FASN inhibition sensitizes metastatic OSCC cells to cisplatin and paclitaxel by downregulating cyclin B1.

OBJECTIVES: To investigate the potential effect of fatty acid synthase (FASN) inhibitor orlistat to enhance the effectiveness of chemotherapy drugs widely used to treat oral squamous cell carcinomas (OSCC), such as 5-fluorouracil, cisplatin, and paclitaxel. METHODS: The OSCC SCC-9 LN-1 metastatic cell line, which expresses high levels of FASN, was used for drug combination experiments. Cell viability was analyzed by crystal violet staining and automatic cell counting. Apoptosis and cell cycle were analyzed by flow cytometry with Annexin-V/7-AAD and propidium iodide staining, respectively. Cyclin B1, Cdc25C, Cdk1, FASN, and ERBB2 levels were assessed by Western blotting. Finally, cell scratch and transwell assays were performed to assess cell migration and invasion. RESULTS: Inhibition of FASN with orlistat sensitized SCC-9 LN-1 cells to the cytotoxic effects of paclitaxel and cisplatin, but not 5-fluorouracil, which was accompanied by a significant reduction in cyclin B1. The suppression of proliferation, migration, and invasion of SCC-9 LN-1 cells induced by orlistat plus cisplatin or paclitaxel was not superior to the effects of chemotherapy drugs alone. CONCLUSION: Our results suggest that orlistat enhances the chemosensitivity of SCC-9 LN-1 cells to cisplatin and paclitaxel by downregulating cyclin B1.

Pharmacological fatty acid synthase inhibitors differently affect the malignant phenotype of oral cancer cells.

OBJECTIVE: Fatty acid synthase levels are associated with aggressiveness, prognosis, and risk of metastasis in oral squamous cell carcinomas. This enzyme contains seven catalytic domains and its inhibition by synthetic or natural drugs has antineoplastic properties such as C75, which is a synthetic inhibitor of the ß- ketoacyl synthase domain, the antibiotic triclosan, ligand of the enoyl reductase domain, and the antiobesity drug orlistat, which inhibits the thioesterase domain. Here, we sought to investigate and compare the in vitro effects of C75, triclosan, and orlistat on malignant phenotypes of the cell line SCC-9: proliferation, cell cycle, apoptosis, adhesion, migration, and invasion. DESIGN: Half-maximal inhibitory concentration (IC50) was determined using cell viability assays. Cell death and cell cycle progression were analyzed by Annexin V-PE/7-ADD-PerCP labeling and propidium iodide staining, respectively. Cell migration and invasion were assayed by transwells assays and cell adhesion using collagen and fibronectin. RESULTS: C75 showed the lowest IC50 and higher inhibition of lipid droplets at low concentrations and reduced cell motility. Triclosan showed the intermediate IC50 value, excellent reduction of lipid bodies at the IC50 when compared with C75 and orlistat. Also, triclosan reduced cell cycle progression, adhesion, migration, and invasion of SCC-9 and induced the highest levels of apoptosis. Orlistat promoted cell cycle arrest, but showed the lowest induction of apoptosis and did not affected invasion and adhesion of SCC-9. CONCLUSION: Altogether, despite the particular effects of the analyzed fatty acid synthase inhibitors, triclosan showed to better interfere in tumorigenic phenotypes of SCC-9 cells.

Immunohistochemical Expression of Fatty Acid Synthase (FASN) is Correlated to Tumor Aggressiveness and Cellular Differentiation in Salivary Gland Carcinomas.

