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Fatty acid metabolism in breast cancer cells: differential inhibitory effects of epigallocatechin gallate (EGCG) and C75

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Abstract

Endogenous fatty acid metabolism is crucial to maintain the cancer cell malignant phenotype. Lipogenesis is regulated by the enzyme fatty acid synthase (FASN); and breakdown of fatty acids is regulated by carnitine palmitoyltransferase-1 (CPT-I). FASN is highly expressed in breast cancer and most common human carcinomas. Several compounds can inhibit FASN, although the degree of specificity of this inhibition has not been addressed. We have tested the effects of C75 and (-)-epigallocatechin-3-gallate (EGCG) on fatty acid metabolism pathways, cellular proliferation, induction of apoptosis and cell signalling in human breast cancer cells. Our results show that C75 and EGCG had comparable effects in blocking FASN activity. Treating cancer cells with EGCG or C75 induced apoptosis and caused a decrease in the active forms of oncoprotein HER2, AKT and ERK1/2 to a similar degree. We observed, in contrast, marked differential effects between C75 and EGCG on the fatty acid oxidation pathway. While EGCG had either no effect or a moderate reduction in CPT-I activity, C75 stimulated CPT-I activity (up to 129%), even in presence of inhibitory levels of malonyl-CoA, a potent inhibitor of the CPT-I enzyme. Taken together, these findings indicate that pharmacological inhibition of FASN occurs uncoupled from the stimulation of CPT-I with EGCG but not with C75, suggesting that EGCG might be free of the CPT-I related in vivo weight-loss that has been associated with C75. Our results establish EGCG as a potent and specific inhibitor of fatty acid synthesis (FASN), which may hold promise as a target-directed anti-cancer drug.

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Acknowledgements

Financial support was provided by Susan G. Komen Breast Cancer Foundation (PDF 0504073) (R. Porta) and Ministerio de Educación y Ciencia (Programa Juan de la Cierva; T. Puig) Grants, and Instituto de Salud Carlos III (FIS PI04/1417; R. Colomer) ISCIII-RETIC RD06 (D. Haro) Projects, and the Spanish Society for Medical Oncology (SEOM) (R. Colomer).

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Authors and Affiliations

  1. Fundació d’Investigació Biomèdica de Girona Dr. Josep Trueta (IdIBGi), Hospital Universitari de Girona Dr. Josep Trueta, Girona, Spain

    Teresa Puig, Alejandro Vázquez-Martín, Javier A. Menéndez, Rut Porta & Gemma Casals

  2. Biochemistry and Molecular Biology, School of Sciences, University of Girona, Girona, Spain

    Teresa Puig

  3. Medical Oncology, Institut Català d’Oncologia de Girona (ICO Girona), Hospital Universitari de Girona Dr. Josep Trueta, Avda. França s/n, 17007, Girona, Spain

    Teresa Puig, Alejandro Vázquez-Martín, Javier A. Menéndez, Rut Porta, Joan Brunet & Ramon Colomer

  4. Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain

    Joana Relat, Pedro F. Marrero & Diego Haro

  5. Biomedical Research Unit, Hospital Vall d’Hebron, Barcelona, Spain

    Jordi Pétriz

  6. MD Anderson Cancer Center España, Madrid, Spain

    Ramon Colomer

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  1. Teresa Puig
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  2. Alejandro Vázquez-Martín
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Correspondence to Teresa Puig.

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10549_2007_9678_MOESM1_ESM.jpg

a. Table 1. Inhibitory effect C75 and EGCG against growth of MDA-MB-231, MCF-7 and SK-Br3 cancer cells. b. Characterization of FASN expression levels in MDA-MB-231, MCF-7 and SK-Br3 breast cancer cells. Cells were grown in complete medium until reaching 80% confluence, then washed twice with PBS, and solubilized in lysis buffer containing phosphatase and protease inhibitors. Equal amounts of protein were analyzed by Western blot and probed with a rabbit anti-FASN monoclonal antibody. Bands in the figure are from one representative experiment. Equivalent results were found in 3 independent experiments. Blots were re-probed with an antibody for b-actin to control for protein loading and transfer. (JPG 33 kb)

10549_2007_9678_MOESM2_ESM.jpg

Treatment of SK-Br3 cancer cells with C75 (30 mM) and EGCG (150 mM) induced cleavage of PARP. Cleavage of poly-ADP-ribose polymerase (PARP) was analysed by Western blot analysis as described in the Material and Methods section. Bands in the figure are from one representative experiment. Equivalent results were found in 3 independent experiments. Blots were re-probed with an antibody for b-actin to control for protein loading and transfer. (JPG 17 kb)

10549_2007_9678_MOESM3_ESM.jpg

Microscopic and immunoblotting analysis of SK-Br3 cells after treatment with C75 and EGCG (48h) or FASN siRNA (72h). a. Compared with control cells (20X) (A) and siRNA negative control cells (200nM) (20X) (B); C75 (30 mM) (20X) (C) and EGCG (150 mM) (20X) (D) induced similar morphological changes in SK-Br3 cells to those induced by FASN inhibition via siRNA (200nM) (20X) (E). b. After transfection time, SK-Br3 cells were collected, washed twice with PBS and solubilized in lysis buffer containing phosphatase and protease inhibitors. Equal amounts of protein were analyzed by Western blot and probed with a rabbit anti-FASN monoclonal antibody. Bands in the figure are from one representative experiment. Equivalent results were found in 3 independent experiments. Blots were re-probed with an antibody for b-actin to control for protein loading and transfer. (JPG 53 kb)

10549_2007_9678_MOESM4_ESM.jpg

C75 stimulates CPT-I activity but EGCG did not. P. pastoris were transformed with the plasmid encoding for the human CPT-Ib (a) and pig (b) CPT-Ia. Mitochondria isolated from these strains were assayed for CPT activity in the presence of DMSO (control) or 200 mM of C75 and EGCG, as described in Material and Methods. Results are an avarage of two separate experiments with two independent mitochondrial preparations. * P< 0.05 versus control, by one-way ANOVA. (JPG 25 kb)

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Puig, T., Vázquez-Martín, A., Relat, J. et al. Fatty acid metabolism in breast cancer cells: differential inhibitory effects of epigallocatechin gallate (EGCG) and C75. Breast Cancer Res Treat 109, 471–479 (2008). https://doi.org/10.1007/s10549-007-9678-5

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