cerulenin and 4-methylene-2-octyl-5-oxofuran-3-carboxylic-acid

Although metabolites and energy balance have long been known to play roles in the regulation of food intake, the potential role of fatty acid metabolism in this process has been considered only recently. Fatty acid synthase (FAS) catalyzes the condensation of acetyl-CoA and malonyl-CoA to generate long-chain fatty acids in the cytoplasm, while the breakdown of fatty acids (beta-oxidation) occurs in mitochondria and is regulated by carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting step for the entry of fatty acids into the mitochondria. Inhibition of FAS using cerulenin or synthetic FAS inhibitors such as C75 reduces food intake and induces profound reversible weight loss. Subsequent studies reveal that C75 also stimulates CPT-1 and increases beta-oxidation. Hypotheses as to the mechanisms by which C75 and cerulenin mediate their effects have been proposed. Centrally, these compounds alter the expression profiles of feeding-related neuropeptides, often inhibiting the expression of orexigenic peptides. Whether through centrally mediated or peripheral mechanisms, C75 also increases energy consumption, which contributes to weight loss. In vitro and in vivo studies demonstrate that at least part of C75's effects is mediated by modulation of AMP-activated protein kinase (AMPK), a known peripheral energy-sensing kinase. Collectively, these data suggest a role for fatty acid metabolism in the perception and regulation of energy balance.

Topics: 4-Butyrolactone; Animals; Body Weight; Carnitine O-Palmitoyltransferase; Cerulenin; Eating; Energy Metabolism; Fatty Acid Synthases; Fatty Acids; Gene Expression; Humans; Models, Biological; Obesity

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.

Topics: 4-Butyrolactone; Apoptosis; Autophagy; Brain; Brain Neoplasms; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cells, Cultured; Cerulenin; Dose-Response Relationship, Drug; Fatty Acid Synthase, Type I; Fatty Acid Synthesis Inhibitors; Glioblastoma; Glioma; Humans; Lactones; Neoplasm Grading; Orlistat; Tissue Culture Techniques

We have examined the potential role of fatty acid oxidation (FAO) in AMP-activated protein kinase (AMPK)-induced meiotic maturation. Etomoxir and malonyl CoA, two inhibitors of carnitine palmitoyl transferase-1 (CPT1), and thus FAO, blocked meiotic induction in dbcAMP-arrested cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) by the AMPK activator, AICAR. C75, an activator of CPT1 and FAO, stimulated meiotic resumption in CEO and DO. This effect was insensitive to the AMPK inhibitor, compound C, indicating an action downstream of AMPK. Palmitic acid or carnitine also promoted meiotic resumption in DO in the presence of AICAR. Since C75 also suppresses the activity of fatty acid synthase (FAS), we tested another FAS inhibitor, cerulenin. Cerulenin stimulated maturation in arrested oocytes, but to a lesser extent, exhibited significantly slower kinetics and was effective in CEO but not DO. Moreover, etomoxir completely blocked C75-induced maturation but was ineffective in cerulenin-treated oocytes, suggesting that the meiosis-inducing action of C75 is through activation of FAO within the oocyte, while that of cerulenin is independent of FAO and acts within the cumulus cells. Finally, we determined that long chain, but not short chain, fatty acyl carnitine derivatives were stimulatory to oocyte maturation. Palmitoyl carnitine stimulated maturation in both CEO and DO, with rapid kinetics in DO; this effect was blocked by mercaptoacetate, a downstream inhibitor of FAO. These results indicate that activation of AMPK stimulates meiotic resumption in mouse oocytes by eliminating a block to FAO.