Fatty acid synthase (FASN) expression is closely related to cancer progression, in particular, tumor aggressiveness and poor prognosis. This study aimed to analyse the expression of FASN in carcinomas of the salivary glands and correlate it with Ki-67 expression. We analysed by immunohistochemistry the expression of FASN and Ki-67 on tissue sections from 7 cases of adenocarcinoma, not otherwise specified (AdNOS), 6 cases of polymorphous adenocarcinoma (PAC), 16 cases of acinic cell carcinoma (AcCC), 19 cases of adenoid cystic carcinoma (AdCC), 15 cases of epithelial-myoepithelial carcinoma (EMC); 10 cases of secretory carcinoma (SC), 13 cases of mucoepidermoid carcinoma (MEC), 10 cases of salivary duct carcinoma (SDC) and 7 cases of myoepithelial carcinoma (MC). These carcinomas were classified into aggressive and indolent regarding their biological behaviour. Additionally, MEC and AdCC were also classified according to the histological grade. High expression of FASN was found in SDC (100%), SC (100%), AcCC (68.7%) and AdNOS (57.2%). No association was found between FASN and Ki-67 expression. Aggressive carcinomas showed a higher rate of Ki-67 proliferation (p < 0.001) and greater expression of FASN when compared to indolent carcinomas (p < 0.05). With regards to carcinomas categorized as indolent, FASN expression was much higher in the lesions that presented cell differentiation (SC and AcCC). Also, FASN expression was significantly higher in high-grade AdCC and MEC when compared to low-grade tumors (p < 0.05). We concluded that FASN expression was correlated to tumor aggressiveness and cellular differentiation in salivary gland carcinomas.

Further evidence for the involvement of mitochondrial aquaporin-8 in hepatocyte lipid synthesis.

We recently provided evidence suggesting that mitochondrial aquaporin-8 (mtAQP8), a channel protein able to conduct H2O2, is involved in the modulation of hepatocyte cholesterogenesis. To expand that study, we cultured human hepatocyte-derived Huh-7 cells in medium with lipoprotein-deficient serum (LPDS) to induce the de novo synthesis of cholesterol and fatty acids. We found that LPDS induced mtAQP8 expression and that AQP8 gene silencing significantly down-regulated the LPDS-induced synthesis of cholesterol and fatty acids as well as the expression of the corresponding key biosynthetic enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase and fatty acid synthase. Our data further support a regulatory role of mtAQP8 in hepatocyte lipid homeostasis.

Inhibition of fatty acid synthase (FASN) affects the proliferation and apoptosis of HepG2 hepatoma carcinoma cells via the ß-catenin/C-myc signaling pathway.

INTRODUCTION AND OBJECTIVES: Research in the last few years has proven that inhibition of fatty acid synthase (FASN) suppresses the migration and invasion of hepatoma carcinoma cells. This study aims to explore the effect of fatty acid synthase knockdown on the apoptosis and proliferation of HepG2 cells. MATERIALS AND METHODS: The human liver cancer cell line HepG2 was cultured and then transfected with FASN-specific siRNA and negative control RNAi. After 48h, cells and protein lysates were used for western blotting, CCK-8 (cell counting kit-8) assays, flow cytometry and other tests. To assess cell apoptosis, Bax, Bcl-2 and caspase-3 were detected; to assess proliferation, CDK4 (cyclin-dependent kinases 4) and P21 were detected; and to determine the signaling pathway involved, ß-catenin and C-myc were also detected. RESULTS: Inhibition of FASN in HepG2 cells can decrease proliferation and promote apoptosis. Flow cytometry and CCK-8 assays demonstrated that the apoptosis rate of FASN-specific siRNA-transfected cells was significantly increased compared to that of the control cells (p<0.01). In addition, the cell cycle analysis revealed that FASN-specific siRNA-transfected cells induced G1 phase arrest (p<0.05), but an increasing trend in G2 (p<0.05). Compared with expression in negative RNAi-transfected cells, the expression of Bcl-2 and CDK-4 was reduced and the expression of Bax, caspase-3 and P21 was increased in FASN-specific siRNA-transfected cells (p<0.05). Regarding the signaling pathway, the expression of ß-catenin and C-myc was significantly reduced when compared to that in negative control cells (p<0.05). CONCLUSIONS: Inhibition of FASN suppressed the cell survival of HepG2 cells by inhibiting the ß-catenin/C-myc pathway. This result suggests the potential treatment value of FASN for hepatoma carcinoma (HCC).

Anticancer properties of the fatty acid synthase inhibitor TVB-3166 on oral squamous cell carcinoma cell lines.

