title: Fatty acid synthase inhibitors induce apoptosis in non-tumorigenic melan-a cells associated with inhibition of mitochondrial respiration.
abstract: 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.
authors: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.
authors: Franco A
authors: FA
authors: Rossato
authors: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil; Departamento de Diagnóstico Oral, Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas (UNICAMP), Piracicaba, SP, Brazil.
authors: Karina G
authors: KG
authors: Zecchin
authors: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.
authors: Paolo G
authors: PG
authors: La Guardia
authors: Departamento de Diagnóstico Oral, Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas (UNICAMP), Piracicaba, SP, Brazil.
authors: Rose M
authors: RM
authors: Ortega
authors: Departamento de Química e Física, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil.
authors: Luciane C
authors: LC
authors: Alberici
authors: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.
authors: Rute A P
authors: RA
authors: Costa
authors: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.
authors: Rodrigo R
authors: RR
authors: Catharino
authors: Departamento de Diagnóstico Oral, Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas (UNICAMP), Piracicaba, SP, Brazil.
authors: Edgard
authors: E
authors: Graner
authors: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.
authors: Roger F
authors: RF
authors: Castilho
authors: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.
authors: Aníbal E
authors: AE
authors: Vercesi
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D019069:Cell Respiration / Q000187:drug effects; D002478:Cells, Cultured; D002569:Cerulenin / Q000494:pharmacology*; D002950:Citrate (si)-Synthase / Q000037:antagonists & inhibitors; D045304:Cytochromes c / Q000378:metabolism; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors*; D054872:Fatty Acid Synthesis Inhibitors / Q000494:pharmacology*; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000201:enzymology / Q000473:pathology*; D008544:Melanocytes / Q000187:drug effects / Q000201:enzymology / Q000473:pathology*; D008545:Melanoma / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D054872:Fatty Acid Synthesis Inhibitors; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D002569:Cerulenin; D045304:Cytochromes c; D064429:Fatty Acid Synthases; D002950:Citrate (si)-Synthase