Visualizing Inhibition Of Fatty Acid Synthase Through Mass Spectrometric Analysis Of Mitochondria From Melanoma Cells

Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid palmitate. In contrast to most normal cells, 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. Recently, we have demonstrated the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells. Herein we compare, via electrospray ionization mass spectrometry (ESI-MS), free fatty acids (FFA) composition of mitochondria isolated from control (EtOH-treated cells) and Orlistat-treated B16-F10 mouse melanoma cells. Principal component analysis (PCA) was applied to the ESI-MS data and found to separate the two groups of samples. Mitochondria from control cells showed predominance of six ions, that is, those of m/z 157 (Pelargonic, 9:0), 255 (Palmitic, 16:0), 281 (Oleic, 18:1), 311 (Arachidic, 20:0), 327 (Docosahexaenoic, 22:6) and 339 (Behenic, 22:0). In contrast, FASN inhibition with Orlistat changes significantly mitochondrial FFA composition by reducing synthesis of palmitic acid, and its elongation and unsaturation products, such as arachidic and behenic acids, and oleic acid, respectively. ESI-MS of mitochondria isolated from Orlistat-treated cells presented therefore three major ions of m/z 157 (Pelargonic, 9:0), 193 (unknown) and 199 (Lauric, 12:0). These findings demonstrate therefore that FASN inhibition by Orlistat induces significant changes in the FFA composition of mitochondria. © 2011 John Wiley & Sons, Ltd.253449452Smith, S., (1994) FASEB J, 8, p. 1248Menendez, J.A., Lupu, R., (2007) Nat. Rev. Cancer, 7, p. 763Weiss, L., Hoffmann, G.E., Schreiber, R., (1986) Biol. Chem. Hoppe Seyler, 367, p. 905Kuhajda, F.P., (2000) Nutrition, 16, p. 202Alo, P.L., Visca, P., Framarino, M.L., (2000) Oncol. Rep., 7, p. 1383Pizer, E.S., Jackisch, C., Wood, F.D., (1996) Cancer Res., 56, p. 2745Pizer, E.S., Wood, F.D., Heine, H.S., (1996) Cancer Res., 56, p. 1189Gansler, T.S., Hardman III, W., Hunt, D.A., (1997) Hum. Pathol., 28, p. 686Dhanasekaran, S.M., Barrette, T.R., Ghosh, D., (2001) Nature, 412, p. 822Swinnen, J.V., Roskams, T., Joniau, S., (2002) Int. J. Cancer, 98, p. 19Innocenzi, D., Alo, P.L., Balzani, A., (2003) J. Cutan. Pathol., 30, p. 23Rossi, S., Graner, E., Febbo, P., (2003) Mol. Cancer Res., 1, p. 707Takahiro, T., Shinichi, K., Toshimitsu, S., (2003) Clin. Cancer Res., 9, p. 2204Visca, P., Sebastiani, V., Botti, C., (2004) Anticancer Res., 24, p. 4169Kapur, P., Rakheja, D., Roy, L.C., (2005) Mod. Pathol., 18, p. 1107Van De Sande, T., Roskams, T., Lerut, E., (2005) J. Pathol., 206, p. 214Rossi, S., Ou, W., Tang, D., (2006) J. Pathol., 209, p. 369Dowling, S., Cox, J., Cenedella, R.J., (2009) Lipids, 44, p. 489Furuya, Y., Akimoto, S., Yasuda, K., (1997) Anticancer Res., 17, p. 4589Pizer, E.S., Chrest, F.J., Digiuseppe, J.A., (1998) Cancer Res., 58, p. 4611Li, J.N., Gorospe, M., Chrest, F.J., (2001) Cancer Res., 61, p. 1493Zhou, W., Han, W.F., Landree, L.E., (2007) Cancer Res., 67, p. 2964Kridel, S.J., Axelrod, F., Rozenkrantz, N., (2004) Cancer Res., 64, p. 2070Carvalho, M.A., Zecchin, K.G., Seguin, F., (2008) Int. J. Cancer, 123, p. 2557Zecchin, K.G., Rossato, F.A., Raposo, H.F., (2010) Lab. Invest., , DOI: 10.1038/labinvest.2010.157Swinnen, J.V., Van Veldhoven, P.P., Timmermans, L., (2003) Biochem. Biophys. Res. Commun., 302, p. 898Catharino, R.R., Haddad, R., Cabrini, L.G., (2005) Anal. Chem., 77, p. 7429Yang, L., Bennett, R., Strum, J., (2009) Anal. Bioanal. Chem., 393, p. 643Fenn, J.B., Mann, M., Meng, C.K., (1989) Science, 246, p. 64Catharino, R.R., Milagre, H.M.S., Saraiva, A.S., (2007) Energy Fuels, 21, p. 3698Ferreira, C.R., Souza, G., Riccio, M.F., (2009) Rapid Commun. Mass Spectrom., 23, p. 1313Santos, L.S., Catharino, R.R., Aguiar, C.L., (2006) J. Radioanal. Nucl. Chem., 269, p. 505Martin, E.B., Morris, A.J., Zhang, J., (1996) IEE Proc. - Control Theory and Applications, 143, p. 132Sumner, L.W., Mendes, P., Dixon, R.A., (2003) Phytochemistry, 62, p. 817Ferreira, C.R., Saraiva, A.S., Catharino, R.R., (2010) J. Lipid Res., , DOI: 10.1194/jlr.D001768Knowles, L.M., Axelrod, F., Browne, C.D., (2004) J. Biol. Chem., 279, p. 30540Abou-Khalil, S., Abou-Khalil, W.H., Planas, L., (1985) Biochem. Biophys. Res. Commun., 127, p. 1039Bligh, E.G., Dyer, W.J., (1959) Can. J. Biochem. Physiol., 37, p. 911Schrauwen, P., Saris, W.H.M., Hesselink, M.K.C., (2001) FASEB J, 15, p. 2497De Freitas, E.R.L., Soares, P.R.O., Santos, R.D., (2008) J. Nanosci. Nanotechnol., 8, p. 2385Liu, J., Shimizu, K., Kondo, R., (2009) Chem. Biodivers., 6, p. 50