Cerulenin, a fatty acid synthase (FAS) inhibitor, induces apoptosis of variety of tumor cells. To elucidate mode of action by cerulenin, we employed the proteomics approach using Schizosaccharomyces pombe. The differential protein expression profile of S. pombe revealed that cerulenin modulated the expressions of proteins involved in stresses and metabolism, including both ade10 and adk1 proteins. The nutrient supplementation assay demonstrated that cerulenin affected enzymatic steps transferring a phosphoribosyl group. This result suggests that cerulenin accumulates AMP and p-ribosyl-s-amino-imidazole carboxamide (AICAR) and reduces other necessary nucleotides, which induces feedback inhibition of enzymes and the transcriptional regulation of related genes in de novo and salvage adenine metabolic pathway. Furthermore, the deregulation of adenine nucleotide synthesis may interfere ribonucleotide reductase and cause defects in cell cycle progression and chromosome segregation. In conclusion, cerulenin induces apoptosis through deregulation of adenine nucleotide biosynthesis resulting in nuclear division defects in S. pombe.

Bacillus subtilis responds to a sudden decrease in temperature by transiently inducing the expression of the des gene encoding for a lipid desaturase, Δ5-Des, which introduces a double bond into the acyl chain of preexisting membrane phospholipids. This Δ5-Des-mediated membrane remodeling is controlled by the cold-sensor DesK. After cooling, DesK activates the response regulator DesR, which induces transcription of des. We show that inhibition of fatty acid synthesis by the addition of cerulenin, a potent and specific inhibitor of the type II fatty acid synthase, results in increased levels of short-chain fatty acids (FA) in membrane phospholipids that lead to inhibition of the transmembrane-input thermal control of DesK. Furthermore, reduction of phospholipid synthesis by conditional inactivation of the PlsC acyltransferase causes significantly elevated incorporation of long-chain FA and constitutive upregulation of the des gene. Thus, we provide in vivo evidence that the thickness of the hydrophobic core of the lipid bilayer serves as one of the stimulus sensed by the membrane spanning region of DesK.

In this study, we determined that cerulenin, a natural product inhibitor of fatty acid synthase, induces mitochondrial injury and apoptosis in human leukemia cells through the mitochondrial translocation of cofilin. Only dephosphorylated cofilin could translocate to mitochondria during cerulenin-induced apoptosis. Disruption of the ROCK1/Akt/JNK signaling pathway plays a critical role in the cerulenin-mediated dephosphorylation and mitochondrial translocation of cofilin and apoptosis. In vivo studies demonstrated that cerulenin-mediated inhibition of tumor growth in a mouse xenograft model of leukemia was associated with mitochondrial translocation of cofilin and apoptosis. These data are consistent with a hierarchical model in which induction of apoptosis by cerulenin primarily results from activation of ROCK1, inactivation of Akt, and activation of JNK. This leads to the dephosphorylation and mitochondrial translocation of cofilin and culminates with cytochrome c release, caspase activation, and apoptosis. Our study has revealed a novel role of cofilin in the regulation of mitochondrial injury and apoptosis and suggests that cerulenin is a potential drug for the treatment of leukemia.

It is fully established that the condensing reaction for the initiation of fatty acid synthesis is essential for viability of many bacteria. In model bacteria such as Escherichia coli, this reaction is exclusively catalyzed by β-ketoacyl-ACP synthase (KAS) III (encoded by fabH) and the FabH loss results in a fatty acid auxotroph. However, such a notion has been under the challenge of recent findings. In an attempt to resolve the conflicting results, in this study, we examined the physiological role of multiple KASIII enzyme homologues in Shewanella oneidensis, an excellent model for researching type II fatty acid synthesis (FASII) and its regulation. We demonstrated that FabH1 and temperature-responsive FabH2 are primarily responsible for initiating synthesis of straight- and branched-chain fatty acids respectively, whereas FabH3 and OleA are dispensable. Cells lacking all these enzymes as a set are viable but carry severe defects in growth. Further analyses revealed that in the absence of KASIII either of FabB (KASI) and FabF2 (KASII) is able to support growth, suggesting that they could initiate FASII. Strikingly, KASIII enzymes and OleA together confer S. oneidensis cells resistance to cerulenin, a selective inhibitor of FabF and FabB. Along with our previous finding that S. oneidensis FabF1 and FabB are fully equivalent with respect to their physiological impacts, these results imply that physiological function promiscuity of bacterial KAS enzymes could be more extensive than previously expected.