OBJECTIVE: Fatty acid synthase (FASN) is overexpressed in several human cancers, including oral squamous cell carcinoma (OSCC). TVB-3166 is a recently described FASN inhibitor with antitumor effects and potential clinical relevance. The objective of this study was to evaluate the effects of TVB-3166 on OSCC cell lines. MATERIALS AND METHODS: The OSCC cell line SCC-9 modified to express ZsGreen (ZsG) (SCC-9 ZsG) and its in vivo selected metastatic derivative LN-1A were used to evaluate anticancer properties of TVB-3166. Cell viability was determined using MTT assays and proliferation determined by cell counting in a Neubauer chamber. Cell death and cell cycle progression were analyzed by Annexin V-PE/7-ADD-PerCP labeling and PI staining, respectively. Cell migration was assayed by scratch assays and cell adhesion using myogel. Production of FASN, p-AKT, CPT1-α, and epithelial-mesenchymal transition (EMT) markers were examined by Western blotting. RESULTS: TVB-3166 significantly reduced cell viability and proliferation, promoted cell cycle arrest and apoptosis, and increased adhesion to myogel in both OSCC cell lines. Finally, the drug reduced SCC-9 ZsG migration. CONCLUSION: Our results demonstrated that TVB-3166 has anticancer effects on both SCC-9 ZsG and its metastatic version LN-1A, which are worthy of investigation in preclinical models for OSCC.

The antimetastatic activity of orlistat is accompanied by an antitumoral immune response in mouse melanoma.

PURPOSE: Fatty acid synthase (FASN), the multifunctional enzyme responsible for endogenous fatty acid synthesis, is highly expressed and associated with poor prognosis in several human cancers, including melanoma. Our group has previously shown that pharmacological inhibition of FASN with orlistat decreases proliferation, promotes apoptosis, and reduces the metastatic spread of B16-F10 cells in experimental models of melanoma. While most of the orlistat antitumor properties seem to be closely related to direct effects on malignant cells, its impact on the host immune system is still unknown. METHODS: The effects of orlistat on the phenotype and activation status of infiltrating leukocytes in primary tumors and metastatic lymph nodes were assessed using a model of spontaneous melanoma metastasis (B16-F10 cells/C57BL/6 mice). Cells from the primary tumors and lymph nodes were mechanically dissociated and immune cells phenotyped by flow cytometry. The expression of IL-12p35, IL-12p40, and inducible nitric oxide synthase (iNOS) was analyzed by qRT-PCR and production of nitrite (NO2-) evaluated in serum samples with the Griess method. RESULTS: Orlistat-treated mice exhibited a 25% reduction in the number of mediastinal lymph node metastases (mean 3.96 ± 0.78, 95% CI 3.63-4.28) compared to the controls (mean 5.7 ± 1.72; 95% CI 5.01-6.43). The drug elicited an antitumor immune response against experimental melanomas by increasing maturation of intratumoral dendritic cells (DC), stimulating the expression of cytotoxicity markers in CD8 T lymphocytes and natural killer (NK) cells, as well as reducing regulatory T cells (Tregs). Moreover, the orlistat-treatment increased serum levels of nitric oxide (NO) concentrations. CONCLUSION: Taken together, these findings suggest that orlistat supports an antitumor response against experimental melanomas by increasing CD80/CD81-positive and IL-12-positive DC populations, granzyme b/NKG2D-positive NK populations, and perforin/granzyme b-positive CD8 T lymphocytes as well as reducing Tregs counts within experimental melanomas.

Potential Anti-obesogenic Effects of Ginkgo biloba Observed in Epididymal White Adipose Tissue of Obese Rats.

Exacerbated expansion of adipose tissue seen in diet-induced obesity leads to endocrine dysfunction and disturbance in adipokine secretion, with such abnormal profile positively associated with type 2 diabetes and other mild chronic inflammatory conditions. Ginkgo biloba extract (GbE), a mixture of polyphenols with antioxidant properties, has been recently investigated in a variety of experimental models of endocrine dysfunction, with several potentially beneficial effects identified, including improvement in insulin sensitivity in obese rats, and reduction of weight gain in ovariectomy-induced obesity and diet-induced obesity. The aim of this study was to investigate in high fat diet-induced obese male rats the effects of GbE supplementation for 2 weeks on adipocyte volume and adipose tissue lipid accumulation. GbE supplementation was effective in reducing energy intake in obese rats compared to the saline-treated placebo group. Epididymal adipocyte volume was reduced in GbE-supplemented rats, as were epididymal [1-14C]-acetate incorporation into fatty acids, perilipin (Plin 1) and fatty acid synthase (Fasn) mRNA, and FAS protein levels. Adipocyte hypertrophy in obesity is associated with insulin resistance, and in the present study we observed a reduction in the adipocyte volume of GbE-supplemented obese rats to dimensions equivalent to adipocytes from non-obese rats. GbE supplementation significantly reduced acetate accumulation and tended to reduce [3H]-oleate incorporation, into epididymal adipose tissue, suggesting a potentially anti-obesogenic effect in longer term therapies. Further studies that investigate the effects of GbE supplementation in other experimental models are required to fully elucidate its suggested beneficial effects on mild chronic inflammatory conditions.