Topics: 4-Butyrolactone; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Carnitine; Carnitine O-Palmitoyltransferase; Cerulenin; Cumulus Cells; Epoxy Compounds; Fatty Acids; Female; Malonyl Coenzyme A; Meiosis; Mice; Mice, Inbred C57BL; Oocytes; Oxidation-Reduction; Palmitic Acid; Ribonucleotides

Coxsackievirus is linked to a large variety of severe human and animal diseases such as myocarditis. The interplay between host factors and virus components is crucial for the fate of the infected cells. However, host proteins which may play a role in coxsackievirus-induced diseases are ill-defined. Two-dimensional gel electrophoresis of protein extracts obtained from coxsackievirus B3 (CVB3)-infected and uninfected HeLa or HepG2 cells combined with spot analysis revealed several proteins which are exclusively up-regulated in infected cells. One of these proteins was identified as the fatty acid synthase (FAS). By using cerulenin and C75, two known inhibitors of FAS we were able to significantly block CVB3 replication. FAS appears to be directly involved in CVB3-caused pathology and is therefore suitable as a therapeutic target in CVB3-induced diseases.

Topics: 4-Butyrolactone; Amino Acid Sequence; Antiviral Agents; Cell Line; Cerulenin; Electrophoresis, Gel, Two-Dimensional; Enterovirus B, Human; Enzyme Inhibitors; Fatty Acid Synthases; Humans; Molecular Sequence Data; Up-Regulation; Virus Replication

Fatty acid synthase (FAS) has been shown previously to be highly expressed in breast and prostate carcinomas, but has low expression level in normal tissues. We also found in this study that FAS was expressed in a number of cancer cell lines of different histotypes. The growth-inhibitory effects of FAS inhibitors cerulenin and C75 were then investigated on these cancer cell lines, particularly the human melanoma A-375. MTT assay revealed that the cancer cell proliferation and viability was reduced dose- and time-dependently by 20.8%-87.1% of the control levels after 24 and 48 h of treatment with 20-160 microM of the inhibitor. Immunoblotting studies showed that both cerulenin and C75 induced poly(ADP-ribose) polymerase (PARP) cleavage in the melanoma cells dose-dependently. Procaspase-3 was also found to be processed into the active and smaller 17 and 19 kDa subunits, and administration of pan-caspase inhibitor Z-VAD-FMK completely rescued the cells from PARP cleavage. This indicated that the cerulenin- and C75-induced apoptosis involved caspase activation. The proapoptotic effects of the FAS inhibitors were further confirmed using confocal microscopy with annexin-V FITC and propidium iodide staining. DNA flow cytometric studies demonstrated that the FAS inhibitors accumulated G2/M cells preceding the elevation of sub G1 or apoptotic cells with fragmented DNA. The induced cell cycle arrest and apoptosis were associated with elevation of p21 and depletion of Bcl-xL and Mcl-1, respectively. Results from this study suggest that FAS inhibitors retard growth of melanoma A-375 cells, involving activation of caspase-dependent apoptosis.

Topics: 4-Butyrolactone; Antineoplastic Agents; Apoptosis; Blotting, Western; Caspases; Cell Cycle; Cell Line, Tumor; Cerulenin; DNA, Neoplasm; Enzyme Inhibitors; fas Receptor; Fatty Acid Synthases; Flow Cytometry; Genes, bcl-2; Humans; Melanoma; Microscopy, Confocal; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Proto-Oncogene Proteins c-bcl-2; Tetrazolium Salts; Thiazoles

Inhibition of fatty acid synthase (FAS) reduces food intake in rodents. As adipose tissue expresses FAS, we sought to investigate the effect of reduced FAS activity on adipocyte differentiation. FAS activity was suppressed either pharmacologically or by siRNA during differentiation of 3T3-L1 cells. Cerulenin (10 microM), triclosan (50 microM), and C75 (50 microM) reduced dramatically visible lipid droplet accumulation, while incorporation of [1-(14C)]acetate into lipids was reduced by 75%, 70%, and 90%, respectively. Additionally, the substances reduced FAS, CEBPalpha, and PPARgamma mRNA by up to 85% compared to that of control differentiated cells. Transient transfection with FAS siRNA suppressed FAS mRNA and FAS activity, and this was accompanied by reduction of CEBPalpha and PPARgamma mRNA levels, and complete prevention of lipid accumulation. CD36, a late marker of differentiation, was also reduced. Together, these results suggest that FAS generated signals may be essential to support preadipocyte differentiation.