Colwellia psychrerythraea 34H is a psychrophilic bacterium able to produce docosahexaenoic acid (DHA). Polyketide synthase pathway is assumed to be responsible for DHA production in marine bacteria.

Currently, very long chain fatty acids (VLCFAs) for oleochemical, pharmaceutical, cosmetic, or food applications are extracted from plant or marine organism resources, which is associated with a negative environmental impact. Therefore, there is an industrial demand to develop sustainable, microbial resources. Due to its ease of genetic modification and well-characterized metabolism, Escherichia coli has established itself as a model organism to study and tailor microbial fatty acid biosynthesis using a concerted genetic engineering approach. In this study, we systematically implemented a plant-derived (Arabidopsis thaliana) enzymatic cascade in Escherichia coli to enable unbranched VLCFA biosynthesis. The four Arabidopsis thaliana membrane-bound VLCFA enzymes were expressed using a synthetic expression cassette. To facilitate enzyme solubilization and interaction of the synthetic VLCFA synthase complex, we applied a self-assembly GFP scaffold. In order to initiate VLCFA biosynthesis, external oleic acid and cerulenin were supplemented to cultures. In this context, we detected the generation of arachidic (20:0), cis-11-eicosenoic (20:1) and cis-13-eicosenoic acid (20:1).

Sheath rot disease caused by Sarocladium oryzae is an emerging threat for rice cultivation at global level. However, limited information with respect to genomic resources and pathogenesis is a major setback to develop disease management strategies. Considering this fact, we sequenced the whole genome of highly virulent Sarocladium oryzae field isolate, Saro-13 with 82x sequence depth.

This study investigated the biological significance of the inhibition of fatty acid synthase (FAS) in multiple myeloma (MM) using the small molecule inhibitor Cerulenin. Cerulenin triggered growth inhibition in both MM cell lines and MM patient cells, and overcame the survival and growth advantages conferred by interleukin-6, insulin-like growth factor-1, and bone marrow stromal cells. It induced apoptosis in MM cell lines with only modest activation of caspase -8, -9, -3 and PARP; moreover, the pan-caspase inhibitor Z-VAD-FMK did not inhibit Cerulenin-induced apoptosis and cell death. In addition, treatment of MM cells with Cerulenin primarily up-regulated apoptosis-inducing factor/endonuclease G, mediators of caspase-independent apoptosis. Importantly, Cerulenin induced endoplasmic reticulum stress response via up-regulation of the Grp78/IRE1alpha/JNK pathway. Although the C-Jun-NH(2)-terminal kinase (JNK) inhibitor SP600215 blocked Cerulenin-induced cytotoxicity, it did not inhibit apoptosis and caspase cleavage. Furthermore, Cerulenin showed synergistic cytotoxic effects with various agents including Bortezomib, Melphalan and Doxorubicin. Our results therefore indicate that inhibition of FAS by Cerulenin primarily triggered caspase-independent apoptosis and JNK-dependent cytotoxicity in MM cells. This report demonstrated that inhibition of FAS has anti-tumour activity against MM cells, suggesting that it represents a novel therapeutic target in MM.