A fatty acid synthase gene (FASN3) from the integument tissue of Rhodnius prolixus contributes to cuticle water loss regulation.

Fatty acid synthase is a multifunctional enzyme involved in the formation of fatty acids. Despite the role of fatty acids in cell signalling and energy metabolism, and as precursors to pheromones and hydrocarbons that waterproof the cuticle, the insect fatty acid synthases have been scarcely studied. Here we perform the molecular characterization of three fatty acid synthase genes (fatty acid synthase RPRC000123, RPRC000269 and RPRC002909) in the Chagas disease vector, Rhodnius prolixus. Gene expression screening by reverse transcription quantitative PCR showed that RPRC000123 and RPRC002909 are expressed almost exclusively in the integument tissue whilst RPRC000269 is mostly expressed in the fat body and also in several body organs. Phylogenetic analysis, together with gene expression results, showed that RPRC000269, RPRC002909 and RPRC000123 are orthologues of Drosophila melanogaster fatty acid synthase 1 (FASN1), FASN2 and FASN3 genes, respectively. After RNA interference-mediated knockdown of RPRC000123, insects died immediately after moulting to the next developmental stage. However, mortality was prevented by placing the insects under saturated humidity conditions, suggesting that dehydration might play a role in the insects' death. Lipid analyses in RPRC000123-silenced insects showed reduced amounts of integument fatty acids and methyl-branched hydrocarbons, compared to controls. These data support an important role for FASN3 in the biosynthesis of the precursors to hydrocarbons that waterproof the insect cuticle.

Fish oil supplementation during adolescence attenuates metabolic programming of perinatal maternal high-fat diet in adult offspring.

Perinatal maternal high-fat diet (HFD) increases susceptibility to obesity and fatty liver diseases in adult offspring, which can be attenuated by the potent hypolipidaemic action of fish oil (FO), an n-3 PUFA source, during adult life. Previously, we described that adolescent HFD offspring showed resistance to FO hypolipidaemic effects, although FO promoted hepatic molecular changes suggestive of reduced lipid accumulation. Here, we investigated whether this FO intervention only during the adolescence period could affect offspring metabolism in adulthood. Then, female Wistar rats received isoenergetic, standard (STD: 9 % fat) or high-fat (HFD: 28·6 % fat) diet before mating, and throughout pregnancy and lactation. After weaning, male offspring received the standard diet; and from 25 to 45 d old they received oral administration of soyabean oil or FO. At 150 d old, serum and hepatic metabolic parameters were evaluated. Maternal HFD adult offspring showed increased body weight, visceral adiposity, hyperleptinaemia and decreased hepatic pSTAT3/STAT3 ratio, suggestive of hepatic leptin resistance. FO intake only during the adolescence period reduced visceral adiposity and serum leptin, regardless of maternal diet. Maternal HFD promoted dyslipidaemia and hepatic TAG accumulation, which was correlated with reduced hepatic carnitine palmitoyl transferase-1a content, suggesting lipid oxidation impairment. FO intake did not change serum lipids; however, it restored hepatic TAG content and hepatic markers of lipid oxidation to STD offspring levels. Therefore, we concluded that FO intake exclusively during adolescence programmed STD offspring and reprogrammed HFD offspring male rats to a healthier metabolic phenotype in adult life, reducing visceral adiposity, serum leptin and hepatic TAG content in offspring adulthood.

Treatment with an inhibitor of fatty acid synthase attenuates bone loss in ovariectomized mice.