Topics: 3T3-L1 Cells; 4-Butyrolactone; Adipocytes; Animals; Cell Differentiation; Cerulenin; Fatty Acid Synthases; Gene Silencing; Mice; Triclosan

Fatty acid synthase (FAS) activity is a potential therapeutic target to treat cancer and obesity. Here, we have identified a molecular link between FAS and HER2 (erbB-2) oncogene, a marker for poor prognosis that is overexpressed in 30% of breast and ovarian cancers. Pharmacological FAS inhibitors cerulenin and C75 were found to suppress p185(HER2) oncoprotein expression and tyrosine-kinase activity in breast and ovarian HER2 overexpressors. Similarly, p185(HER2) expression was dramatically down-regulated when FAS gene expression was silenced by using the highly sequence-specific mechanism of RNA interference (RNAi). Pharmacological and RNAi-mediated silencing of FAS specifically down-regulated HER2 mRNA and, concomitantly, caused a prominent up-regulation of PEA3, a transcriptional repressor of HER2. A cytoplasmic redistribution of p185(HER2) was associated with marked morphological changes of FAS RNAi-transfected cells, whereas chemical inhibitors of FAS promoted a striking nuclear accumulation of p185(HER2). The simultaneous targeting of FAS and HER2 by chemical FAS inhibitors and the humanized antibody directed against p185(HER2) trastuzumab, respectively, was synergistically cytotoxic toward HER2 overexpressors. Similarly, concurrent RNAi-mediated silencing of FAS and HER2 genes synergistically stimulated apoptotic cell death in HER2 overexpressors. p185(HER2) was synergistically down-regulated after simultaneous inhibition of FAS and HER2 by either pharmacological inhibitors or small interfering RNA. These findings provide evidence of an active role of FAS in cancer evolution by specifically regulating oncogenic proteins closely related to malignant transformation, strongly suggesting that HER2 oncogene may act as the key molecular sensor of energy imbalance after the perturbation of tumor-associated FAS hyperactivity in cancer cells.

Topics: 4-Butyrolactone; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Line; Cell Size; Cell Survival; Cerulenin; Fatty Acid Synthases; Female; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Humans; Ovarian Neoplasms; Receptor, ErbB-2; RNA, Small Interfering; Signal Transduction; Transcription Factors; Trastuzumab

Topics: 4-Butyrolactone; Animals; Anti-Obesity Agents; Cerulenin; Enzyme Inhibitors; Fatty Acid Synthases; Humans; Hypothalamus; Mice; Mice, Obese; Models, Biological; Obesity

Topics: 4-Butyrolactone; Adipose Tissue; Animals; Carnitine O-Palmitoyltransferase; Cell Size; Cerulenin; Energy Intake; Fatty Acids; Ghrelin; Humans; Malonyl Coenzyme A; Obesity; Peptide Hormones; Peptides

Many common human cancer tissues express high levels of fatty acid synthase (FAS), the primary enzyme for the synthesis of fatty acids, and the differential expression of FAS between normal and neoplastic tissues has led to the consideration of FAS as a target for anticancer therapy. To investigate the potential of targeting FAS for the treatment of pleural mesothelioma, we first determined whether FAS is overexpressed in human mesothelioma. By immunohistochemistry, we found 22 of 30 human mesothelioma tissue samples tested to express significantly increased levels of FAS compared with normal tissues, including mesothelium. To further explore FAS as a therapeutic target in mesothelioma, we established a nude mouse xenograft model for human mesothelioma using the H-Meso cell line. The i.p. xenografts of this cell line have high levels of FAS expression and fatty acid synthesis pathway activity and grow along mesothelial surfaces in a manner similar to the growth pattern of human mesothelioma. Growth of these tumor xenografts was essentially abolished in mice treated with weekly i.p. injections of C75, a synthetic, small molecule inhibitor of FAS, at levels that resulted in no significant systemic toxicity except for reversible weight loss. These results suggest that FAS may be an effective target for pharmacological therapy in a high proportion of human mesotheliomas.

Topics: 4-Butyrolactone; Animals; Antifungal Agents; Antineoplastic Agents; Cerulenin; Disease Models, Animal; Fatty Acid Synthases; Humans; Immunoenzyme Techniques; Mesothelioma; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Mesothelial; Paraffin Embedding; Prognosis; Tumor Cells, Cultured