High levels of fatty acid synthase (FAS) expression have been observed in several cancers, including breast, prostate, colon and lung carcinoma, compared with their respective normal tissue. We present data that show high levels of FAS protein in human and rat glioma cell lines and human glioma tissue samples, as compared to normal rat astrocytes and normal human brain. Incubating glioma cells with the FAS inhibitor cerulenin decreased endogenous fatty acid synthesis by approximately 50%. Cell cycle analysis demonstrated a time- and dose-dependent increase in S-phase cell arrest following cerulenin treatment for 24 h. Further, treatment with cerulenin resulted in time- and dose-dependent decreases in glioma cell viability, as well as reduced clonogenic survival. Increased apoptotic cell death and PARP cleavage were observed in U251 and SNB-19 cells treated with cerulenin, which was independent of the death receptor pathway. Overexpressing Bcl-2 inhibited cerulenin-mediated cell death. In contrast, primary rat astrocytes appeared unaffected. Finally, RNAi-mediated knockdown of FAS leading to reduced FAS enzymatic activity was associated with decreased glioma cell viability. These findings suggest that FAS might be a novel target for antiglioma therapy.

Bone and fat cells have an antagonistic relationship. Adipocytes exert a toxic effect on bone cells in vitro through the secretion of fatty acids, which are synthesized by fatty acid synthase (FAS). Inhibition of FAS in vitro rescues osteoblasts from fat-induced toxicity and cell death. In this study, we hypothesized that FAS inhibition would mitigate the loss of bone mass in ovariectomized (OVX) mice. We treated OVX C57BL/6 mice with cerulenin (a known inhibitor of FAS) for 6 weeks and compared their bone phenotype with vehicle-treated controls. Cerulenin-treated mice exhibited a significant decrease in body weight, triglycerides, leptin, and marrow and subcutaneous fat without changes in serum glucose or calciotropic hormones. These effects were associated with attenuation of bone loss and normalization of the bone phenotype in the cerulenin-treated OVX group compared to the vehicle-treated OVX group. Our results demonstrate that inhibition of FAS enhances bone formation, induces uncoupling between osteoblasts and osteoclasts, and favors mineralization, thus providing evidence that inhibition of FAS could constitute a new anabolic therapy for osteoporosis.

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.

Sheath rot is an emerging rice disease that leads to considerable yield losses. The main causal agent is the fungus Sarocladium oryzae. This pathogen is known to produce the toxins cerulenin and helvolic acid, but their role in pathogenicity has not been clearly established. S. oryzea isolates from different rice-producing regions can be grouped into three phylogenetic lineages. When grown in vitro, isolates from these lineages differed in growth rate, colour and in the ability to form sectors. A diverse selection of isolates from Rwanda and Nigeria, representing these lineages, were used to further study their pathogenicity and toxin production. Liquid chromatography high-resolution mass spectrometry analysis was used to measure cerulenin and helvolic acid production in vitro and in planta. The three lineages clearly differed in pathogenicity on the japonica cultivar Kitaake. Isolates from the least pathogenic lineage produced the highest levels of cerulenin in vitro. Helvolic acid production was not correlated with the lineage. Sectorisation was observed in isolates from the two least pathogenic lineages and resulted in a loss of helvolic acid production. In planta, only the production of helvolic acid, but not of cerulenin, correlated strongly with disease severity. The most pathogenic isolates all belonged to one lineage. They were phenotypically stable, shown by the lack of sectorisation, and therefore maintained high helvolic acid production in planta.

Cisplatin-resistant A549CisR and H157CisR cell lines were developed by treating parental A549 (A549P) and H157 (H157P) cells. These cisplatin-resistant cells showed slight growth retardation, but exhibited higher epithelial-mesenchymal transition (EMT) and increased metastatic potential compared to parental cells. We observed a highly up-regulated fatty acid synthase (FASN) level in A549CisR and H157CisR cells compared to parental cells and the up-regulation of FASN was also detected in A549P and H157P cells after short time treatment with cisplatin, suggesting that the high level of FASN in cisplatin-resistant cells may be from the accumulated cellular responses during cisplatin-resistance developmental process. We next investigated whether the inhibition of FASN by using a specific FASN inhibitor, cerulenin, can influence growth and EMT/metastatic potential of A549CisR and H157CisR cells. There was slight growth inhibition, but significantly reduced EMT/metastatic potential in cisplatin-resistant cells upon inhibitor treatment. The in vitro result was further investigated in orthotopic xenograft mouse models established with luciferase-tagged H157P and H157CisR cells. Mice were injected with cerulenin or vehicle after tumors were developed. No significant tumor regression was detected at the end of cerulenin treatment, but IHC staining showed higher expression of EMT/metastasis markers in H157CisR cell-derived tumors than H157P cell-derived tumors, and showed dramatic reduction of these markers in tumor tissues of cerulenin-treated mice, confirming the in vitro results. In mechanism dissection studies, we revealed the existence of the FASN-TGF-β1-FASN positive loop in A549CisR and H157CisR cells, but not in parental cells, which is believed to augment the FASN function in cisplatin-resistant cells.

