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.

Reciprocal regulation of acetyl-CoA carboxylase 1 and senescence in human fibroblasts involves oxidant mediated p38 MAPK activation

“…Although the source of oxidant species was not explored, mitochondria appear as likely candidates in this setting, since absence of ACC1 could result in an increase in fatty acid oxidation and concomitantly mitochondrial superoxide formation [44]. A decrease in endogenous fatty acid synthesis could also affect phospholipid and mitochondrial membrane composition, impacting on the formation of superoxide by the mitochondrial complexes [45, 46]. …”

Quantitative label-free mass spectrometry analysis of formalin-fixed, paraffin-embedded tissue representing the invasive cutaneous malignant melanoma proteome

“…A second metabolic-associated protein discovered to be significantly elevated in invasive melanoma is FASN, a key protein involved in the metabolism of cancer cells (30). In order to validate this finding and other selected proteins, the expression levels of FASN and 14-3-3ε were further investigated in melanoma samples.…”

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“…Fatty acid synthase (FASN) is responsible for the production of palmitate and is minimally expressed in normal cells. In neoplastic cells, however, FASN has a much higher expression [65]. Inhibition of FASN by cerulenin or orlistat increased percentage of cells in G0/ G1 phase as well as expression of tumor suppressor genes p21 and p53 [66].…”

“…Since tumor cells synthesize the majority of fatty acids de novo, stabilization of this protein has been shown to increase cell survival in prostate cancer cells. Destabilization of FASN, such as through ubiquitination after curcumin treatment may, therefore, induce cancer cell apoptosis [65,67].…”