Available online at www.sciencedirect.com Journal of Nutritional Biochemistry 99 (2022) 108833 RESEARCH PAPER A high extra-virgin olive oil diet induces changes in metabolic pathways of experimental mammary tumors Maite Garcia-Guasch a, Lourdes Navarro a, Vanessa Rivero a, Irmgard Costa b, Eduard Escrich a, Raquel Moral a,∗ a Department of Cell Biology, Physiology and Immunology, Physiology Unit, Faculty of Medicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain b Department of Pathology, Corporació Parc Taulí-UDIAT, Sabadell, Barcelona, Spain Received 31 July 2020; received in revised form 22 February 2021; accepted 6 July 2021 Abstract Breast cancer is the most common malignancy in women worldwide, and environmental factors, especially diet, have a role in the etiology of this disease. This work aimed to investigate the influence of high fat diets (rich in corn oil or extra virgin olive oil -EVOO-) and the timing of dietary intervention (from weaning or after induction) on tumor metabolism in a seven,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer model in rat. The effects of lipids (oils and fatty acids) have also been investigated in MCF-7 cells. The results have confirmed different effects on tumor progression depending on the type of lipid. Molecular analysis at mRNA, protein and activity level of enzymes of the main metabolic pathways have also shown differences among groups. Thus, the animals fed with the EVOO-enriched diet developed tumors with less degree of clinical and morphological malignancy and showed modified glucose and mitochondrial metabolism when compared to the animals fed with the corn oil-enriched diet. Paradoxically, no clear influence on lipid metabolism by the high fat diets was observed. Considering previous studies on proliferation and apoptosis in the same samples, the results suggest that metabolic changes have a role in the molecular context that results in the modulation of different signaling pathways. Moreover, metabolic characteristics, without the context of other pathways, may not reflect tumor malignancy. The time of dietary intervention plays also a role, suggesting the importance of metabolic plasticity and the relation with mammary gland status when the tumor is induced. © 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Keywords: Dietary lipids; Extra virgin olive oil; Breast cancer; Metabolism; Carcinogenesis; Glucose metabolism. 1. Introduction carbohydrates, fiber, antioxidant substances, polyphenols and other micronutrients, fish, and olive oil as the principal source of fat [6]. Breast cancer is the most common cancer in women world- Some epidemiological [2,4,5] and especially experimental wide with high incidence (24.2%), prevalence (30.1%) and mortal- studies have reported an influence of dietary lipids on breast ity (15%) rates [1]. This disease is a multifactorial neoplastic pro- cancer risk that depends on the type and the quantity of fat, cess in which genetic, epigenetic and endocrine factors are in- and the timing of dietary intervention [7]. In general, saturated volved. Moreover, the environment also has a role in its etiology, fatty acids and n-6 polyunsaturated fatty acids (PUFA) show a being diet one of the most important factors [2]. In this sense, the positive association with breast cancer risk, whereas n-3 PUFA, Mediterranean diet is known as a healthy eating pattern associated as well as the ratio n-3/n-6 PUFA, decrease such risk [8,9,10]. with an improvement in health status and with a protective effect Concerning monounsaturated fatty acids (MUFA), epidemiological in the development of chronic diseases, including breast cancer evidence remains unclear [10] while experimental studies have re- [3,4,5]. This diet is characterized by high consumption of vegetal ported from a weak-promoting to a protective effect on mammary products resulting in high intake of monounsaturated fat, complex carcinogenesis [7,11,12]. Several mechanisms underlying such Abbreviations: DMBA, 7,12-dimethylbenz(a)anthracene; EVOO, extra-virgin olive oil; FA, fatty acids; HCO, high corn oil; HOO, high extra-virgin olive oil; LF, low-fat; MUFA, monounsaturated fatty acids; PPP, pentose phosphate