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Mitochondrial free fatty acid ␤-oxidation supports oxidative phosphorylation and proliferation in cancer cells

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Sara Rodríguez-Enríquez a,b,∗ , Luz Hernández-Esquivel a , Alvaro Marín-Hernández a , Mohammed El Hafidi c , Juan Carlos Gallardo-Pérez a , Ileana Hernández-Reséndiz a , José S. Rodríguez-Zavala a , Silvia C. Pacheco-Velázquez a , Rafael Moreno-Sánchez a

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Departamento de Bioquímica, Instituto Nacional de Cardiología, Juan Badiano No. 1, Col. Sección 16, Tlalpan, México D.F. 14080, Mexico Laboratorio de Medicina Traslacional, Instituto Nacional de Cancerología, Ciudad de Mexico, D.F., Mexico c Departamento de Medicina Cardiovascular, Instituto Nacional de Cardiología, Ciudad de México, D.F., Mexico b

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a r t i c l e

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Article history: Received 2 February 2015 Received in revised form 29 May 2015 Accepted 8 June 2015 Available online xxx

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Keywords: Cancer cells Tumor mitochondria Perhexeline ␤-Oxidation Anti-mitochondrial therapy

Oxidative phosphorylation (OxPhos) is functional and sustains tumor proliferation in several cancer cell types. To establish whether mitochondrial ␤-oxidation of free fatty acids (FFAs) contributes to cancer OxPhos functioning, its protein contents and enzyme activities, as well as respiratory rates and electrical membrane potential ( m) driven by FFA oxidation were assessed in rat AS-30D hepatoma and liver (RLM) mitochondria. Higher protein contents (1.4–3 times) of ␤-oxidation (CPT1, SCAD) as well as proteins and enzyme activities (1.7–13-times) of Krebs cycle (KC: ICD, 2OGDH, PDH, ME, GA), and respiratory chain (RC: COX) were determined in hepatoma mitochondria vs. RLM. Although increased cholesterol content (9-times vs. RLM) was determined in the hepatoma mitochondrial membranes, FFAs and other NAD-linked substrates were oxidized faster (1.6–6.6 times) by hepatoma mitochondria than RLM, maintaining similar  m values. The contents of ␤-oxidation, KC and RC enzymes were also assessed in cells. The mitochondrial enzyme levels in human cervix cancer HeLa and AS-30D cells were higher than those observed in rat hepatocytes whereas in human breast cancer biopsies, CPT1 and SCAD contents were lower than in human breast normal tissue. The presence of CPT1 and SCAD in AS-30D mitochondria and HeLa cells correlated with an active FFA utilization in HeLa cells. Furthermore, the ␤-oxidation inhibitor perhexiline blocked FFA utilization, OxPhos and proliferation in HeLa and other cancer cells. In conclusion, functional mitochondria supported by FFA ␤-oxidation are essential for the accelerated cancer cell proliferation and hence anti-␤-oxidation therapeutics appears as an alternative promising approach to deter malignant tumor growth. © 2015 Published by Elsevier Ltd.

1. Introduction

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Abbreviations: AAT, aspartate aminotransferase; AcAc, acetoacetate; ANT, adenine nucleotide translocase; COX, cytochrome c oxidase; CPT1, carnitine palmitoyl transferase I; LCHAD, long-chain 3-hydroxyacyl CoA dehydrogenase; GA, glutaminase; GDH, glutamate dehydrogenase; Gln, glutamine; Glut, glutamate; ICD, isocitrate dehydrogenase; LDH, lactate dehydrogenase; Mal, malate; ME, malic enzyme; ND, NADH-ubiquinone oxidoreductase; OxPhos, oxidative phosphorylation; PDH, pyruvate dehydrogenase; Pyr, pyruvate; RLM, rat liver mitochondria; ROS, radical oxygen species; SCAD, short-chain acyl CoA dehydrogenase; SDH, succinate dehydrogenase; 2OG, 2-oxoglutarate; 2OGDH, 2-oxoglutarate dehydrogenase; ␤ OHBut, ␤-hydroxybutyrate. ∗ Corresponding author at: Instituto Nacional de Cardiología, Departamento de Bioquímica, Juan Badiano No. 1, Col. Sección 16, Tlalpan, 14080 Mexico, Mexico. Q4 Tel.: +52 55 55 73 29 11. E-mail address: [email protected] (S. Rodríguez-Enríquez).

Enhanced glycolysis is one of the most important cancer metabolic hallmarks (Cantor and Sabatini, 2012; Hanahan and Weinberg, 2011). It has been suggested that tumor cells permanently maintain an impaired oxidative phosphorylation (OxPhos) which promotes an increased glycolysis (Warburg, 1956). In consequence, OxPhos flux, and mitochondrial enzyme activities and contents are not usually determined in studies of cancer energy metabolism (Owens et al., 2011; Putignani et al., 2012). Several proposals on the possible mechanisms associated with the OxPhos impairment in cancer cells have emerged. Some of these proposed mechanisms are: mutations in the Krebs cycle enzymes (fumarate hydratase, succinate dehydrogenase and isocitrate dehydrogenases) (Xekouki and Stratakis, 2012; Yang et al., 2012); absence (Mayr et al., 2008) of one of the principal enzyme controlling

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Please cite this article in press as: Rodríguez-Enríquez, S., et al., Mitochondrial free fatty acid ␤-oxidation supports oxidative phosphorylation and proliferation in cancer cells. Int J Biochem Cell Biol (2015), http://dx.doi.org/10.1016/j.biocel.2015.06.010

