triclosan also enhances tobramycin killing of multiple Burkholderia cenocepacia and Staphylococcus aureus clinical isolates grown as biofilms. Additionally, triclosan showed synergy with other aminoglycosides, such as gentamicin or streptomycin. Triclosan is a well-tolerated aminoglycoside adjuvant shown to be safe for human use that could improve the treatment of biofilm-based infections.

N-Acylated Derivatives of Sulfamethoxazole Block Chlamydia Fatty Acid Synthesis and Interact with FabF.

The type II fatty acid synthesis (FASII) pathway is essential for bacterial lipid biosynthesis and continues to be a promising target for novel antibacterial compounds. Recently, it has been demonstrated that Chlamydia is capable of FASII and this pathway is indispensable for Chlamydia growth. Previously, a high-content screen with Chlamydia trachomatis-infected cells was performed, and acylated sulfonamides were identified to be potent growth inhibitors of the bacteria. C. trachomatis strains resistant to acylated sulfonamides were isolated by serial passage of a wild-type strain in the presence of low compound concentrations. Results from whole-genome sequencing of 10 isolates from two independent drug-resistant populations revealed that mutations that accumulated in fabF were predominant. Studies of the interaction between the FabF protein and small molecules showed that acylated sulfonamides directly bind to recombinant FabF in vitro and treatment of C. trachomatis-infected HeLa cells with the compounds leads to a decrease in the synthesis of Chlamydia fatty acids. This work demonstrates the importance of FASII for Chlamydia development and may lead to the development of new antimicrobials.

Effects of fatty acid synthase inhibitors on lymphatic vessels: an in vitro and in vivo study in a melanoma model.

Fatty acid synthase (FASN) is responsible for the endogenous production of fatty acids from acetyl-CoA and malonyl-CoA. Its overexpression is associated with poor prognosis in human cancers including melanomas. Our group has previously shown that the inhibition of FASN with orlistat reduces spontaneous lymphatic metastasis in experimental B16-F10 melanomas, which is a consequence, at least in part, of the reduction of proliferation and induction of apoptosis. Here, we sought to investigate the effects of pharmacological FASN inhibition on lymphatic vessels by using cell culture and mouse models. The effects of FASN inhibitors cerulenin and orlistat on the proliferation, apoptosis, and migration of human lymphatic endothelial cells (HDLEC) were evaluated with in vitro models. The lymphatic outgrowth was evaluated by using a murine ex vivo assay. B16-F10 melanomas and surgical wounds were produced in the ears of C57Bl/6 and Balb-C mice, respectively, and their peripheral lymphatic vessels evaluated by fluorescent microlymphangiography. The secretion of vascular endothelial growth factor C and D (VEGF-C and -D) by melanoma cells was evaluated by ELISA and conditioned media used to study in vitro lymphangiogenesis. Here, we show that cerulenin and orlistat decrease the viability, proliferation, and migration of HDLEC cells. The volume of lymph node metastases from B16-F10 experimental melanomas was reduced by 39% in orlistat-treated animals as well as the expression of VEGF-C in these tissues. In addition, lymphatic vessels from orlistat-treated mice drained more efficiently the injected FITC-dextran. Orlistat and cerulenin reduced VEGF-C secretion and, increase production of VEGF-D by B16-F10 and SK-Mel-25 melanoma cells. Finally, reduced lymphatic cell extensions, were observed following the treatment with conditioned medium from cerulenin- and orlistat-treated B16-F10 cells. Altogether, our results show that FASN inhibitors have anti-metastatic effects by acting on lymphatic endothelium and melanoma cells regardless the increase of lymphatic permeability promoted by orlistat.

The stringent response plays a key role in Bacillus subtilis survival of fatty acid starvation.