pathway; PUFA, polyunsaturated fatty acids; TCA, tricarboxylic acid. ∗ Corresponding author at: Department of Cell Biology, Physiology and Immunology, Physiology Unit, Faculty of Medicine, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Tel.: 34-93-581-13-41. E-mail addresses: mariateresa.garcia.guasch@uab.cat (M. Garcia-Guasch), Lourdes.Navarro@uab.cat (L. Navarro), Vanessa.Rivero@uab.cat (V. Rivero), icosta@tauli.cat (I. Costa), Eduard.Escrich@uab.cat (E. Escrich), Raquel.Moral@uab.cat (R. Moral). https://doi.org/10.1016/j.jnutbio.2021.108833 0955-2863/© 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 2 M. Garcia-Guasch, L. Navarro, V. Rivero et al. / Journal of Nutritional Biochemistry 99 (2022) 108833 effects of lipids on breast cancer have been investigated [8]. We have previously reported that dietary lipids influence clinical and morphological features of experimental mammary tumors through different complex molecular mechanisms, including modifications in expression profiles of metabolism genes [7]. Cancer cells accumulate alterations in the main metabolic path- ways that sustain elevated rates of proliferation. Such metabolic reprogramming is considered a cancer hallmark [13]. The first metabolic alteration in cancer described was the Warburg Effect, a shift of glucose metabolism to its fermentation into lactate in aerobic conditions. This effect is thought to confer an advantage by producing more building blocks for cell growth and assuring NADPH production for the antioxidant defense [14,15]. Another im- Fig. 1. Experimental design. portant metabolic change affects glutamine availability, a key ni- Female Sprague-Dawley rats were fed the low fat control diet (LF), the high corn trogen and carbon supplier in several reactions [16]. Tumor cells oil diet from weaning (HCO) or from induction (LF-HCO), and the high extra-virgin also present increased lipid synthesis which is closely coupled to olive oil diet from weaning (HOO) or from induction (LF-HOO), n = 20 animals / glucose metabolism and may balance lipogenesis, lipid uptake and group. Animals were induced with 5 mg of 7,12-dimethylbenz[α ]anthracene (DMBA) at 53 days of age and euthanized at 236-256 days. lipolysis to maintain lipid homeostasis [17]. In previous works, we have reported different effects of high fat diets on mammary carcinogenesis. A diet high in corn oil had were 67.9% w/w in LF and 45.9% in both high fat diets. The diets also contained pro- a clear promoting effect while a diet high in extra virgin olive tein in the form of casein (18% w/w in the LF diet and 23% in the high fat diets) and oil (EVOO) had a weak influence [7,11,12]. Extra virgin olive oil 5% w/w cellulose, 5.9% w/w salt mixture and 0.24% w/w vitamin mixture. In order and seed oils composition differ on their fatty acid profile and the to maintain the normal lipidic metabolism, they were supplemented with methio- minor compounds. EVOO is rich in MUFA (oleic acid represents nine (0.51% w/w in LF and 0.66% in the high fat diets), choline (1800 mg/kg diet) and folic acid (5 mg/kg diet). The characteristics of lipids, minor compounds and 60-85% of total fatty acids, depending on the oil variety). More- energy composition of diets are given in Supplemental Table 1. Diets were prepared over, EVOO is rich in minor bioactive compounds (more than 230 weekly and stored under nitrogen in the dark at 4°C. have been identified), including polyphenols such as hydroxyty- rosol, secoiridoids or flavonoids. Corn oil is rich in n-6 PUFA (50- 2.3. RNA extraction, reverse transcription and real-time PCR 60% of total fatty acids) and contains some minor compounds such as sterols and tocopherols. The differential effects that high corn oil Total RNA from tumor samples was isolated using the RNeasy Extraction Kit and high EVOO diets exert on mammary carcinogenesis could be (QiaGen, Hilden, Germany) and quantified with Nanodrop 1,0 0 0 (ThermoFisher mediated, at least in part, by affecting the expression of different Scientific Inc, Waltham, MA, USA). Two micrograms of total RNA were reverse- transcribed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosys- genes involved in proliferation, apoptosis and metabolism. Thus, tems; Foster