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tumor OxPhos. i.e. respiratory complex I (Rodríguez-Enríquez et al., 2000); and activation of several oncogenes (K-RAS), and transcription factors (HIF1-␣), that may lead to lower OxPhos functioning (Chiaradonna et al., 2006). However, this view has been challenged by numerous recent studies demonstrating that in several metastatic cancers, OxPhos is the predominant ATP cellular supplier in strong preference over glycolysis (Moreno-Sánchez et al., 2007; Ralph et al., 2010; Zu and Guppy, 2004). Thus, active OxPhos and mitochondrial remodeling and ROS production have been associated with invasiveness onset (Zhao et al., 2013), metastatic and malignant phenotype acquisition (Sotgia et al., 2012; Ralph et al., 2015), cellular cycle activation and autophagy resistance (Salem et al., 2012), and other mechanisms implicated in drug resistance (Indran et al., 2011; Lu and Chao, 2012). As a consequence of the misunderstanding on the role of OxPhos in cancer cells, scarcely any bioenergetics information has been reported using tumor mitochondria. In this regard, pioneer works (Moreadith and Lehninger, 1984; Rivera et al., 1988; RodríguezEnríquez et al., 2000) have shown that fast growing tumor cells oxidizes glutamine at high rates even when glucose is available (Fan et al., 2013; Guppy et al., 2002; Lazo, 1981; Reitzer et al., 1979). The oxidation of other substrates (ketone bodies, amino and imino-acids as well as free fatty acids) in isolated tumor mitochondria has not been extensively studied or contradictory results have been described (Cederbaum and Rubin, 1976; Ciapaite et al., 2011; Dietzen and Davis, 1993; Parlo and Coleman, 1984; Ralph et al., 2010; Rossignol et al., 2004). Among the different mitochondrial pathways evaluated, the ␤oxidation was of particular interest because it has been reported that the consumption of octanoyl-, myristoyl-, palmitoyl- and stearoyl-carnitine was significantly lower in AS-30D mitochondria than in RLM (Dietzen and Davis, 1993). In contrast, in human hepatocarcinoma HepG2 cells, ␤-oxidation is active and may replenish the mitochondrial acetyl-CoA pool required for OxPhos (Wong et al., 2004). High carnitine-acyl-transferase 1 (CPT1) mRNA contents have also been determined in ovary, colon, esophageal, prostate and colorectal carcinomas, as well as Zadjela hepatoma and K-RAS transformed fibroblasts (Alfonso et al., 2005; Capuano et al., 1997; Herrmann et al., 2003). Therefore, inhibition of ␤oxidation has been suggested as potential target to diminish tumor growth (Samudio et al., 2010; Tirado-Vélez et al., 2012). However, for other ␤-oxidation enzymes such as acyl CoA dehydrogenase, the mRNA content was significantly decreased in colorectal carcinoma vs. colon normal tissue (Birkenkamp-Demtroder et al., 2002). To characterize the mitochondrial ␤-oxidation in cancer cells, the first part of this work examined, in mitochondria isolated from AS-30D carcinoma grown in rats fed ad libitum, (i) the OxPhos rate and  m driven by FFAs comparing them to those driven by Krebs cycle intermediates, imino acids and ketone bodies; (ii) the ␤-oxidation and other OxPhos protein contents and activities; and (iii) the mitochondrial cholesterol content. In parallel, studies were also performed in mitochondria isolated from AS-30D carcinoma grown in 24h-fasted rats, in order to determine the changes in mitochondrial function and energy status induced by the nutritional state. For comparative purposes mitochondria isolated from rat liver (RLM), the organ from which the tumor originated, were also used (Orrick et al., 1973). In the second part of the present study, mitochondrial ␤oxidation was analyzed in intact tumor cells and human cancer biopsies (i) to resolve the existing discrepancies about the enzyme/transporter contents of this pathway; (ii) to establish whether this pathway is used for driving OxPhos and whether it has a functional role supporting tumor proliferation, which may help in the design of targeted strategies abolishing oxidative-type tumors; and (iii) to identify potential new metabolic tumor biomarkers.

2. Material and methods

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AS-30D tumor cells were propagated by intraperitoneal injection of 2–5 × 108 cells into female Wistar rats of 200–250 g weight. The cells were harvested, washed with Krebs–Ringer buffer and stored in ice at a density of 2–5 × 108 cells/mL until use as described previously (Rodríguez-Enríquez et al., 2001). Tumor cells from human carcinomas from cervix (HeLa), colon (Colo-205), lung (A-549) and breast (MCF-7, MDA-MB231, MDA-MB468) as well as 3T3 (mouse) and CCD25Lu (human) fibroblasts (American Type Culture Collection, ATCC; Rockville, MD, USA) were grown (1 × 107 cells/dish) in Dulbecco-MEM medium supplemented with 10% fetal bovine serum (Gibco; Rockville, MD, USA) plus 10,000 U penicillin/streptomycin (Sigma, Steinheim, Germany) and placed under a humidified atmosphere of 5% CO2 /95% air at 37 ◦ C until 80–90% confluence was reached. The genotyping (National Institute of Genomic Medicine, INMEGEN, México) of the HeLa, Colo-205, A-549, MCF-7, MDA-MB231, MDA-MB468 and 3T3 cell lines used in the present study revealed genotypes identical to those of the original ATCC tumor clones. 2.2. Isolation of rat hepatocytes Rat hepatocytes were isolated by the method of liver digestion using portal vein collagenase perfusion as previously described (Berry and Friend, 1969). Hepatocytes were resuspended in sterile phosphate-buffered saline (PBS), diluted (v/v) with an isosmotic Percoll solution (45 mL Percoll/4.5 mL of 10× Hank’s balanced salt solution) and centrifuged at 800 rpm for 5 min. Cell viability ≥85% was estimated by trypan blue exclusion. 2.3. Human breast cancer biopsies Five infiltrating ductal breast carcinoma samples were collected from female patients at Instituto Nacional de Cancerología, México, following the handling protocols approved by the Institutional Ethics Committee and supported by patient’s informed consents according to the Declaration of Helsinki (Pacheco-Velázquez et al., 2014). For normal breast tissue, 5 samples were used as a control. Statistical analysis of human tumor and non-tumor samples was performed by using Student’s t-test analyses as described elsewhere (Pacheco-Velázquez et al., 2014). 2.4. Isolation of mitochondria from rat AS-30D hepatoma and liver (RLM) The digitonin permeabilization procedure was used to isolate AS-30D mitochondria (Moreadith and Fiskum, 1984). The final concentration of digitonin (Sigma Aldrich, CA, USA) used for plasma membrane solubilization was 10–40 ␮g/mg cellular protein (Rodríguez-Enríquez et al., 2001). The mitochondrial pellet was then washed with SHE (Sucrose 250 mM, HEPES 10 mM, EGTA 1 mM pH 7.4) buffer and incubated with 0.5% (w/v) fatty acid free-albumin and 1 mM ADP for 15 min at 4 ◦ C before final centrifugation. RLM were isolated as described previously (Moreno-Sánchez, 1985). Female Wistar rat (250–300 g) liver was extracted and homogenized in cold SHE buffer, pH 7.3. The cellular homogenate was centrifuged at 700 × g and 4 ◦ C for 10 min. The supernatant of the first low-speed centrifugation was collected and further centrifuged at 7000 × g. The mitochondrial pellet was resuspended in cold SHE buffer and pre-incubated by 10 min in ice with 0.1% BSA plus1 mM ADP with occasional stirring; subsequently, the mixture was diluted 20 times and centrifuged at 7000 × g and 4 ◦ C for