The stringent response is a universal adaptive mechanism to protect bacteria from nutritional and environmental stresses. The role of the stringent response during lipid starvation has been studied only in Gram-negative bacteria. Here, we report that the stringent response also plays a crucial role in the adaptation of the model Gram-positive Bacillus subtilis to fatty acid starvation. B. subtilis lacking all three (p)ppGpp-synthetases (RelBs , RelP and RelQ) or bearing a RelBs variant that no longer synthesizes (p)ppGpp suffer extreme loss of viability on lipid starvation. Loss of viability is paralleled by perturbation of membrane integrity and function, with collapse of membrane potential as the likely cause of death. Although no increment of (p)ppGpp could be detected in lipid starved B. subtilis, we observed a substantial increase in the GTP/ATP ratio of strains incapable of synthesizing (p)ppGpp. Artificially lowering GTP with decoyinine rescued viability of such strains, confirming observations that low intracellular GTP is important for survival of nutritional stresses. Altogether, our results show that activation of the stringent response by lipid starvation is a broadly conserved response of bacteria and that a key role of (p)ppGpp is to couple biosynthetic processes that become detrimental if uncoordinated.

FASN, ErbB2-mediated glycolysis is required for breast cancer cell migration.

Both fatty acid synthase (FASN) and ErbB2 have been shown to promote breast cancer cell migration. However, the underlying molecular mechanism remains poorly understood and there is no reported evidence that directly links glycolysis to breast cancer cell migration. In this study, we investigated the role of FASN, ErbB2-mediated glycolysis in breast cancer cell migration. First, we compared lactate dehydrogenase A (LDHA) protein levels, glycolysis and cell migration between FASN, ErbB2-overexpressing SK-BR-3 cells and FASN, ErbB2-low-expressing MCF7 cells. Then, SK-BR-3 cells were treated with cerulenin (Cer), an inhibitor of FASN, and ErbB2, LDHA protein levels, glycolysis, and cell migration were detected. Next, we transiently transfected ErbB2 plasmid into MCF7 cells and detected FASN, LDHA protein levels, glycolysis and cell migration. Heregulin-ß1 (HRG-ß1) is an activator of ErbB2 and 2-deoxyglucose (2-DG) and oxamate (OX) are inhibitors of glycolysis. MCF7 cells were treated with HRG-ß1 alone, HRG-ß1 plus 2-DG, OX or cerulenin and glycolysis, and cell migration were measured. We found that FASN, ErbB2-high-expressing SK-BR-3 cells displayed higher levels of glycolysis and migration than FASN, ErbB2-low-expressing MCF7 cells. Inhibition of FASN by cerulenin impaired glycolysis and migration in SK-BR-3 cells. Transient overexpression of ErbB2 in MCF7 cells promotes glycolysis and migration. Moreover, 2-deoxyglucose (2-DG), oxamate (OX), or cerulenin partially reverses heregulin-ß1 (HRG-ß1)-induced glycolysis and migration in MCF7 cells. In conclusion, this study demonstrates that FASN, ErbB2-mediated glycolysis is required for breast cancer cell migration. These novel findings indicate that targeting FASN, ErbB2-mediated glycolysis may be a new approach to reverse breast cancer cell migration.

Development of a Self-Assembled Nanoparticle Formulation of Orlistat, Nano-ORL, with Increased Cytotoxicity against Human Tumor Cell Lines.

Fatty acid synthase (FASN), the enzyme that catalyzes de novo synthesis of fatty acids, is expressed in many cancer types. Its potential as a therapeutic target is well recognized, but inhibitors of FASN have not yet been approved for cancer therapy. Orlistat (ORL), an FDA-approved lipase inhibitor, is also an effective inhibitor of FASN. However, ORL is extremely hydrophobic and has low systemic uptake after oral administration. Thus, new strategies are required to formulate ORL for cancer treatment as a FASN inhibitor. Here, we report the development of a nanoparticle (NP) formulation of ORL using amphiphilic bioconjugates that are derived from hyaluronic acid (HA), termed Nano-ORL. The NPs were loaded with up to 20 wt % weight of ORL at greater than 95% efficiency. The direct inhibition of the human recombinant thioesterase domain of FASN by ORL extracted from Nano-ORL was similar to that of stock ORL. Nano-ORL demonstrated a similar ability to inhibit cellular FASN activity when compared to free ORL, as demonstrated by analysis of (14)C-acetate incorporation into lipids. Nano-ORL treatment also disrupted mitochondrial function similarly to ORL by reducing adenosine triphosphate turnover in MDA-MB-231 and LNCaP cells. Nano-ORL demonstrated increased potency compared to ORL toward prostate and breast cancer cells. Nano-ORL decreased viability of human prostate and breast cancer cell lines to 55 and 57%, respectively, while free ORL decreased viability to 71 and 79% in the same cell lines. Moreover, Nano-ORL retained cytotoxic activity after a 24 h preincubation in aqueous conditions. Preincubation of ORL dramatically reduced the efficacy of ORL as indicated by high cell viability (>85%) in both breast and prostate cell lines. These data demonstrate that NP formulation of ORL using HA-derived polymers retains similar levels of FASN, lipid synthesis, and ATP turnover inhibition while significantly improving the cytotoxic activity against cancer cell lines.