City, CA, USA). For the real-time PCR, 25 ng of cDNA were amplified this work aims to investigate the influence of such diets on tumor with the TaqMan methodology in the iCycler iQ Real-Time PCR detection system metabolism in the rat model of 7,12-dimethylbenz(a)anthracene (Bio-Rad Laboratories, Hercules, CA, USA). Specific Gene TaqMan Assays for glut1, (DMBA)-induced breast cancer. To this end, we analyzed the main hk2, pfkl, pfkm, g6pdh, pgd, gls and Hprt as the housekeeping control gene were metabolic pathways to gain insight into the role of dietary lipids in obtained from Applied Biosystems. PCRs were performed at 10min at 95°C followed by 15s at 95°C and 60s at 60°C for 40 cycles. Cycle thresholds (Ct) for each sample breast cancer development and to provide scientific knowledge to were obtained and 2−Ct calculated. establish healthy dietary advice for the population. 2.4. Protein extraction 2. Materials and methods Total protein was extracted homogenizing tumor samples in an extraction 2.1. Animals and experimental design buffer (50 mM Tris–HCl pH 7.2, 250 mM sucrose, 2 mM EDTA, 1 mM EGTA, 5 mM MgCl2, 50 μM NaF, 100 μM Na3VO4, 10 μl/ml protease inhibitor cocktail (Sigma- Animals received humane care under a protocol approved following the legis- Aldrich), 10 mM β -mercaptoethanol and 1% Triton X-100). For cytosolic, mitochon- lation applicable in this country. Female Sprague-Dawley rats (Charles River Lab, drial and membrane extracts samples were fractioned by cold centrifugations at L’Arbresle Cedex, France) at 23 days of age were distributed in five groups depend- 105.0 0 0g for one hour following standard protocols. Protein quantification was de- ing on the diet administered and the timing of dietary intervention (n=20 each termined by Lowry method using the commercial DC Protein Assay Kit (Bio-Rad). group), and were maintained under standard conditions (Fig. 1). Animals were fed with a control low fat diet (LF), with a high corn oil diet from weaning (HCO) or after induction (LF-HCO), or with a high extra virgin olive oil diet from weaning 2.5. Western blot (HOO) or after induction (LF-HOO). Mammary tumors were induced by oral gav- age with a single dose (5 mg) of 7,12-dimethylbenz(a)anthracene (DMBA) (Sigma- The different protein extracts (15-20 ug) were subjected to SDS-PAGE elec- Aldrich, St. Louis, MO, USA) at 53 days of age. Rats were euthanized at 236-256 trophoresis on an acrylamide gel (Mini-Protean TGX Stain Free Gels, Bio-Rad) days by decapitation. Tumors were removed, the three diameters were measured, and transferred to a PVDF membrane with the Trans-Blot Turbo Transfer System and volume was calculated (V = 4/3π 4/3π [d1/2] x [d2/2] x [d3/2]). A portion of (Bio-Rad). Membranes were blocked with 5%BSA TBS-Tween 0.1% or 5% skimmed each tumor was fixed in 4% formalin for anatomopathological analysis, and the rest milk TBS-Tween 0.1% for 1h at room temperature and incubated with the spe- was flash-frozen and stored at -80°C for molecular analysis. cific primary antibody overnight at +4°C. Primary antibodies used were anti-Glut1 The morphological degree of tumor malignancy was characterized by applying (1:1,0 0 0), anti-PFKL (1:50 0), anti-ALDOA (1:1,0 0 0), anti-G6PDH (1:3,0 0 0), anti-PGD the Scarf-Bloom-Richardson grading method (SBR3, scoring 1-3), and the modified (1:5,0 0 0), anti-ASCT2 (1:3,0 0 0), anti-ACC (1:1,0 0 0), anti-pACC (1:3,0 0 0), anti-FASN method adapted to rat mammary adenocarcinomas (SBR11, scoring 3-11) that we (1:20,0 0 0), anti-SCD (1:2,0 0 0), anti-AMPK (1:2,0 0 0), anti-pAMPK (1:2,0 0 0) from have previously described [18]. The highest categories indicated the most morpho- cell signaling; anti-LDHA (1:30,0 0 0), anti-CPT1a (1:1,0 0 0), anti-ACDL (1:3,0 0 0), logically aggressive tumors. anti-UCP2 (1:5,0 0 0), anti-ACLY (1:5,0 0 0) from abcam; and anti-GLS (1:1,0 0 0) from Thermofisher scientific. After incubation with the secondary antibody (anti-rabbit, anti-mouse or anti-goat, Sigma-Aldrich) for 1h at room temperature, the mem- 2.2. Diets branes were incubated with Luminata Forte Western HRP Substrate (EMD Millipore) luminogen for 3-5 minutes. Proteins were visualized using the Chemidoc XRS+ Three semi-synthetic