Please cite this article in press as: Rodríguez-Enríquez, S., et al., Mitochondrial free fatty acid ␤-oxidation supports oxidative phosphorylation and proliferation in cancer cells. Int J Biochem Cell Biol (2015), http://dx.doi.org/10.1016/j.biocel.2015.06.010

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10 min. The mitochondrial pellet was resuspended in SHE buffer at 50–80 mg protein/mL.

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were then added at the indicated concentrations without (State 4) or with 600 nmol of ADP (State 3). At the end of each experiment, the uncoupler CCCP (5 ␮M) was added. 2.7. Enzyme activity assays

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Mitochondrial and cellular fractions from rat liver and AS30D hepatoma and HeLa cells as well as human breast biopsies were dissolved in RIPA (PBS 1× pH 7.2, 1% IGEPAL NP40, SDS 0.1% and sodium deoxycholate 0.05%) lysis buffer plus protease inhibitors cocktail (Roche, Mannheimm, Germany) and centrifuged at 2000 × g and 4 ◦ C for 30 min. Protein samples of mitochondria, cells or biopsies (40 ␮g) were re-suspended in loading buffer plus 5% ␤-mercaptoethanol and loaded onto 10–12.5% polyacrylamide gel under denaturalizing conditions (Gallardo-Pérez et al., 2009). Electrophoretic transfer to PVDF membranes (BioRad; Hercules, CA, USA) was followed by overnight immunoblotting with CPT-1, SCAD and peroxisomal LCHAD (1:500 dilution) antibodies; non-phosphorylated PDH-E1␣, mitochondrial ICD␭, 2OGDH, SDHC, mitochondrial AAT, GAL, ND1, ANT1, COXIV, ATPS-5B, LDH-A, PPAR␣, PPAR␥, ALDH2 and ␣-tubulin (1:1000 dilution) antibodies (Santa Cruz; Santa Cruz, CA, USA) at 4 ◦ C. The hybridization bands were developed with the corresponding secondary antibodies conjugated with horseradish peroxidase (Santa Cruz). The signal was detected by using a chemi-luminiscence ECL-Plus detection system (Amersham Bioscience; Little Chalfont, Buckinghamshire, UK). Densitometry analysis of all samples was performed using the Scion Image Software (Scion; Bethesda, MD, USA). Densitometries were normalized with ␣-tubulin (cells and biopsies) or ND1 (mitochondria). Percentage of each isoform represents the mean ± S.D. of at least three independent experiments. 2.6. Determination of oxygen consumption and changes in mitochondrial membrane potential ( m) in AS-30D and liver mitochondria Respiration of AS-30D and liver mitochondria (1 mg protein/mL) was assayed oxygraphically with a Clark-type O2 electrode in KME (120 mM KCl, 20 mM Mops, 1 mM EGTA) buffer pH 7.2 plus 2 mM KH2 PO4 , and different oxidizable substrates (as indicated in Results). For state 3 onset 600 nmol ADP were added. Changes in the electrical membrane potential ( m) were determined (a) quantitatively by measuring the tetraphenylphosphonium ([3 H]-TPP+ ) distribution across the inner mitochondrial membrane (Moreno-Sánchez et al., 1995, 1999); and (b) qualitatively with the permeant cationic dye safranine O (Akerman and Wikström, 1976). For TPP+ assay, mitochondria (2 mg) were incubated in 0.5 mL of KME medium under orbital shaking at 37 ◦ C, plus 0.8 ␮M [3H]-TPP+ (specific activity 0.06–0.07 ␮Ci/nmol), 5 mM KH2 PO4 and the different oxidizable substrates (as indicated in Results) without (State 4) or with 2 mM ADP (State 3). After 4 min incubation, mitochondria were centrifuged at 16,000 × g and 4 ◦ C for 2 min. The supernatant was quickly removed and the mitochondrial pellet was dissolved in 0.5% sodium dodecyl sulfate; aliquots of supernatant and pellet were used for determination of radioactivity by liquid scintillation counting in a Packard liquid scintillation counter (TRI-CARB 2100TR, Meriden, CT, USA). The changes in the membrane potential were calculated using the Nernst equation and corrected for non-specific TPP+ binding as described elsewhere (Rottenberg, 1984; Moreno-Sánchez et al., 1995).  m was also estimated by following the change in absorbance of safranin O at 554 minus 520 nm in a dual-wavelength spectrophotometer (2501PC Shimadzu; Japan) (Rodríguez-Enríquez et al., 2012). Mitochondria (0.5 mg protein/mL) were pre-incubated for 5 min at 37 ◦ C in KME buffer with 5 mM Pi plus 5 ␮M safranin O under smooth stirring for the complete endogenous substrates oxidation (Marín-Hernández et al., 2003). The different oxidizable substrates