NFκB is a central mediator of inflammation. Present inhibitors of NFκB are mostly based on inhibition of essential machinery such as proteasome and protein kinases, or activation of nuclear receptors; as such, they are of limited therapeutic use due to severe toxicity. Here we report an LPS-induced NFκB enhanceosome in which TonEBP is required for the recruitment of p300. Increased expression of TonEBP enhances the NFκB activity and reduced TonEBP expression lowers it. Recombinant TonEBP molecules incapable of recruiting p300 do not stimulate NFκB. Myeloid-specific deletion of TonEBP results in milder inflammation and sepsis. We discover that a natural small molecule cerulenin specifically disrupts the enhanceosome without affecting the activation of NFκB itself. Cerulenin suppresses the pro-inflammatory activation of macrophages and sepsis without detectable toxicity. Thus, the NFκB enhanceosome offers a promising target for useful anti-inflammatory agents.

Dinoflagellates are unicellular organisms that are implicated in harmful algal blooms (HABs) caused by potent toxins that are produced through polyketide synthase (PKS) pathways. However, the exact mechanisms of toxin synthesis are unknown due to a lack of genomic segregation of fat, toxins, and other PKS-based pathways. To better understand the underlying mechanisms, the actions and expression of the PKS proteins were investigated using the toxic dinoflagellate Amphidinium carterae as a model. Cerulenin, a known ketosynthase inhibitor, was shown to reduce acetate incorporation into all fat classes with the toxins amphidinol and sulpho-amphidinol. The mass spectrometry analysis of cerulenin-reacted synthetic peptides derived from ketosynthase domains of A. carterae multimodular PKS transcripts demonstrated a strong covalent bond that could be localized using collision-induced dissociation. One multi-modular PKS sequence present in all dinoflagellates surveyed to date was found to lack an AT domain in toxin-producing species, indicating trans-acting domains, and was shown by Western blotting to be post-transcriptionally processed. These results demonstrate how toxin synthesis in dinoflagellates can be differentiated from fat synthesis despite common underlying pathway.

Inhibition of fatty acid synthesis (FAS) stimulates tumor cell death and reduces angiogenesis. When SH-SY5Y cells or primary neurons are exposed to hypoxia only, inhibition of FAS yields significantly enhanced cell injury. The pathophysiology of stroke, however, is not only restricted to hypoxia but also includes reoxygenation injury. Hence, an oxygen-glucose-deprivation (OGD) model with subsequent reoxygenation in both SH-SY5Y cells and primary neurons as well as a murine stroke model were used herein in order to study the role of FAS inhibition and its underlying mechanisms. SH-SY5Y cells and cortical neurons exposed to 10 h of OGD and 24 h of reoxygenation displayed prominent cell death when treated with the Acetyl-CoA carboxylase inhibitor TOFA or the fatty acid synthase inhibitor cerulenin. Such FAS inhibition reduced the reduction potential of these cells, as indicated by increased NADH2 +/NAD+ ratios under both in vitro and in vivo stroke conditions. As observed in the OGD model, FAS inhibition also resulted in increased cell death in the stroke model. Stroke mice treated with cerulenin did not only display increased brain injury but also showed reduced neurological recovery during the observation period of 4 weeks. Interestingly, cerulenin treatment enhanced endothelial cell leakage, reduced transcellular electrical resistance (TER) of the endothelium and contributed to poststroke blood-brain barrier (BBB) breakdown. The latter was a consequence of the activated NF-κB pathway, stimulating MMP-9 and ABCB1 transporter activity on the luminal side of the endothelium. In conclusion, FAS inhibition aggravated poststroke brain injury as consequence of BBB breakdown and NF-κB-dependent inflammation.