diets were designed, as described before [19,20,21]: a low hardware associated with Image Lab Software 5.1-Beta (Bio-Rad). Densitometric val- fat diet (LF, 3% corn oil w/w), a high corn oil diet (HCO, 20% corn oil) and a high ues were normalized with the total protein loaded [22] and relativized to an inter- olive oil diet (HOO, 3% corn oil+17% EVOO). Carbohydrates in the form of dextrose nal control sample loaded in duplicates in all blots [7]. M. Garcia-Guasch, L. Navarro, V. Rivero et al. / Journal of Nutritional Biochemistry 99 (2022) 108833 3 Table 1 Clinical and anatomopaghological characteristics of tumors LF HCO LF-HCO HOO LF-HOO Carcinogenesis parameters (clinical malignancy) Tumor-bearing animals (%) 80a 100b 100b 75a 85a,b Tumor yield (total number of tumors) 47a 100b 87b,c 58a,c 82b,c Anatomopathological degree (morphological malignancy) n 47 98 84 50 81 SBR3, number of tumors 1/2/3 27/14/6 a 23/43/32b,c 17/29/38 b 19/22/9 a 34/28/19 a,c degree 1 (%) 57.4 23.4 20.3 38.0 42.0 degree 2 (%) 29.8 43.9 34.5 44.0 34.6 degree 3 (%) 12.8 32.7 45.2 18.0 23.4 adapted SBR11, median 5a 7 b,c 8c 7 a,b,c 6.5 a,b Characteristics of tumors included in the molecular study n 14 26 24 21 25 SBR3, number of tumors 1/2/3 10/3/1a 8/10/8 b,c 3/9/12 c 8/11/2 a,b,c 7/14/4b degree 1 (%) 71.4 30.8 12.5 38.1 28.0 degree 2 (%) 21.4 38.4 37.5 52.4 56.0 degree 3 (%) 7.2 30.8 50.0 9.5 16.0 adapted SBR11, median 5a 7.5 b,c 8.5 b 6 a,c 7c Tumor volume, median (cm3) 0.60a 1.80b,c 3.89c 1.99a,b,c 2.31a,b Values within a row with different superscript letters are significantly different (P<.05). Quantitative data (adapted SBR11, tumor vol- ume): Mann-Whitney U test. Qualitative data (tumor-bearing animals, tumor yield, distribution of tumors according to SBR1, SBR2, SBR3): chi-squared test. 2.6. Enzymatic activity assays 3. Results For determination of citrate synthase and isocitrate dehydrogenase activity, we 3.1. Clinical and morphological malignancy of tumors are influenced used the Citrate Synthase Activity Assay Kit (MAK193, Sigma Aldrich) and Isoci- trate Dehydrogenase Activity Assay Kit (MAK062, Sigma Aldrich). Ten mg of tumor by dietary lipids tissue were homogenized in 100uL of ice-cold extraction buffer, kept on ice for 10 minutes and centrifuged at 10,0 0 0xg for 5 minutes. Supernatants were trans- Carcinogenesis parameters and the morphological degree of tu- ferred to fresh tubes and protein concentrations were determined. Activity as- mor aggressiveness are shown in Table 1. The groups fed the high says were assessed according to the manufacturer’s instructions. The enzymatic activities were determined with coupled enzymes reactions in which resultant corn oil diet had a higher incidence (percentage of tumor-bearing products are proportional to activity and are measured spectrophotometrically at animals) and tumor yield (total number of tumors) than the con- 412 nm. trol LF group. The HCO group had also higher incidence and tumor yield than the HOO group. Morphological degree of malignancy was also clearly higher in the groups fed the high corn oil diet (as shown by the percentage of tumors with high degree of SBR3, 2.7. Cell culture treatment and the SBR11 median), especially in the LF-HCO group, whereas MCF-7 breast cancer cells were obtained from the American Type Culture the groups fed the EVOO diet displayed intermediate values. More- Collection (ATCC) and grown in EMEM (Gibco) supplemented with 10% fetal over, the subgroup of tumors used in the molecular analyses pre- bovine serum (FBS) and 0.01 mg/mL of insulin. Cells were maintained at 37°C sented in this work also showed a higher degree of malignancy in in a humidified atmosphere of 95% air and 5% CO2 , and tested for mycoplasma periodically. the groups fed the high corn oil diet, especially in the LF-HCO one MCF-7 cells were seeded 48 hours prior to treatments in p100 plates with (significantly higher degree in comparison to LF and EVOO groups). EMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) and 0.01 mg/mL Tumor volume was significantly higher in the high corn oil diet of insulin. For fatty acid treatments, oleic or linoleic acid-albumin from bovine groups versus LF, and in LF-HCO versus LF-HOO. serum (Sigma Aldrich) were used at 0mM (control) or 1 mM concentration. All the solutions contained 0.1 mg/mL of BSA. For oils treatments, extra virgin olive oil (EVOO) or corn oil solutions were prepared at 0% (control) or 0.1% oil solubi- 3.2. Dietary lipids influence the mRNA expression of enzymes of the lized in DMSO (0.3%). The EMEM medium was supplemented with 5% FBS and 0.01 main metabolic pathways mg/mL of insulin. All treatments (fatty acid and oils) were carried out for 24h, 48h, 72h and 96h, and protein was extracted following the standard protein extraction protocols. The expression levels of metabolism-related genes were an- alyzed by RT-PCR and are shown in Fig. 2. In general, tumors from rats fed with the high fat diets showed an increase in mRNA expression of glucose uptake and glycolysis-related genes (P<.01, 2.8. Statistical analysis Fig. 2A) and pentose phosphate pathway (PPP) genes (P<.001, Statistical analyses were performed using R Deducer. The statistical test was Fig. 2B) in comparison to tumors from the low fat-fed rats. Gene determined depending on the sample distribution (Shapiro – Wilk test) and the expression patterns also revealed differences due to the type of equality of variances (Levene’s test). Quantitative parametrical data were analyzed dietary lipid. Higher levels of hk2, pfkl and pfkm mRNA were with one-way ANOVA test (t-test equal variance). Non-parametrical data were an- observed in the LF-HCO group compared to the LF-HOO group, alyzed with Kruskal Wallis test – wilcoxon method. The chi-squared test was used for the qualitative data (carcinogenesis parameters and distribution of mammary while g6pdh and pgd expression was increased in HOO versus adenocarcinomas according to the degree of morphological malignancy). The level HCO. We also observed differences related to the time of dietary of significance was established at P<.05. intervention, with higher levels of glut1, hk2, pfkl and pgd genes 4 M. Garcia-Guasch, L. Navarro, V. Rivero et al. / Journal of Nutritional Biochemistry 99 (2022) 108833 Fig. 2. Effect of diets on relative mRNA expression levels in mammary tumors. (A) Glucose transport and glycolysis-related genes. (B) Pentose phosphate pathway (PPP)-related genes. (C) Glutaminolysis-related gene. Boxplots (median, box 25 and 75 percentile, whisker 10 and 90 percentile). Solid lines connecting groups indicate statistically significant differences (P<.05), dashed lines indicate differences close to significance (P<.1), Kruskal Wallis test. in LF-HCO compared to the group fed with the same diet from Analysis of UCP2 was carried out in mitochondrial fractions weaning (HCO). The mRNA levels of hk2 and pfkm were also (Fig. 4C). Results showed higher UCP2 protein levels in the LF-HOO different between LF-HOO and HOO. group compared to the control and LF-HCO groups. The study of glutaminolysis (Fig. 2C) revealed a decrease in gls mRNA in the LF-HOO group in comparison to all other experimen- tal groups. 3.4. The diet rich in EVOO increases the activity of the main tricarboxylic acid (TCA) cycle enzymes 3.3. Dietary lipids have an effect on glucose metabolism, glutaminolysis and UCP2 protein expression but not on lipid The results of the enzymatic activity of citrate synthase (CS) metabolism-related enzymes and isocitrate dehydrogenase isoenzymes (IDH1-3) are shown in Fig. 5. An increase in CS activity was obtained in both high EVOO The enzymes of the main metabolic pathways were analyzed by diet groups in comparison to LF and high corn oil diet groups. western blot. Fig. 3 depicts the results of enzymes with a role in IDH1/2 activity was significantly increased in the LF-HOO group. glucose transport and glycolysis (Fig. 3A), Warburg effect (Fig. 3B) No differences were observed in IDH3 activity. and pentose phosphate pathway -PPP- (Fig. 3C). Changes in protein expression by the effect of dietary lipids were observed in the glu- cose transporter Glut 1, the glycolysis key enzyme PFKL, and the 3.5. In vitro effects of dietary lipids PPP enzymes G6PDH and PGD, with significant higher protein lev- els in the HOO group compared to the HCO group. The same trend MCF-7 cells were treated with fatty acids (oleic acid and linoleic