Sonic hedgehog inhibitors prevent colitis-associated cancer via orchestrated mechanisms of IL-6/gp130 inhibition, 15-PGDH induction, Bcl-2 abrogation, and tumorsphere inhibition.

Sonic hedgehog (SHH) signaling is essential in normal development of the gastrointestinal (GI) tract, whereas aberrantly activated SHH is implicated in GI cancers because it facilitates carcinogenesis by redirecting stem cells. Since colitis-associated cancer (CAC) is associated with inflammatory bowel diseases, in which SHH and IL-6 signaling, inflammation propagation, and cancer stem cell (CSC) activation have been implicated, we hypothesized that SHH inhibitors may prevent CAC by blocking the above SHH-related carcinogenic pathways. In the intestinal epithelial cells IEC-6 and colon cancer cells HCT-116, IL-6 expression and its signaling were assessed with SHH inhibitors and levels of other inflammatory mediators, proliferation, apoptosis, tumorsphere formation, and tumorigenesis were also measured. CAC was induced in C57BL/6 mice by administration of azoxymethane followed by dextran sodium sulfate administration. SHH inhibitors were administered by oral gavage and the mice were sacrificed at 16 weeks. TNF-α-stimulated IEC-6 cells exhibited increased levels of proinflammatory cytokines and enzymes, whereas SHH inhibitors suppressed TNF-α-induced inflammatory signaling, especially IL-6/IL-6R/gp130 signaling. SHH inhibitors significantly induced apoptosis, inhibited cell proliferation, suppressed tumorsphere formation, and reduced stemness factors. In the mouse model, SHH inhibitors significantly reduced tumor incidence and multiplicity, decreased the expression of IL-6, TNF-α, COX-2, STAT3, and NF-κB, and significantly induced apoptosis. In colosphere xenografts, SHH inhibitor significantly suppressed tumorigenesis by inhibiting tumorsphere formation. Taken together, our data suggest that administration of SHH inhibitors could be an effective strategy to prevent colitis-induced colorectal carcinogenesis, mainly by targeting IL-6 signaling, ablating CSCs, and suppressing oncogenic inflammation, achieving chemoquiescence ultimately.

Progress in the development of fatty acid synthase inhibitors as anticancer targets.

Fatty acid synthase (E.C. 2.3.1.85; FASN) is a multifunctional enzyme system that catalyzes the formation of fatty acids from acetyl-CoA, malonyl-CoA, and NADPH and plays a central role in lipid biosynthesis. Two classes of FASN exist: FASN I in animals and fungi, and FASN II in plants and prokaryotes. Animal FASN I is a homodimeric protein found in the cytosol of lipogenic tissues such as the liver and brain. Many human carcinomas exhibit elevated levels of FASN I, though the benefit to cancer cells is still unclear. Inhibition of FASN I selectively effects apoptosis in cancer cells, and the role of FASN I in chemotherapy is a growing area of research with the use of natural products and small molecule inhibitors.

Triclosan induces Fas receptor-dependent apoptosis in mouse neocortical neurons in vitro.