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 polymorphisms of genes associated with the profile of fatty acids of sheep / Os polimorfismos de genes associados ao perfil de ácidos graxos de ovinos

The present study aimed to evaluate the occurrence of polymorphisms in Diacylglycerol acyltransferase (DGTA-1 and 2), Fatty acid synthase (FASN), Stearoyl-CoA desaturase (SCD) genes and the Thioesterase domain of FASN (TE-FASN) gene that may be related to the lipid profile. In the experiment, a total of 84 sheep from different genetic groups were used. For the evaluation of the polymorphism of the genes, PCR-Single Strand Conformation Polymorphism (SSCP) technique and subsequent sequencing were used. In DGAT-2 gene, four genotypes were identified with the presence of 6 polymorphisms, with two (c.229T> C; c.255T> C) that resulted into the exchange of phenylalanine by leucine. In FASN gene, two genotypes were identified. In TE-FASN gene, three genotypes and 17 polymorphisms were identified. DGAT-1 and SCD genes did not reveal the occurrence of polymorphism. There was difference in relation to C14: 0, C18: 0 fatty acids and Δ9-desaturase C18 for DGAT-2 gene and of C18: 2ω6t for TE-FASN. There were differences among the genetic groups for C10: 0, C12: 0, C17: 0, C18: 2ω6t, C18: 3ω3, C20: 2, total of ω3, ω3/ω6 and atherogenicity index. There is occurrence of polymorphism of DGAT-2 and TE-FASN genes and these should be further studied in sheep since they revealed influence of the genotypes on the fatty acid profile.(AU)

O presente estudo teve o objetivo de avaliar a ocorrência de polimorfismos nos genes Diacilglicerol aciltransferase (DGTA1 e 2), Ácido graxo sintase (FASN), Estearoil-CoA dessaturase (SCD) e o Domínio da tioesterase do gene FASN (TE-FASN), que possam estar relacionados ao perfil lipídico. No experimento, foram utilizados um total de 84 ovinos de diferentes grupos genéticos. Para avaliação do polimorfismo dos genes, foi utilizada a técnica de polimorfismo de conformação de cadeia simples (PCR-SSCP) e posterior sequenciamento. No gene DGAT-2, foram identificados quatro genótipos com a presença de seis polimorfismos, com dois (c.229T>C; c.255T>C) que resultaram na troca da fenilalanina por leucina. No gene FASN, foram identificados dois genótipos. No gene TE-FASN, foram identificados três genótipos e 17 polimorfismos. Os genes DGAT-1 e SCD não revelaram a ocorrência de polimorfismo. Houve diferença em relação aos ácidos graxos C14:0, C18:0 e ∆9-desaturaseC18 para o gene DGAT-2 e de C18:2ω6t para TE-FASN. Houve diferença entre os grupos genéticos para C10:0, C12:0, C17:0, C18:2ω6t, C18:3ω3, C20:2, total de ω3, ω3/ω6 e índice de aterogenicidade. Há ocorrência de polimorfismo dos genes DGAT-2 e TE-FASN, e estes devem ser mais estudados em ovinos, pois revelaram influência dos genótipos sobre o perfil de ácidos graxos.(AU)

The polymorphisms of genes associated with the profile of fatty acids of sheep / Os polimorfismos de genes associados ao perfil de ácidos graxos de ovinos

The present study aimed to evaluate the occurrence of polymorphisms in Diacylglycerol acyltransferase (DGTA-1 and 2), Fatty acid synthase (FASN), Stearoyl-CoA desaturase (SCD) genes and the Thioesterase domain of FASN (TE-FASN) gene that may be related to the lipid profile. In the experiment, a total of 84 sheep from different genetic groups were used. For the evaluation of the polymorphism of the genes, PCR-Single Strand Conformation Polymorphism (SSCP) technique and subsequent sequencing were used. In DGAT-2 gene, four genotypes were identified with the presence of 6 polymorphisms, with two (c.229T> C; c.255T> C) that resulted into the exchange of phenylalanine by leucine. In FASN gene, two genotypes were identified. In TE-FASN gene, three genotypes and 17 polymorphisms were identified. DGAT-1 and SCD genes did not reveal the occurrence of polymorphism. There was difference in relation to C14: 0, C18: 0 fatty acids and Δ9-desaturase C18 for DGAT-2 gene and of C18: 2ω6t for TE-FASN. There were differences among the genetic groups for C10: 0, C12: 0, C17: 0, C18: 2ω6t, C18: 3ω3, C20: 2, total of ω3, ω3/ω6 and atherogenicity index. There is occurrence of polymorphism of DGAT-2 and TE-FASN genes and these should be further studied in sheep since they revealed influence of the genotypes on the fatty acid profile.(AU)