was observed in PGD when comparing LF-HOO and LF-HCO groups. acid) and oils (EVOO and corn oil) for 24, 48, 72 and 96 hours. In relation to lipid metabolism, the expression levels of the We analyzed the proteins found as differentially expressed in the main proteins of fatty acid transport (FATP1), β -oxidation (CPT1a, DMBA-induced tumors (PFKL, GAPDH, PGD, UCP2) in the fatty ACADL) and de novo synthesis (ACLY, FASN, pACC and ACC, SCD) acid- and oil-treated cells (supplemental Figure 1). Results showed have been analyzed (Fig. 4A). In general, no differences have been heterogeneous data depending on lipid, concentration and time of found by the effect of the experimental diets. exposures, with no clear trends along time (e.g. decreased PFKL For the study of glutaminolysis, we have analyzed the expres- by oleic acid at 48h but increased levels at 72h). Proteins re- sion of the glutamine transporter ASCT2 and GLS enzyme (Fig. 4B). lated to lipid metabolism in oil-treated cells showed an effect Expression of GLS was increased in HCO and HOO groups in com- of EVOO increasing the lipid transporter CD36 and decreasing parison to the LF group. FASN. M. Garcia-Guasch, L. Navarro, V. Rivero et al. / Journal of Nutritional Biochemistry 99 (2022) 108833 5 Fig. 3. Effect of diets on protein expression of main carbohydrate metabolic pathways in mammary tumors. (A) Glucose transport and glycolysis related-enzymes. (B) Warburg effect related-enzyme. (C) PPP related-enzymes. Boxplots (median, box 25 and 75 percentile, whisker 10 and 90 percentile). Solid lines connecting groups indicate statistically significant differences (P<.05), dashed lines indicate differences close to significance (P<.1), Kruskal Wallis test. 4. Discussion Results showed that tumors from rats fed with the high fat diets, especially from the high corn oil diet groups, displayed morpho- In this work, we have studied the effect of high fat diets logical characteristics of higher degree of malignancy. These results on metabolic enzymes (at mRNA, protein or activity level) in a suggest that diets rich in fat may have an unspecific promoting carcinogen-induced breast cancer model, as a mechanism of the effect on tumorigenesis, but for the same amount of fat there is influence of diets on the progression of this disease. The results a different specific effect depending on the main type of dietary obtained show an effect of the high EVOO diet on glucose and ox- lipid. idative metabolism and suggest a complex interconnection among To get insight into the molecular mechanisms by which dietary metabolism and signaling pathways. lipids influence mammary carcinogenesis we previously performed We have previously observed a strong promoter effect of the transcriptomic analyses in tumors from the different experimen- diet high in corn oil while a weak effect of the diet high in ex- tal groups. Gene expression profiles and enriched functional cate- tra virgin olive oil, both in the clinical and morphological man- gories indicated an effect of high fat diets on tumor metabolism, ifestation of mammary tumorigenesis [7]. In this study, we have increasing the expression of glycolytic enzymes [7]. In this work, also observed, in the subgroup of tumors where molecular analyses we have validated and extended those results, analyzing the effect have been carried out, a differential effect of dietary lipids on the of these two different high fat diets on key metabolism pathways. anatomopathological degree of malignancy. Tumor phenotype was At mRNA level, we found changes in glycolysis and PPP enzymes in characterized by both the Scarff–Bloom–Richardson (SBR) grading the high fat diet groups related to the control, but these differences method (SBR3) and the one adapted to this rat model (SBR11) [23]. were not significant at the protein level. Taking in mind that the 6 M. Garcia-Guasch, L. Navarro, V. Rivero et al. / Journal of Nutritional Biochemistry 99 (2022) 108833 Fig. 4. Effect of diets on the expression of proteins related to lipid metabolism and glutaminolysis, and UCP2, in mammary tumors. (A) Lipid metabolism-related proteins. (B) Glutaminolysis-related proteins. (C) UCP2. Boxplots (median, box 25 and 75 