Triclosan (TCS) is a commonly used antimicrobial agent in personal care and sanitizing products, as well as in household items. Numerous studies have demonstrated the presence of TCS in various human tissues. Several studies have reported the accumulation of TCS in fish and human brain tissue. The aim of the present study was to investigate the effect of TCS on apoptosis in mouse neocortical neurons after 7 days of culture in vitro following 3, 6 and 24 h of exposure. To explore the mechanism underlying the effects of TCS in neurons, we studied the activation and protein expression of the Fas receptor (FasR) and caspase-8, caspase-9 and caspase-3, as well as DNA fragmentation in TCS-treated cells. Cultures of neocortical neurons were prepared from Swiss mouse embryos on day 15/16 of gestation. The cells were cultured in phenol red-free Neurobasal medium with B27 and glutamine. The cultures were treated with concentrations of TCS ranging from 1 nM to 100 µM for 3, 6 and 24 h. The level of lactate dehydrogenase (LDH) was measured in the culture medium to exclude the cytotoxic concentrations. The cytotoxic effects were only observed when the highest concentrations of TCS were used (50 and 100 µM). To study apoptosis, the activities of caspase-8, caspase-9 and caspase-3 were measured, and DNA fragmentation was evaluated. Our results are the first time to demonstrate that TCS can induce an apoptotic process in neocortical neurons in vitro. The data demonstrated that TCS caused caspase-3 activation, DNA fragmentation and apoptotic body formation. Non-cytotoxic concentrations of TCS activated the extrinsic apoptotic signaling pathway, which is dependent on FasR and caspase-8 activation. However, it is also possible that TCS may activate the intrinsic apoptotic pathway after long-term exposure. Therefore, further studies on the mechanism underlying the effects of TCS on the nervous system are needed.

The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer.

Inhibition of FASN has emerged as a promising therapeutic target in cancer, and numerous inhibitors have been investigated. However, severe pharmacological limitations have challenged their clinical testing. The synthetic FASN inhibitor triclosan, which was initially developed as a topical antibacterial agent, is merely affected by these pharmacological limitations. Yet, little is known about its mechanism in inhibiting the growth of cancer cells. Here we compared the cellular and molecular effects of triclosan in a panel of eight malignant and non-malignant prostate cell lines to the well-known FASN inhibitors C75 and orlistat, which target different partial catalytic activities of FASN. Triclosan displayed a superior cytotoxic profile with a several-fold lower IC50 than C75 or orlistat. Structure-function analysis revealed that alcohol functionality of the parent phenol is critical for inhibitory action. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Importantly, triclosan, C75 and orlistat induced distinct changes to morphology, cell cycle, lipid content and the expression of key enzymes of lipid metabolism, demonstrating that inhibition of different partial catalytic activities of FASN activates different metabolic pathways. These finding combined with its well-documented pharmacological safety profile make triclosan a promising drug candidate for the treatment of prostate cancer.

Inhibition of fatty acid synthase induces pro-survival Akt and ERK signaling in K-Ras-driven cancer cells.

Cancer cells with constitutive phosphatidylinositol 3-kinase (PI3K)/Akt pathway activation have been associated with overexpression of the lipogenic enzyme fatty acid synthase (FAS) as a means to provide lipids necessary for cell growth. In contrast, K-Ras-driven cancer cells suppress utilization of de novo synthesized fatty acids and rely on exogenously supplied fatty acids for cell growth and membrane phospholipid biosynthesis. Consistent with a differential need for de novo fatty acid synthesis, cancer cells with activated PI3K signaling were sensitive to suppression of FAS; whereas mutant K-Ras-driven cancer cells continued to proliferate with suppressed FAS. Surprisingly, in response to FAS suppression, we observed robust increases in both Akt and ERK phosphorylation. Akt phosphorylation was dependent on the insulin-like growth factor-1 receptor (IGF-1R)/PI3K pathway and mTOR complex 2. Intriguingly, K-Ras-mediated ERK activation was dependent on N-Ras. Pharmacological inhibition of PI3K and MEK in K-Ras-driven cancer cells resulted in increased sensitivity to FAS inhibition. These data reveal a surprising sensitivity of K-Ras-driven cancer cells to FAS suppression when stimulation of Akt and ERK was prevented. As K-Ras-driven cancers are notoriously difficult to treat, these findings have therapeutic implications.

Fatty acid synthase inhibitors induce apoptosis in non-tumorigenic melan-a cells associated with inhibition of mitochondrial respiration.