O presente estudo teve o objetivo de avaliar a ocorrência de polimorfismos nos genes Diacilglicerol aciltransferase (DGTA1 e 2), Ácido graxo sintase (FASN), Estearoil-CoA dessaturase (SCD) e o Domínio da tioesterase do gene FASN (TE-FASN), que possam estar relacionados ao perfil lipídico. No experimento, foram utilizados um total de 84 ovinos de diferentes grupos genéticos. Para avaliação do polimorfismo dos genes, foi utilizada a técnica de polimorfismo de conformação de cadeia simples (PCR-SSCP) e posterior sequenciamento. No gene DGAT-2, foram identificados quatro genótipos com a presença de seis polimorfismos, com dois (c.229T>C; c.255T>C) que resultaram na troca da fenilalanina por leucina. No gene FASN, foram identificados dois genótipos. No gene TE-FASN, foram identificados três genótipos e 17 polimorfismos. Os genes DGAT-1 e SCD não revelaram a ocorrência de polimorfismo. Houve diferença em relação aos ácidos graxos C14:0, C18:0 e ∆9-desaturaseC18 para o gene DGAT-2 e de C18:2ω6t para TE-FASN. Houve diferença entre os grupos genéticos para C10:0, C12:0, C17:0, C18:2ω6t, C18:3ω3, C20:2, total de ω3, ω3/ω6 e índice de aterogenicidade. Há ocorrência de polimorfismo dos genes DGAT-2 e TE-FASN, e estes devem ser mais estudados em ovinos, pois revelaram influência dos genótipos sobre o perfil de ácidos graxos.(AU)

Molecular dynamics simulation and binding free energy studies of novel leads belonging to the benzofuran class inhibitors of Mycobacterium tuberculosis Polyketide Synthase 13.

In this work, the binding mechanism of new Polyketide Synthase 13 (Pks13) inhibitors has been studied through molecular dynamics simulation and free energy calculations. The drug Tam1 and its analogs, belonging to the benzofuran class, were submitted to 100 ns simulations, and according to the results obtained for root mean square deviation, all the simulations converged from approximately 30 ns. For the analysis of backbone flotation, the root mean square fluctuations were plotted for the Cα atoms; analysis revealed that the greatest fluctuation occurred in the residues that are part of the protein lid domain. The binding free energy value (ΔGbind) obtained for the Tam16 lead molecule was of -51.43 kcal/mol. When comparing this result with the ΔGbind values for the remaining analogs, the drug Tam16 was found to be the highest ranked: this result is in agreement with the experimental results obtained by Aggarwal and collaborators, where it was verified that the IC50 for Tam16 is the smallest necessary to inhibit the Pks13 (IC50 = 0.19 µM). The energy decomposition analysis suggested that the residues which most interact with inhibitors are: Ser1636, Tyr1637, Asn1640, Ala1667, Phe1670, and Tyr1674, from which the greatest energy contribution to Phe1670 was particularly notable. For the lead molecule Tam16, a hydrogen bond with the hydroxyl of the phenol not observed in the other analogs induced a more stable molecular structure. Aggarwal and colleagues reported this hydrogen bonding as being responsible for the stability of the molecule, optimizing its physic-chemical, toxicological, and pharmacokinetic properties.

Lipid metabolism alterations in the neuronal response to A53T α-synuclein and Fe-induced injury.