The activities of AAT, ME, GDH, GA, PDH and ALDH were determined at 37 ◦ C following the formation or consumption of NAD(P)H at 340 nm in either the UV–visible diode array (Agilent; Santa Clara, CA, USA) and Shimadzu UV–spectrophotometers (Shimadzu, Kyoto, Japan), and the Aminco-Bowman Series 2 spectrofluorometer (Rochester, NY, USA). AAT activity was assayed in 50 mM triethanolamine, pH 7.4 plus 50 mM aspartate, 0.2 mM NADH, MDH (17 U), 2 ␮M rotenone and 30 ␮g mitochondrial protein/mL and 0.02% Triton X-100. The reaction was started with 10 mM 2OG. ME determination was assayed in 25 mM Tris/HCl, pH 7.6. The reaction mixture contained 0.3 mM NADP+ , 10 mM MgCl2 , 0.05–0.1 mg mitochondrial protein/mL and 0.02% Triton X-100. The reaction was started with 10 mM malate (MacDonald et al., 2009). GDH activity was carried out in 50 mM trietanolamine, pH 7.5, plus 0.15 mM NADH, 100 mM (NH4 )2 SO4 , 10 mM ADP, 2 ␮M rotenone, 0.1 mg of mitochondrial protein/mL and 0.02% Triton X-100. The reaction was started with 20 mM 2OG (Shashidharan et al., 1997). GA was measured in 250 mM hydroxylamine, pH 8.0, plus 1 mM NAD+ , 0.5–30 mM glutamine, 1 U GDH, 2 ␮M rotenone and 0.02% Triton X-100. The reaction was started by adding 0.1 mg of mitochondrial protein/mL (Hartman, 1971). ALDH activity was assayed in SHE buffer plus 0.04% Triton X-100, 1 mM NAD+ and 1 mg mitochondrial protein/mL. The reaction was started by the addition of 0.4 mM acetaldehyde or 0.4 mM propionaldehyde or 15 ␮M malondialdehyde or 5 ␮M 4-hydroxy-2-nonenal (YovalSánchez et al., 2011). PDH activity was assayed in 25 mM Tris/HCl, pH 7.6, plus 1 mM DTT, 5 mM NAD+ , 5 mM MgCl2 , 0.02% Triton X100, 0.4 mM AMP, 3 mM pyruvate, 4 ␮M rotenone and 0.1–0.5 mg of mitochondrial protein/mL. The reaction was initiated with 0.1 mM CoA (Lazo and Sols, 1980). Except where stated otherwise, enzyme activity determinations were performed with saturating substrate concentrations under initial-rate conditions, and correcting for the absorbance baseline obtained in the absence of one of the specific substrates. No reaction was detected in the absence of mitochondrial protein. COX activity was determined polarographically by using a Clark-type O2 electrode as previously described (Moreno-Sánchez et al., 1991). Mitochondria (0.025–0.05 mg protein/mL) were incubated at 37 ◦ C in air-saturated ME buffer (MOPS 25 mM, EGTA 1 mM) plus 7 mM fresh ascorbate, 1 ␮M antimycin, 50 ␮M horse heart cytochrome c. The reaction was started by adding 1.4 mM N,N,N ,N -tetramethyl-p-phenylenediamine (TMPD); further addition of 10 mM azide inhibited 90 ± 5% the TMPD-dependent COX activity. 2.8. Determination of cholesterol content in isolated AS-30D and liver mitochondria membranes Cholesterol extraction was performed using 2–5 mg of mitochondrial protein/mL in Krebs-Ringer medium plus butylhydroxytoluene (BHT) 0.002% (v/v) and stored at −70 ◦ C as described previously (El Hafidi et al., 2001). For cholesterol esters determination, lipid fractions were separated by thin layer chromatography on silica gel 60G using a mixture of hexane-etherformic acid (80:20:2, v/v) and with cholesterol-3-heptadecanoate as internal standard. Afterwards, cholesterol was identified in the transesterified lipid fraction by heating at 80 ◦ C for 2 h in the presence of BHT. Methyl esters were separated and identified by gas liquid chromatography (Carlo Erba Model 2300, Milan, Italy) fitted with a pre-coated (CP-Sil 88) 25 m × 0.25 mm

Please cite this article in press as: Rodríguez-Enríquez, S., et al., Mitochondrial free fatty acid ␤-oxidation supports oxidative phosphorylation and proliferation in cancer cells. Int J Biochem Cell Biol (2015), http://dx.doi.org/10.1016/j.biocel.2015.06.010

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fused-silica capillary column (Agilent). Analysis was performed at 195 ◦ C by using helium gas to maintain a steady flow rate of 1 mL/min.

Table 1 Mitochondrial enzyme activities and cholesterol content in AS-30D tumor and rat liver (RLM) isolated mitochondria. mU/mg protein

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2.9. Determination of free fatty acids (FFAs) in AS-30D and HeLa cells For FFAs extraction, 4 mg protein/mL of AS-30D or HeLa cells were resuspended in Krebs-Ringer medium containing BHT (0.002%, v/v). Afterwards, samples were incubated with 20–25 ␮g of heptadecanoic acid as internal standard and diluted with chloroform/methanol plus BHT 0.002% buffer (Folch et al., 1957). Samples were centrifuged at 1800 g for 2 min and the chloroform/ethanol phase was concentrated by evaporation under a N2 stream at room temperature. For FFAs esterification to their corresponding methyl esters, concentrated FFAs were dissolved in 1 mL of ethanol plus 0.1 mL 2,2-dimethoxypropane and 0.01 mL H2 SO4 (Tserng et al., 1981). The lipid residue was extracted with hexane-diethylether (1:1, V/V) and evaporated by gassing with N2 at room temperature. Samples were kept at −70 ◦ C ˜ until their use (El Hafidi and Banos, 1997). The identification of FFAs was performed with a Shimadzu gas chromatograph (GC 2010, Kyoto, Japan) equipped with an auto-injector/autosampler (AOC.20i) modem. Analysis of peak areas was performed with the Shimadzu GC solution software (version 2.3, Shimadzu, Kyoto Japan).