The gene encoding phosphopantetheinyl transferase (PPTase), pfaE, a component of the polyketide synthase (PKS) pathway, is crucial for the production of docosahexaenoic acid (DHA, 22:6ω3), along with the other pfa cluster members pfaA, pfaB, pfaC and pfaD. DHA was produced in Escherichia coli by co-expressing pfaABCD from DHA-producing Colwellia psychrerythraea 34H with one of four pfaE genes from bacteria producing arachidonic acid (ARA, 20:4ω6), eicosapentaenoic acid (EPA, 20:5ω3) or DHA, respectively. Substitution of the pfaE gene from different strain source in E. coli did not influence the function of the PKS pathway producing DHA, although they led to different DHA yields and fatty acid profiles. This result suggested that the pfaE gene could be switchable between these strains for the production of DHA. The DHA production by expressing the reconstituted PKS pathway was also investigated in different E. coli strains, at different temperatures, or with the treatment of cerulenin. The highest DHA production, 2.2 mg of DHA per gram of dry cell weight or 4.1% of total fatty acids, was obtained by co-expressing pfaE(EPA) from the EPA-producing strain Shewanella baltica with pfaABCD in DH5α. Incubation at low temperature (10-15°C) resulted in higher accumulation of DHA compared to higher temperatures. The addition of cerulenin to the medium increased the proportion of DHA and saturated fatty acids, including C12:0, C14:0 and C16:0, at the expense of monounsaturated fatty acids, including C16:1 and C18:1. Supplementation with 1 mg/L cerulenin resulted in the highest DHA yield of 2.4 mg/L upon co-expression of pfaE(DHA) from C. psychrerythraea.

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.

Fatty-acid synthesis in bacteria is of great interest as a target for the discovery of antibacterial compounds. The addition of a new acetyl moiety to the growing fatty-acid chain, an essential step in this process, is catalyzed by beta-ketoacyl-ACP synthase (KAS). It is inhibited by natural antibiotics such as cerulenin and thiolactomycin; however, these lack the requirements for optimal drug development. Structure-based biophysical screening revealed a novel synthetic small molecule, 2-phenylamino-4-methyl-5-acetylthiazole, that binds to Escherichia coli KAS I with a binding constant of 25 microM as determined by fluorescence titration. A 1.35 A crystal structure of its complex with its target reveals noncovalent interactions with the active-site Cys163 and hydrophobic residues of the fatty-acid binding pocket. The active site is accessible through an open conformation of the Phe392 side chain and no conformational changes are induced at the active site upon ligand binding. This represents a novel binding mode that differs from thiolactomycin or cerulenin interaction. The structural information on the protein-ligand interaction offers strategies for further optimization of this low-molecular-weight compound.

Lipid Droplets (LD) are dynamic organelles that originate in the Endoplasmic Reticulum and mostly bud off toward the cytoplasm, where they store neutral lipids for energy and protection purposes. LD also have diverse proteins on their surface, many of which are necessary for the their correct homeostasis. However, these organelles also act as reservoirs of proteins that can be made available elsewhere in the cell. In this sense, they act as sinks that titrate key regulators of many cellular processes. Among the specialized factors that reside on cytoplasmic LD are proteins destined for functions in the nucleus, but little is known about them and their impact on nuclear processes. By screening for nuclear proteins in publicly available LD proteomes, we found that they contain a subset of nucleoporins from the Nuclear Pore Complex (NPC). Exploring this, we demonstrate that LD act as a physiological reservoir, for nucleoporins, that impacts the conformation of NPCs and hence their function in nucleo-cytoplasmic transport, chromatin configuration, and genome stability. Furthermore, our in silico modeling predicts a role for LD-released fatty acids in regulating the transit of nucleoporins from LD through the cytoplasm and to nuclear pores.