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.
Highlights
The metabolic enzyme fatty acid synthase (FASN) is responsible for the production of saturated fatty acids, such as palmitate, through the condensation of acetyl-CoA and malonyl-CoA [1,2,3,4,5,6,7]
Death in the melan-a cell line occurred mainly by apoptosis, as we previously demonstrated for the B16-F10 melanoma cells [47], while the necrosis rates remained unchanged by these treatments (DMSO: 2.460.5%, 22 mM cerulenin: 2.460.5%; EtOH: 1.560.8%; 30 mM orlistat: 2.460.4%)
The role of FASN in nonmalignant cells remains uncertain, it is known that the FASN inhibitor cerulenin promotes a reduction in the proliferation of normal fibroblasts in primary cultures [52,53], and orlistat has been shown to have antiproliferative effects in human umbilical vein endothelial cells (HUVEC) [26,48,54]
Summary
The metabolic enzyme fatty acid synthase (FASN) is responsible for the production of saturated fatty acids, such as palmitate, through the condensation of acetyl-CoA and malonyl-CoA [1,2,3,4,5,6,7]. FASN inhibition reduces cell proliferation and induces apoptosis in vitro and decreases the size of prostate, ovarian and breast cancer xenografts [39,40,41]. Mitochondrial involvement in apoptosis, as evidenced by increased levels of the pro-apoptotic protein Bax and the release of cytochrome c, has been found in several tumor cell lines, including neuroblastoma, melanoma, colon carcinoma, breast cancer and skin carcinoma, following pharmacological FASN inhibition [37,44]. We showed that FASN inhibition activates the intrinsic apoptotic pathway, as evidenced by the release of cytochrome c and the activation of caspases-9 and -3; this activation is preceded by increased production of reactive oxygen species and elevated cytosolic calcium concentrations in these melanoma cells [47]. Orlistat treatment of B16-F10 cells resulted in significant changes in the mitochondrial free fatty acid (FFA) composition, as demonstrated by electrospray ionization mass spectrometry (ESIMS) [49]
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