2.10. Cancer cell proliferation assays, cellular and mitochondrial oxygen consumption, cellular  m and FFA contents in the presence of ˇ-oxidation inhibitors For proliferation assays, HeLa (cervical), Colo-205 (colon), A-549 (lung) and MCF-7, MDA-MB231 and MDA-MB468 (breast) carcinomas as well as 3T3 (mouse) and CCD25Lu (human) fibroblasts (20,000 cells) were grown in 96 multi-well plates. For cell proliferation assays, cells were allowed to grow for 24 h and then perhexiline (0.1–50 ␮M) was added for further 24 h culture; the proliferation rate was determined with 3-(4,5-dimethylthiazol-2yl)-2,5-diphenytetrazolium bromide (MTT; Riss et al., 2004). For oxygen consumption, AS-30D cells, HeLa cells and rat hepatocytes (4–6 mg cellular protein/mL) were incubated in Krebs–Ringer medium (125 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.4 mM CaCl2 , 1 mM H2 PO4 , 25 mM HEPES, pH 7.4) at 37 ◦ C for 60 min in the absence or in the presence of perhexiline (10, 50, 100 ␮M). Thereafter, oligomycin-sensitive respiration was determined with a Clark-type O2 electrode (Rodríguez-Enríquez et al., 2000). For cellular  m determination, the permeant cation dye rhodamine 6G (250 nM) was incubated in Krebs-Ringer buffer at 37 ◦ C for 15 min. The assay was started by adding tumor cells or rat hepatocytes (0.25 mg protein/mL) previously pre-incubated by 60 min in the absence or in the presence of perhexiline (10, 50, 100 ␮M). Changes in the fluorescence were registered at em = 565 nm using exc = 480 nm in a Shimadzu RF-5301PC spectrofluorometer (Shimadzu, Kyoto, Japan). At the end of each experiment, 5 ␮M CCCP was added. For mitochondrial assays, respiration of isolated mitochondria from AS-30D cells and rat liver (1 mg protein/mL) were determined in KME buffer in the presence of 1, 10 and 100 ␮M perhexiline and in the presence of either 10 ␮M palmitoyl DL carnitine plus 0.1 mM malate; 5 mM glutamate plus 0.1 mM malate; or 5 mM succinate plus 1 ␮M rotenone. For state 3 onset 600 nmol ADP were added. For intracellular FFAs contents, HeLa and AS-30D cells were incubated for 60 min as previously indicated in the presence of 50 ␮M perhexiline.

PDH ME GA GDH ALDH AAT COX Cholesterol

AS-30D mitochondria

RLM

16 ± 11 (3) 8.3 ± 1 (4) 60 ± 15 (4)* 0.7 ± 1 (4)* 4.2 ± 0.2 (3) U/mg protein 4.5 ± 0.9 (3) 1.5 ± 0.4 (4) ␮g/mg protein 36 ± 10 (4)*

1.2 ± 1 (3) 90%) and intracellular  m (100%) in both AS-30D and HeLa cells as well as in rat hepatocytes without affecting cellular viability (>96%) after 60 min pre-incubation (Table 6). Higher perhexeline doses (100 ␮M) also abolished OxPhos but cell death was high (>60%; data not shown). At 10 ␮M perhexeline, an OxPhos flux inhibition of 30–40% in both HeLa and AS-30D cells correlated with a severe decrease (>75%) in their  m (Table 6). Other ␤oxidation inhibitors such as etomoxir did not affect cellular OxPhos and viability at 50–100 ␮M in 60 min incubations of AS-30D cells.

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The growth of metastatic HeLa cells was highly sensitive to perhexiline. However, other metastatic (Colo 205, MDA-MB321 and MDA-MB468) and non-metastatic (MCF-7 and A-549) cancer cell lines, as well as normal mouse and human fibroblasts, were much less sensitive, exhibiting 3–9 times higher IC50 values vs. HeLa (Table 6). At 50 ␮M perhexeline, growth and viability were abolished for all cell types (data not shown).

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4.1.1. High contents and activities of tumor OxPhos and ˇ-oxidation enzymes and transporters The activities of several OxPhos enzymes (ME, GA, COX and AAT) were determined in AS-30D mitochondria (cf. Table 1). These were similar to values previously reported for the same tumor mitochondria as well as Ehrlich ascites and rat liver mitochondria (Dietzen and Davis, 1993; Kovacevic´ et al., 1991; McGivan et al., 1974, 1980; Teller et al., 1992). On the other hand, PDH activity was significantly lower (3-times) in our study compared to the value reported by Dietzen and Davis (1993).

Table 5 Effect of OxPhos and ␤-oxidation inhibitors on free fatty acid (FFA) contents in AS-30D and HeLa cells. +Rotenone(1 ␮M)

+Perhexeline (50 ␮M)

AS-30D

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HeLa

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HeLa

Saturated FFAs

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t60

t0

t60

Myristic (14:0) Palmitic (16:0) Stearic (18:0) Unsaturated FFAs Palmitoleic (16:1) Oleic (18:1, ␻-9) Vaccenic (18:1, ␻-7) Linoleic (18:2, ␻-3) Arachidonic (20:4, ␻-6)

1.4 ± 1 8.1 ± 2 6 ± 1a

2±1 5 ± 0.7 4 ± 0.4c

0.8 ± 0.9 7.4 ± 1.5 2.8 ± 0.6

0.6 ± 0.3 7.6 ± 2 3 ± 0.6

1.2 ± 0.3 9 ± 3.7 6.5 ± 2

2.3 ± 0.7 13 ± 10 10.5 ± 5d

3 ± 2.2 7±1 3.1 (2)

1.4 ± 0.7 8.5 ± 3.5 3 ± 0.7

0.2 ± 0.05a 2.5 ± 0.3 2.2 (2) 2.3 ± 0.2b ND

0.27 ± 0.17c 2.2 ± 0.9 0.2 (2) 2.1 ± 0.3c ND

1.5 ± 0.1 3.7 ± 0.9 1.8 ± 0.9 1 ± 0.5 ND

1.3 ± 0.4 4 ± 1.5 1.5 ± 0.8 1.4 ± 0.3 ND

3.4 ± 4.5 3.5 ± 1.7 1 ± 0.9 4.9 ± 2.6 1.9 ± 0.95

0.5 ± 0.3d 4 ± 1.2 1 ± 0.4d 4.5 ± 0.6 1.6 ± 0.35

1 ± 0.6 3.3 ± 1.5 2.2 ± 1 2.3 ± 1 1 ± 0.7

1.7 ± 0.6 4±1 2.3 ± 0.6 1 ± 0.5 0.9 ± 0.4

FFA contents is expressed in nmol/mg cellular protein. The values shown represent the mean ± SD of 3 independent cell preparations, except for AS-30D vaccenic acid and HeLa stearic t0 . In general, there were no significant differences at p < 0.05 between rotenone t0 and t60 . N.D., not determined. a p < 0.01 vs. HeLa t0+rote . b p < 0.05 vs. HeLa t0+rote . c p < 0.05 vs. HeLa t60+rote . d p < 0.05 vs. HeLa t60+perh .