The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent of FASN inhibition.

How lipidomics provides new insight into drug discovery.

INTRODUCTION: Automated lipidomic methods based on mass spectrometry (MS) are now proposed to screen a large variety of candidate drugs available that inhibit de novo lipid synthesis and replace tedious methods based on radiotracer incorporation. A major new interest in inhibitors of de novo lipogenesis is their proapoptotic effect observed in cancerous cells. AREAS COVERED: In this review, the authors focus on the screening methods of antilipogenic inhibitors targeting the synthesis of malonylCoA (carbonic anhydrase, acetylCoA carboxylase), palmitylCoA (fatty acid synthase condensing and thioesterase subunits) and monounsaturated fatty acids (Δ9-desaturase). The consequences of inhibition depend on how the pathway deviates above the blockade: accelerated mitochondrial fatty acid oxidation following the decreased malonylCoA level, accumulation of ketone bodies and increased cholesterol synthesis following the increased acetylCoA level. Side effects such as anorexia and skin defects may critically decrease therapeutic indices in the long term. The authors emphasize the need for assessment of toxicity in short-term treatments inducing proapoptotic effects observed in aggressive hormone-dependent malignancies. EXPERT OPINION: The activity of lipogenesis inhibitors can be recognised in lipid profiles established by a combination of MS-based measurements and multivariate analysis processing hundreds of lipid molecular species. Because the method can be automated, it is suitable for screening large chemical libraries, with particular focus on anticancer activities.

Overexpression of fatty acid synthase in human gliomas correlates with the WHO tumor grade and inhibition with Orlistat reduces cell viability and triggers apoptosis.

Fatty acid synthase (FASN), catalyzing the de novo synthesis of fatty acids, is known to be deregulated in several cancers. Inhibition of this enzyme reduces tumor cell proliferation. Unfortunately, adverse effects and chemical instability prevent the in vivo use of the best-known inhibitors, Cerulenin and C75. Orlistat, a drug used for obesity treatment, is also considered as a potential FASN inhibitor, but its impact on glioma cell biology has not yet been described. In this study, we analyzed FASN expression in human glioma samples and primary glioblastoma cell cultures and the effects of FASN inhibition with Orlistat, Cerulenin and C75. Immunohistochemistry followed by densitometric analysis of 20 glioma samples revealed overexpression of FASN that correlated with the WHO tumor grade. Treatment of glioblastoma cells with these inhibitors resulted in a significant, dose-dependent reduction in tumor cell viability and fatty acid synthesis. Compared to Cerulenin and C75, Orlistat was a more potent inhibitor in cell cultures and cell lines. In LN229, cell-growth was reduced by 63.9 ± 8.7 % after 48 h and 200 µM Orlistat compared to controls; in LT68, the reduction in cell growth was 76.3 ± 23.7 %. Nuclear fragmentation assay and Western blotting analysis after targeting FASN with Orlistat demonstrated autophagy and apoptosis. Organotypic slice cultures treated with Orlistat showed reduced proliferation after Ki67 staining and increased caspase-3 cleavage. Our results suggest that FASN may be a therapeutic target in malignant gliomas and identify Orlistat as a possible anti-tumor drug in this setting.

Structural insights into bacterial resistance to cerulenin.

