Fatty acid synthase inhibition with Orlistat promotes apoptosis and reduces cell growth and lymph node metastasis in a mouse melanoma model

Abstract

Fatty acid synthase (FASN) is the enzyme responsible for the endogenous synthesis of the saturated fatty acid palmitate. In contrast to most normal cells, malignant cells depend on FASN activity for growth and survival. In fact, FASN is overexpressed in a variety of human cancers including cutaneous melanoma, in which its levels of expression are associated with a poor prognosis and depth of invasion. Here, we show that the specific inhibition of FASN activity by the antiobesity drug Orlistat or siRNA is able to significantly reduce proliferation and promote apoptosis in the mouse metastatic melanoma cell line B16-F10. These results prompted us to verify the effect of FASN inhibition on the metastatic process in a model of spontaneous melanoma metastasis, in which B16-F10 cells injected in the peritoneal cavity of C57BL/6 mice metastasize to the mediastinal lymph nodes. We observed that mice treated with Orlistat 48 hr after the inoculation of B16-F10 cells exhibited a 52% reduction in the number of mediastinal lymph node metastases, in comparison with the control animals. These results suggest that FASN activity is essential for B16-F10 melanoma cell proliferation and survival while its inactivation by Orlistat significantly reduces their metastatic spread. The chemical inhibition of FASN activity could have a potential benefit in association with the current chemotherapy for melanoma. ' 2008 Wiley-Liss, Inc.Key words: melanoma; fatty acid synthase; metastasis; Orlistat; B16-F10 cells Endogenous fatty acid synthesis from the small carbon precursors acetyl-CoA and malonyl-CoA is dependent on the activity of fatty acid synthase (FASN, EC2.3.1.85). In most of the cells, FASN is downregulated by the dietary fatty acids, with exception of lipogenic tissues as liver, lactating breast, fetal lung and adipose tissue.1,2 Recent studies provide compelling evidence that neoplastic lipogenesis is essential for cancer cell survival. In fact, several human epithelial malignancies, such as those of prostate, breast, ovary, bladder, lung, stomach and oral cavity, melanoma as well as soft tissue sarcomas overexpress FASN. [3][4][5][6][7][8][9][10][11][12][13][14][15][16] For some of these tumors, such as prostate, breast and ovarian cancers and melanoma, 4,5,9,12 FASN overexpression has also been associated with a poor prognosis. Experimental studies have shown that FASN inhibition reduces cell proliferation by blocking DNA replication during S-phase, induces apoptosis, [17][18][19][20] and decrease the size of prostate, ovarian and breast cancer xenografts. 3,21,22 In addition, the inhibition of FASN activity has a chemopreventive effect in the breast cancer transgenic neu-N mouse model. 23 Orlistat (tetrahydrolipstatin) is an irreversible inhibitor of pancreatic and gastric lipases clinically used because of its antiobesity properties that also blocks the activity of the thioesterase domain of FASN. 24 In fact, FASN inhibition by Orlistat reduces proliferation and promotes apoptosis in prostate, breast and stomach cancer cel...

Inhibition of fatty acid synthase in melanoma cells activates the intrinsic pathway of apoptosis

“…4,29,41 In the present study, we demonstrated that FASN inhibition induced the intrinsic pathway of apoptosis in B16-F10 mouse melanoma cells, which was preceded by an increase in both ROS production and cytosolic calcium concentration. In addition, this apoptotic cell death was not dependent on p53 activation or MPT.…”

“…12,16 We have recently shown that the inhibition of FASN activity with the anti-obesity drug orlistat significantly reduced proliferation and promoted apoptosis in the mouse metastatic melanoma cell line B16-F10. 29 In this study, we show that FASN inhibition activates the intrinsic pathway of apoptosis in B16-F10 cells by cytochrome c release and caspase-9 and -3 activation, which were preceded by increased reactive oxygen species (ROS) production and cytosolic calcium concentration. p53 activation and the mitochondrial permeability transition (MPT) were not involved in the orlistat-or cerulenin-induced apoptotic cell death.…”

“…The combined chloroform extracts were evaporated and the lipogenesis determined in 1 ml of scintillation solution, as previously described. 29 Analysis of Cell Death and Cell Cycle Samples were analyzed in a FACSCalibur flow cytometer (BD Biosciences, USA) equipped with an argon laser and CellQuest software (version 4.1). Seven to ten thousand events were acquired for each sample.…”

“…29 B16-F10 cells were seeded in 6-well culture plates (3-4 Â 10 4 cells) and after 24 h serum starved for the same period of time. The medium was replaced by fresh medium containing FASN inhibitors and cells incubated for more 24 or 48 h, harvested and fixed in cold 70% ethanol.…”

“…After a washing step according to the manufacturer instructions, cells were resuspended in the same medium and analyzed by flow cytometry, as described before. 29 Detection of Caspase-9 and -8 Activities Treated cells (3 Â 10 6 cells) were resuspended in 0.2 ml of chilled lysis buffer (20 mM HEPES pH 7.5, 10 mM KCl, 250 mM sucrose, 2 mM MgCl, and 1 mM EDTA) containing 0.5 mM DTT. Cell suspensions were sonicated (Mosonix Sonicator S-3000, New Highway Farmingdale, USA) and frozen at À801C.…”