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561 562 563 564 565 566 567 568 569 570

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Table 6 Effect of perhexiline on cellular OxPhos, mitochondrial state 3 respiratory rates and cancer cell proliferation. Cells 60 min incubation OxPhos, ng atoms oxygen min−1 (mg cell protein)−1 HeLa (n = 4) AS-30D (n = 4) Rat hepatocytes (n = 2)  m (AFU) HeLa (n = 2) AS-30D (n = 2) Rat hepatocytes (n = 2) a p < 0.01, b p < 0.05 vs. no drug; c p < 0.01 vs. 10 ␮M Per.

0 8.5 ± 1.4 5.7 ± 1.2 5.2 68 68.5 54.5

+10 ␮M Per 5 ± 0.8 3.9 ± 1.3b –

+50 ␮M Per 0.5 ± 0.4a,c 0.25 ± 0.5a,c 2.7

2.5 18 7.5

0 0 0

Isolated mitochondria State 3, natoms oxygen min−1 (mg protein)−1

1–2 min incubation

AS-30D G + M (n = 5) S + R(n = 5) PC + M (n = 5) RLM G + M (n = 5) S + R (n = 5) PC + M (n = 5) a p < 0.01; b p < 0.05 vs. no drug; c p < 0.01; d p < 0.02 vs. 10 ␮M Per.

+10 ␮M Per 289 ± 45 199 ± 60 247 ± 39

0 270 ± 65 220 ± 56 274 ± 51 260 ± 48 270 ± 79 290 ± 91

300 ± 70 301 ± 45 336 ± 78a

+100 ␮M Per 184 ± 66b,d 128 ± 65 128 ± 47c,b 219 ± 121 106 ± 83c 224 ± 197

Cellular growth Perhexiline IC50 (␮M) after 24 h Metastatic CA HeLa (cervix) (n = 7) Colo 205 (colon) (n = 4) MDA-MB231 (breast) (n = 4) MDA-MB468 (breast) (n = 4) Non metastatic CA MCF-7 (breast) (n = 4) A-549 (lung) (n = 4) Non-cancer cells 3T3 (n = 4) CDD25Lu (n = 3)

3.4 ± 1.7a , c 16 ± 5c 11 ± 0.6b , c 15 ± 0.8b , c 20 ± 10 22 ± 1.4c 19 ± 3 30 ± 2.5

For cells, perhexeline was added 60 min before the determination of respiratory rates and  m. For isolated mitochondria, the drug was added 1–2 min before activation of OxPhos (i.e., addition of ADP). Abbreviations: Per, perhexeline; G, 5 mM glutamate; M, 0.1 mM or 5 mM malate for AS-30D mitochondria or RLM, respectively; PC, 10 ␮M palmitoyl-dl-carnitine; S, 0.5 mM or 5 mM succinate for AS-30D mitochondria and RLM, respectively; R, 1 ␮M rotenone; AFU, arbitrary fluoresce units. n represents the number of individual experiments. CA, carcinoma. a p < 0.01. b p < 0.05 vs. 3T3 fibroblast. c p < 0.01 vs. CDD25Lu fibroblasts. 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593

Nevertheless, the tumor PDH activity was still 13-times higher than their normal counterparts (cf. Table 1). In addition, increased tumor PDH activity correlated with a non-significant higher content of the active (non-phosphorylated) PDH vs. RLM, and with an active pyruvate oxidation (cf. Table 2). For GDH, tumor activity was significantly lower (2-times) in AS-30D mitochondria than in RLM but similar to that reported for other ascites cells mitochondria (Kovacevic´ et al., 1991; McGivan et al., 1974, 1980; Teller et al., 1992). Other studies have also shown that the contents of several OxPhos proteins (ND1␣, ECHS1, citrate synthase, MDH, fumarate hydratase, COXII and SDHB) are similar, or slightly higher, in HRAS-transformed breast MCF10A cells, compared with their normal non-transformed cells (Shaw et al., 2013). The higher GA content detected in HeLa and AS-30D cells compared to hepatocytes (cf. Fig. 2A) has also been reported for colorectal carcinoma (Huang et al., 2014). In contrast to the present study (cf. Fig. 1), the content of ATPS ␤ subunit has been found to be diminished in isolated mitochondria from human hepatocellular biopsies (Capuano et al., 1997). There are few studies with isolated tumor mitochondria, cells or tumor biopsies in which mitochondrial ␤-oxidation enzymes contents, activities as well as OxPhos flux sustained by FFAs have

been examined. The data presented here indicated the expression in human breast cancer biopsies (cf. Fig. 2B) of significant levels of several proteins involved in FFAs mitochondrial ␤-oxidation, as also documented for breast and colorectal (CPT1), prostate (ECHS) carcinoma and hepatocarcinoma (PPAR␣) (Kurokawa et al., 2011; Lin et al., 2007; Mazzarelli et al., 2007). The lower contents of some of these proteins vs. non-cancer biopsies (cf. Fig. 2B) indicated that mitochondrial ␤-oxidation may not be an adequate biomarker for breast tumor identification and prognosis, as it has been proposed for leukemia and myeloma (Samudio et al., 2010; Tirado-Vélez et al., 2012). However, the breast cancer biopsy samples used in the present study were of the HR+ subtype, the dominant type of breast carcinoma in México (Pacheco-Velázquez et al., 2014; Salazar et al., 1996). Thus, it cannot be discarded that mitochondrial ␤-oxidation might still be a suitable biomarker when other breast cancer subtypes are analyzed. For instance, the mitochondrial matrix enzyme complex 2OGDH has been proposed as biomarker for triple negative breast cancer subtype (Pacheco-Velázquez et al., 2014). 4.1.2. High oxidation and OxPhos rates and  m supported by FFAs and other NAD+ -linked substrates in cancer mitochondria There are several studies in which exogenous substrate oxidation rates have been analyzed in mitochondria isolated from