UNLABELLED: Cerulenin is a fungal toxin that inhibits both eukaryotic and prokaryotic ketoacyl-acyl carrier protein synthases or condensing enzymes. It has been used experimentally to treat cancer and obesity, and is a potent inhibitor of bacterial growth. Understanding the molecular mechanisms of resistance to cerulenin and similar compounds is thus highly relevant for human health. We have previously described a Bacillus subtilis cerulenin-resistant strain, expressing a point-mutated condensing enzyme FabF (FabF[I108F]) (i.e. FabF with isoleucine 108 substituted by phenylalanine). We now report the crystal structures of wild-type FabF from B. subtilis, both alone and in complex with cerulenin, as well as of the FabF[I108F] mutant protein. The three-dimensional structure of FabF[I108F] constitutes the first atomic model of a condensing enzyme that remains active in the presence of the inhibitor. Soaking the mycotoxin into preformed wild-type FabF crystals allowed for noncovalent binding into its specific pocket within the FabF core. Interestingly, only co-crystallization experiments allowed us to trap the covalent complex. Our structure shows that the covalent bond between Cys163 and cerulenin, in contrast to that previously proposed, implicates carbon C3 of the inhibitor. The similarities between Escherichia coli and B. subtilis FabF structures did not explain the reported inability of ecFabF[I108F] (i.e. FabF from Escherichia coli with isoleucine 108 substituted by phenylalanine) to elongate medium and long-chain acyl-ACPs. We now demonstrate that the E. coli modified enzyme efficiently catalyzes the synthesis of medium and long-chain ketoacyl-ACPs. We also characterized another cerulenin-insensitive form of FabF, conferring a different phenotype in B. subtilis. The structural, biochemical and physiological data presented, shed light on the mechanisms of FabF catalysis and resistance to cerulenin. DATABASE: Crystallographic data (including atomic coordinates and structure factors) have been deposited in the Protein Data Bank under accession codes 4LS5, 4LS6, 4LS7 and 4LS8.

Fatty acid synthase mediates the epithelial-mesenchymal transition of breast cancer cells.

This study aimed to investigate the role of fatty acid synthase (FASN) in the epithelial-mesenchymal transition (EMT) of breast cancer cells. MCF-7 cells and MCF-7 cells overexpressing mitogen-activated protein kinase 5 (MCF-7-MEK5) were used in this study. MCF-7-MEK5 cells showed stable EMT characterized by increased vimentin and decreased E-cadherin expression. An In vivo animal model was established using the orthotopic injection of MCF-7 or MCF-7-MEK5 cells. Real-time quantitative PCR and western blotting were used to detect the expression levels of FASN and its downstream proteins liver fatty acid-binding protein (L-FABP) and VEGF/VEGFR-2 in both in vitro and in vivo models (nude mouse tumor tissues). In MCF-7-MEK5 cells, significantly increased expression of FASN was associated with increased levels of L-FABP and VEGF/VEGFR-2. Cerulenin inhibited MCF-7-MEK5 cell migration and EMT, and reduced FASN expression and down-stream proteins L-FABP, VEGF, and VEGFR-2. MCF-7-MEK5 cells showed higher sensitivity to Cerulenin than MCF-7 cells. Immunofluorescence revealed an increase of co-localization of FASN with VEGF on the cell membrane and with L-FABP within MCF-7-MEK5 cells. Immunohistochemistry further showed that increased percentage of FASN-positive cells in the tumor tissue was associated with increased percentages of L-FABP- and VEGF-positive cells and the Cerulenin treatment could reverse the effect. Altogether, our results suggest that FASN is essential to EMT possibly through regulating L-FABP, VEGF and VEGFR-2. This study provides a theoretical basis and potential strategy for effective suppression of malignant cells with EMT.

The inhibition of oleic acid induced hepatic lipogenesis and the promotion of lipolysis by caffeic acid via up-regulation of AMP-activated kinase.

BACKGROUND: Caffeic acid (CA) can inhibit toxin-induced liver injury. In this study, CA is assessed for its lipid lowering potential when oleic acid is used to induce non-alcoholic fatty liver disease in human HepG2 cells. RESULTS: The results showed that both the triglyceride and cholesterol content are decreased in the HepG2 cells by using the enzymatic colorimetric method. CA enhances the phosphorylation of AMP-activated protein kinase (AMPK) and its primary downstream targeting enzyme, acetyl-CoA carboxylase. CA down-regulates the lipogenesis gene expression of sterol regulatory element-binding protein-1 and its target genes, fatty acid synthase in the presence of oleic acid. In addition, CA significantly decreases cholesterol and triglyceride production via inhibition the expression of both 3-hydroxy-3-methyglutary coenzyme A reductase and glycerol-3-phosphate acyltransferase. These effects are eliminated by pretreatment with compound C, an AMPK inhibitor. CONCLUSIONS: These results demonstrate that CA inhibits oleic acid induced hepatic lipogenesis and the promotion of lipolysis via up-regulation of AMP-activated kinase.