Pathological α-synuclein (α-syn) overexpression and iron (Fe)-induced oxidative stress (OS) are involved in the death of dopaminergic neurons in Parkinson's disease (PD). We have previously characterized the role of triacylglycerol (TAG) formation in the neuronal response to Fe-induced OS. In this work we characterize the role of the α-syn variant A53T during Fe-induced injury and investigate whether lipid metabolism has implications for neuronal fate. To this end, we used the N27 dopaminergic neuronal cell line either untransfected (UT) or stably transfected with pcDNA3 vector (as a transfection control) or pcDNA-A53T-α-syn (A53T α-syn). The overexpression of A53T α-syn triggered an increase in TAG content mainly due to the activation of Acyl-CoA synthetase. Since fatty acid (FA) ß-oxidation and phospholipid content did not change in A53T α-syn cells, the unique consequence of the increase in FA-CoA derivatives was their acylation in TAG moieties. Control cells exposed to Fe-induced injury displayed increased OS markers and TAG content. Intriguingly, Fe exposure in A53T α-syn cells promoted a decrease in OS markers accompanied by α-syn aggregation and elevated TAG content. We report here new evidence of a differential role played by A53T α-syn in neuronal lipid metabolism as related to the neuronal response to OS.

Orlistat as a FASN inhibitor and multitargeted agent for cancer therapy.

INTRODUCTION: Cancer cells have increased glycolysis and glutaminolysis. Their third feature is increased de novo lipogenesis. As such, fatty acid (FA) synthesis enzymes are over-expressed in cancer and their depletion causes antitumor effects. As fatty acid synthase (FASN) plays a pivotal role in this process, it is an attractive target for cancer therapy. AREAS COVERED: This is a review of the lipogenic phenotype of cancer and how this phenomenon can be exploited for cancer therapy using inhibitors of FASN, with particular emphasis on orlistat as a repurposing drug. EXPERT OPINION: Disease stabilization only has been observed with a highly selective FASN inhibitor used as a single agent in clinical trials. It is too early to say whether the absence of tumor responses other than stabilization results because even full inhibition of FASN is not enough to elicit antitumor responses. The FASN inhibitor orlistat is a 'dirty' drug with target-off actions upon at least seven targets with a proven role in tumor biology. The development of orlistat formulations suited for its intravenous administration is a step ahead to shed light on the concept that drug promiscuity can or not be a virtue.

Toxic effects of phytol and retinol on human glioblastoma cells are associated with modulation of cholesterol and fatty acid biosynthetic pathways.

Glioblastoma (GBM) is the most common primary brain tumor. Genetic mutations may reprogram the metabolism of neoplastic cells. Particularly, alterations in cholesterol and fatty acid biosynthetic pathways may favor biomass synthesis and resistance to therapy. Therefore, compounds that interfere with those pathways, such as phytol (PHY) and retinol (RET), may be appropriate for cytotoxic approaches. We tested the effect of PHY or RET on the viability of human GBM cell lines (U87MG, A172 and T98G). Since the compounds showed a dose-dependent cytotoxic effect, additional analyses were performed with IC50 values. Transcriptome analyses of A172 cells treated with PHY IC50 or RET IC50 revealed down-regulated genes involved in cholesterol and/or fatty acid biosynthetic pathways. Thus, we investigated the expression of proteins required for cholesterol and/or fatty acid synthesis after treating all lineages with PHY IC50 or RET IC50 and comparing them with controls. Sterol regulatory element-binding protein 1 (SREBP-1) expression was reduced by PHY in U87 and T98G cells. However, fatty acid synthase (FAS) protein expression, which is regulated by SREBP-1, was down-regulated in all lineages after both treatments. Moreover, farnesyl-diphosphate farnesyltransferase (FDFT1) levels, a protein associated with cholesterol synthesis, were reduced in all lineages by PHY and in U87MG and A172 cells by RET. Our results suggest that SREBP-1, FAS and FDFT1 are potential target(s) for future in vivo approaches against GBM and support the use of inhibitors of their synthesis, including PHY and RET, for such approaches.

Maternal conjugated linoleic acid modulates TAG metabolism in adult rat offspring.