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594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611

612 613 614 615

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different murine (AS-30D, Ehrlich, Morris) and human (HT29 colon adenocarcinoma, lymphoblast, prostate PC-3) cancer cells (Briscoe et al., 1994; Carpentieri and Sordahl, 1975; De Bari et al., 2013; Dietzen and Davis, 1994; Gauthier et al., 1990; Kovacevic´ et al., 1991; Parlo and Coleman, 1984). However, in the majority of these studies exogenous substrate oxidation was accompanied by a high malate concentration (0.3–5 mM), which activates the tumor ME (cf. Table 2). Because ME has high affinity for malate (KmL-Mal = 130 ␮M; Chang et al., 1992) and oxidizes it at high rates, state 3 respiration driven by other exogenous substrates in tumor mitochondria is masked when malate is present in high concentrations (Carpentieri and Sordahl, 1975; Dietzen and Davis, 1994; Kovacevic´ et al., 1991; Parlo and Coleman, 1984). Therefore, in the present study care was taken to not overestimate the oxidation rates driven by each oxidizable substrate (cf. Tables 2 and 3). To circumvent ME activation, Briscoe et al. (1994) used low [malate] (100 ␮M) to drive glutamate, ␤-OHbut and AcAc oxidation in AS-30D mitochondria. In that study, states 3 and 4 respiratory rates as well as respiratory controls for the mentioned substrates were similar to those shown in Table 2 of the present study, except for AcAc. Unfortunately, other mitochondrial substrates such as glutamine, pyruvate, proline or FFAs were not evaluated under low [malate] conditions (Briscoe et al., 1994). Here, we demonstrated that several substrates are actively oxidized by tumor mitochondria (cf. Tables 2 and 3) to provide Krebs cycle intermediaries such as 2OG (i.e., glutamate, glutamine) and acetyl-CoA (FFAs, propionate, pyruvate) (Rodríguez-Enríquez et al., 2011). In AS-30D hepatoma mitochondria, oxidations of proline, propionate and ketone bodies were not apparent despite 24 h fasting of the tumor-bearing animals (cf. Supplemental Table 2). Probably a more severe fasting is required to induce in cancer cells a significant expression of the enzymes involved in the oxidations of proline (proline oxidase), propionate (propionylCoA synthetase, propionylCoA carboxylase), and ketone bodies (␤-hydroxybutyrate dehydrogenase). Proline and propionate oxidations generate glutamate and succinyl CoA, respectively, whereas ketone bodies oxidation forms acetyl-CoA. Although proline oxidase and succinylCoA acetoacetyl transferase (SCAAT) activities are 4 and 40-times higher in some tumor mitochondria vs. normal ones (reviewed in Rodríguez-Enríquez et al., 2011), it was shown that both enzyme activities were low in AS-30D cells. In fact, there are no studies analyzing AS-30D mitochondrial POX content. However, this enzyme is present in Morris hepatoma 5123 but it is undetectable in Novikoff hepatoma (Pitot et al., 1961). It has been documented for AS-30D mitochondria that SCAAT is 40-times higher than in RLM, thus contributing to an accelerated AcAc oxidation (4-fold higher vs. RLM) whereas for ␤-OHbut, a low substrate oxidation correlated with low activity of ␤-hydroxybutyrate dehydrogenase (Briscoe et al., 1994). Thus, it seems possible that the contents of SCAAT and ␤hydroxybutyrate dehydrogenase are low in AS-30D cells and in mitochondria, an observation that explains the negligible oxidation of AcAc and ␤-OHbut (cf. Table 2). AS-30D mitochondria were not able to oxidize l-lactate (cf. Table 2), as occurs in HepG2 hepatocarcinoma mitochondria, where an active l-lactate /pyruvate shuttle transport from cytosol to mitochondrial matrix has been described (Pizzuto et al., 2012). The active oxidation of mitochondrial substrates by hepatoma mitochondria was accompanied by the generation and utilization of  m (cf. Table 3). Indeed, the magnitude of  m generated by AS-30D mitochondria with 2OG, glutamate and pyruvate (cf. Table 3) was similar to values previously reported (Dietzen and Davis, 1993; Marín-Hernández et al., 2003) but slightly lower to those found in RLM (cf. Table 3). Unfortunately, for the rest of the assayed oxidizable substrates, no  m data are available for comparative purposes.

4.1.3. High cholesterol content does not perturb mitochondrial functionality in tumor cells One particular metabolic distinctive feature in tumor cells is their elevated cholesterol content in their plasma and both mitochondrial membranes (Dietzen and Davis, 1994; Feo et al., 1975; Parlo and Coleman, 1984; Pedersen, 1978; Woldegiorgis and Shrago, 1985). It has been reported that cholesterol content is 4–10 times higher in both inner and outer mitochondrial membrane of several hepatomas such as AS-30D, Morris 7777, 7800, 3924A and 5123 as well as in Yoshida ascites AH-130 compared to non-tumor mitochondria (Dietzen and Davis, 1994; Feo et al., 1975; Parlo and Coleman, 1984; Woldegiorgis and Shrago, 1985). It has been determined that elevated cholesterol (and other sterols) levels affect the activity of several respiratory chain enzymes including COX (Mohan et al., 2009; Sauer et al., 2011). Furthermore, a mechanistic relationship has been proposed between its mitochondrial localization and the higher content of the vitamin D receptor with an increased biosynthesis of cholesterol and decreased transcription of nuclear and mitochondrial COX subunits (Consiglio et al., 2014). However, in marked contrast, COX assays in isotonic medium (KME buffer) showed significantly higher activity in cholesterol-rich AS-30D mitochondria vs. RLM (data not shown). When assayed under optimal conditions (in hypotonic ME medium), COX activity in both types of mitochondria was 2–4 times higher, but non-significant difference between them was found (Table 1). Thus, although dysfunction of mitochondrial processes has been associated with high cholesterol/phospholipid ratios in plasma and mitochondrial membranes (Parlo and Coleman, 1984), the data of the present work, and other studies in normal and tumor mitochondria (Dietzen and Davis, 1994; Echegoyen et al., 1993; Feo et al., 1975; Woldegiorgis and Shrago, 1985), indicate that enhanced cholesterol levels do not cause failures in OxPhos. On the contrary, increased cholesterol in tumor mitochondria decreases membrane fluidity thus lowering the H+ passive diffusion across the inner mitochondrial membrane (Baggetto and Testa-Parussini, 1990). In this regard, it has been documented in different human tumor cell lines (colon CX-1, breast MCF-7) and in mouse H-RAS and c-MYC-initiated tumors that  m is 2–27-times higher than normal cells (Fantin et al., 2002; Modica-Napolitano and Aprille, 1987; Rodríguez-Enríquez et al., 2009) which may drive higher rates of ATP synthesis. In contrast to these reports, state 4  m in RLM was slightly higher than in AS-30D mitochondria (see Table 3), indeed suggesting that the increased cholesterol content affects tumor  m, or alternatively that the digitonin permeabilization step in the isolation procedure damages AS-30D mitochondria. In support of this last explanation, AS-30D cells and rat hepatocytes showed similar  m values (cf. Table 3) indicating similar availability of H+ gradients for the ATP synthase in in situ mitochondria.