Conjugated linoleic acid (CLA) might regulate the lipid depots in liver and adipose tissue. As there is an association between maternal nutrition, fat depots and risk of offspring chronic disease, the aim was to investigate the effect of maternal CLA consumption on TAG regulation and some inflammatory parameters in adult male rat offspring receiving or not receiving CLA. Female Wistar rats were fed control (C) or CLA-supplemented (1 %, w/w) diets during 4 weeks before and throughout pregnancy and lactation. After weaning, male offspring of CLA rats were fed C or CLA diets (CLA/C and CLA/CLA groups, respectively), whereas C male rat offspring were fed a C diet (C/C group) for 9 weeks. Serum TAG levels were increased in the CLA/CLA and CLA/C groups, associated with a reduction of lipoprotein lipase activity and weights of adipose tissue. The liver TAG levels were decreased in the CLA/CLA group, related to a significant reduction of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and glucose-6-phosphate dehydrogenase enzyme activities, as well as to the mRNA levels of FAS, ACC, stearoyl-CoA desaturase-1 and sterol regulatory element-binding protein-1c. Even though normal TAG levels were found in the liver of CLA/C rats, a reduction of lipogenesis was also observed. Thus, these results demonstrated a programming effect of CLA on the lipid metabolic pathways leading to a preventive effect on the TAG accretion in adipose tissue and the liver of male rat offspring. This knowledge could be important to develop some dietary strategies leading to a reduced incidence of obesity and fatty acid liver disease in humans.

(-)-UB006: A new fatty acid synthase inhibitor and cytotoxic agent without anorexic side effects.

C75 is a synthetic anticancer drug that inhibits fatty acid synthase (FAS) and shows a potent anorexigenic side effect. In order to find new cytotoxic compounds that do not impact food intake, we synthesized a new family of C75 derivatives. The most promising anticancer compound among them was UB006 ((4SR,5SR)-4-(hydroxymethyl)-3-methylene-5-octyldihydrofuran-2(3H)-one). The effects of this compound on cytotoxicity, food intake and body weight were studied in UB006 racemic mixture and in both its enantiomers separately. The results showed that both enantiomers inhibit FAS activity and have potent cytotoxic effects in several tumour cell lines, such as the ovarian cell cancer line OVCAR-3. The (-)-UB006 enantiomer's cytotoxic effect on OVCAR-3 was 40-fold higher than that of racemic C75, and 2- and 38-fold higher than that of the racemic mixture and its opposite enantiomer, respectively. This cytotoxic effect on the OVCAR-3 cell line involves mechanisms that reduce mitochondrial respiratory capacity and ATP production, DDIT4/REDD1 upregulation, mTOR activity inhibition, and caspase-3 activation, resulting in apoptosis. In addition, central and peripheral administration of (+)-UB006 or (-)-UB006 into rats and mice did not affect food intake or body weight. Altogether, our data support the discovery of a new potential anticancer compound (-)-UB006 that has no anorexigenic side effects.

A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism.

Unlike most bacteria, mycobacteria rely on the multi-domain enzyme eukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo. These metabolites are precursors of the biosynthesis of most of the lipids present both in the complex mycobacteria cell wall and in the storage lipids inside the cell. In order to study the role of the type I FAS system in Mycobacterium lipid metabolism in vivo, we constructed a conditional mutant in the fas-acpS operon of Mycobacterium smegmatis and analysed in detail the impact of reduced de novo fatty acid biosynthesis on the global architecture of the cell envelope. As expected, the mutant exhibited growth defect in the non-permissive condition that correlated well with the lower expression of fas-acpS and the concomitant reduction of FAS I, confirming that FAS I is essential for survival. The reduction observed in FAS I provoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA, and a strong reduction of C12 to C18 acyl-CoAs, but not of long-chain acyl-CoAs (C19 to C24). The most intriguing result was the ability of the mutant to keep synthesizing mycolic acids when fatty acid biosynthesis was impaired. A detailed comparative lipidomic analysis showed that although reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis, mycolic acids were still being synthesized in the mutant, although with a different relative species distribution. However, when triacylglycerol degradation was inhibited, mycolic acid biosynthesis was significantly reduced, suggesting that storage lipids could be an intracellular reservoir of fatty acids for the biosynthesis of complex lipids in mycobacteria. Understanding the interaction between FAS I and the metabolic pathways that rely on FAS I products is a key step to better understand how lipid homeostasis is regulated in this microorganism and how this regulation could play a role during infection in pathogenic mycobacteria.