4.2. Active mitochondrial ˇ-oxidation in tumor cells 4.2.1. Inhibition of ˇ-oxidation abolishes OxPhos flux and tumor proliferation It has been proposed that mitochondrial ␤-oxidation is non functional in several tumor carcinomas (PC3, HCT15, HepG2), because the prevalent high levels of malonyl-CoA, a lipogenesis product, strongly inhibits CPT1, and the activation of the P13K/Akt signal transduction pathway down-regulates CPT1A (Deberardinis et al., 2006) under nutritional stress. However, ␤-oxidation was not evaluated in these tumor cell lines to validate such proposal. In addition, it has been also documented that several hepatocarcinoma cell lines and chronic lymphocytic leukemia cells activate their mitochondrial ␤-oxidation consuming palmitate and stearate (Wong et al., 2004; Spaner et al., 2013).

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681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729

730

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791

Here, it was determined that palmitate, estearate and linolenate were actively consumed by HeLa cells (cf. Table 4), in a process sensitive to rotenone and perhexeline (cf. Table 5), direct and indirect inhibitors respectively of OxPhos. These data correlated with (i) the high capacity of AS-30D tumor mitochondria to oxidize exogenous FFAs (cf. Tables 2 and 3) and (ii) high contents of ␤-oxidation enzymes/transporters in both isolated AS-30D mitochondria (cf. Fig. 1) and AS-30D and HeLa cells (cf. Fig. 2B). These observations also correlated with the enhanced PPAR␣ and PPAR␥ levels found in tumor cells (cf. Fig. 2B). PPARs are involved in the up-regulation of several genes encoding mitochondrial ␤-oxidation enzymes and transporters (reviewed in Ashrafian et al., 2007). Over-expression of these transcriptional factors has also been reported for several carcinomas of liver, prostate, colon, breast and brain and chronic lymphocytic leukemia cells (Krishnan et al., 2007; Kurokawa et al., 2011; Spaner et al., 2013). The CPT1 and ␤-oxidation inhibitor perhexeline (Ashrafian et al., 2007), a drug commonly used for angina treatment (Inglis and Stewart, 2006), showed inhibitory effect on (i) FFA cellular consumption (cf. Table 5), (ii) OxPhos activity (cf. Tables 2, 3 and 5) and (iii) cancer cell growth (cf. Table 6). Similar results were reported for colon carcinoma (HT29) and HepG2 cells, where perhexiline at similar doses diminished cellular growth (Batra and Alenfall, 1991). It should be noted that growth of the metastatic cancer cell line HeLa was significantly more sensitive to perhexiline, but not that of metastatic MDA-MB231, MDA-MB468 and Colo205 cells, and non-metastatic cancer cell lines MCF-7 and A549, and the non-cancer CDD25Lu and 3T3 fibroblasts. These results suggested higher dependence on FFA oxidation for ATP supply in only some metastatic tumor cells (Batra and Alenfall, 1991). Perhexeline has been indeed considered as potential anticancer treatment (Agren et al., 2014). However, several side effects has been documented (NADPH oxidase inhibitor; Ca2+ and K+ channels inhibitor; Ashrafian et al., 2007) which may limit its use for clinical trials. Here, it was described that this inhibitor also affected oxidation of NAD+ -linked substrates which does not require CPT1 and ␤-oxidation activity (cf. Table 6). Therefore, quantitative biochemical studies of the OxPhos sensitivity to the drug in several cancer cell types are required to solidly establish whether it can be safely used as anticancer drug. In conclusion, the present study shows by using different experimental approaches that mitochondrial FFA ␤-oxidation is certainly functional in cancer cells, driving OxPhos-dependent ATP supply for cell proliferation. Therefore, anti-mitochondrial ␤-oxidation therapy may be added to the list of promising selective anti-cancer strategies.

Acknowledgements

The authors gratefully thank Prof. Franklin D. Rumjanek for his observations and critical reading of the manuscript. The present 793 Q7 work was partially supported by CONACyT-México grants Nos. 794 107183 to SRE, 180322 to AMH, 80534 and 123636 to RMS. Insti795 tuto de Ciencia y Tecnología del Distrito Federal grant No. PICS08. 796 IHR (290596) and SCPV (269212) were supported by Posgrado en 797 Ciencias Biomédicas-UNAM CONACyT Fellowships. 798 792

799

800 801 802

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biocel.2015.06. 010

11

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804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887

G Model BC 4647 1–13 12 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973

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Mitochondrial free fatty acid β-oxidation supports oxidative phosphorylation and proliferation in cancer cells.

Oxidative phosphorylation (OxPhos) is functional and sustains tumor proliferation in several cancer cell types. To establish whether mitochondrial β-o...
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