percentile, whisker 10 and 90 percentile). Solid lines connecting groups indicate statistically significant differences (P<.05), dashed lines indicate differences close to significance (P<.1), Kruskal Wallis test. Fig. 5. Effect of diets on the activity of TCA cycle enzymes in mammary tumors. Enzymatic activity of citrate synthase (CS) and isocitrate dehydrogenase (IDH1, IDH2 and IDH3). Boxplots (median, box 25 and 75 percentile, whisker 10 and 90 percentile). Solid lines connecting groups indicate statistically significant differences (P<.05), dashed lines indicate differences close to significance (P<.1), Kruskal Wallis test. M. Garcia-Guasch, L. Navarro, V. Rivero et al. / Journal of Nutritional Biochemistry 99 (2022) 108833 7 Fig. 6. Effects of the EVOO-rich diet on tumor metabolic pathways. (A) Effect of the administration of the EVOO-rich diet from weaning (comparison of HOO versus HCO). (B) Effect of the administration of the EVOO-rich diet after induction (comparison of LF-HOO versus LF-HCO). Green arrows represent significant higher levels of protein expression or enzymatic activity in the EVOO-rich diet group. Color version of figure is available online. two high fat diets were isocaloric and that the type of oil was the role in glucose uptake (Glut1), glycolysis (PFKL) and PPP (G6PDH, only difference in their composition, we compared among high fat PGD) and increased the activity of the TCA cycle enzyme citrate diet groups. As summed up in Fig. 6, our results suggest that the synthase (CS). The group fed with the same diet after induction EVOO-enriched diet provided from weaning (observed in the HOO (LF-HOO) showed increased UCP2 protein levels and enzymatic ac- group) increased the mRNA and protein levels of enzymes with a tivity of TCA cycle enzymes (CS and IDH). 8 M. Garcia-Guasch, L. Navarro, V. Rivero et al. / Journal of Nutritional Biochemistry 99 (2022) 108833 No differences were found among groups when beta-oxidation, and pro-apoptotic proteins expression in such LF-HOO group. The lipid synthesis or protein metabolism were studied. On the other tumor suppressor p53 is involved in key cellular processes such hand, results obtained in MCF-7 cells treated with fatty acid or as cell cycle arrest, cellular senescence, apoptosis or cell oxidative oils showed subtle and heterogeneous effects on the expression balance, in addition to the control of metabolism by limiting gly- of metabolism proteins. Although the in vitro results do not show colysis and promoting mitochondrial respiration [33]. On the other clear trends in many proteins, treatment with oils suggests changes hand, the same level of oxidative stress may have different con- such as in the lipid transporter CD36 or an increase in FASN (a key sequences depending on the cell context, e.g. evading or induc- protein in fatty acid synthesis) by the effect of corn oil. Therefore, ing apoptosis depending on the activated signaling pathways [34]. the tumor cells would respond to the exogenous source of lipids, Thus, our results suggest that UCP2 is increased in the LF-HOO as also suggested by the results of gene expression in tumors. This group as cell protection from oxidative stress, concomitantly with effect of lipids, although subtle, can be of great importance in the an increase in apoptotic pathways that would lead to cell death, early stages of carcinogenesis. The chronic effect of the dietary in- which would be consistent with the lower morphological malig- tervention may be different in established tumors, such as those nancy of tumors in this group. Other studies have also reported analyzed in this work, as many characteristics have already been an effect of EVOO components (polyphenols and oleic acids) on selected and cannot be separated from the context of other cellular apoptosis in cancer [35]. In tongue squamous cell carcinomas, oleic processes. Thus, although caution must be applied when extrapo- acid treatment induced apoptosis and autophagy [32], and hydrox- lating in vitro and in vivo effects, both n-6 PUFA and n-9 MUFA ytyrosol regulated proliferation and apoptosis-related pathways in may affect tumor metabolism depending on dose or time variables. DMBA-induced mammary tumors [36]. There is evidence of the modulation of metabolism genes by di- Results less conclusive were obtained from the HOO group. This etary lipids in tissues like liver but reported data is less conclusive group showed an increase in glycolysis and PPP, which has been for tumor tissues [24]. Tumor cells undergo uncontrolled growth related to sustained cell growth and a decrease in apoptosis [37]. that has an impact on the regulation of metabolic pathways, ei- This metabolic profile favors NADPH generation which may lead to ther as a cause or as a consequence [14]. Nowadays, it is known inhibit caspase activation [38]. Thus, an increase in NADPH could that tumor cells use glycolysis, oxidative phosphorylation, or both, be a cause that apoptosis is not increased in this group fed with depending on the cell conditions. Moreover, cancer cells can shift EVOO. On the other hand, the HOO group showed less activation their metabolic profile during tumorigenesis, tumor progression of proliferation pathways when compared to HCO [29] as well as and metastasis to provide the requirements needed for cell growth a lower clinical and anatomopathological degree of malignancy. and survival. Actually, metabolic changes and adaptation to main- Therefore, the metabolic profile is not a direct reflection of tumor tain cell growth is considered a cancer hallmark [14,25]. Upregu- malignancy since it is interconnected with the whole cell network. lation of glycolysis, PPP, the Warburg effect or IDH1/2 activity in More studies are required to deep in and clarify the importance of malignant cells may be related to ROS detoxification and increased cell metabolic profiles and its relation to tumor malignancy. redox potential via NADPH generation [26,27]. Also, the function of In summary, in this work we have studied the effect of dietary UCP2 (an uncoupling protein family located in the mitochondrial fats on breast cancer tumorigenesis pointing out the importance membrane) is thought to be an adaptive response limiting ROS of the type of lipid. Thus, high fat diets lead to a more malignant in breast cancer cells and avoiding ROS-mediated apoptosis [28]. phenotype when compared with a low fat diet, but the degree of Hence, according to reported data in the literature, the metabolic malignancy is clearly associated with the type of fat. Tumors from profiles we found in tumors from rats fed the EVOO-enriched diet animals fed with the EVOO-enriched diet showed a lower degree (in comparison to those from the groups fed the high corn oil of malignancy and changes in metabolic pathways, in comparison diet), may reflect an adaptative response to sustain cell growth or to those from rats fed with the corn oil-enriched diet. The results prevent cell death, but this is not in accordance with the clini- obtained, together with previous studies in these same samples, cal and morphological behavior of tumors that we have observed. suggest that these metabolic changes have a role in the molecular Thus, anatomopathological and clinical parameters clearly showed context that results in the modulation of different signaling path- a lower degree of aggressiveness in the groups fed the EVOO diet ways in the tumors from the animals fed with EVOO. Therefore, in comparison to the ones fed the high corn oil diet. This appar- the metabolic characteristics, without the context of other path- ently contradictory fact may be explained by the interconnection ways such as proliferation or apoptosis, may not reflect tumor ma- of signaling pathways required for cell growth and survival. There- lignancy. Moreover, we have also observed that the time of dietary fore, the results obtained can be part of a complex and intercon- intervention has a role in metabolic plasticity and that may be re- nected cell net. In this sense, we have previously reported that lated to the status of the mammary gland when the tumor is in- the EVOO-rich diet influences the molecular context in tumor cells duced. Future studies in metabolomics will be conducted to elu- resulting in increased apoptosis and decreased proliferation when cidate the effects of dietary lipids on the molecular basis and in- compared to the effects of the high corn oil diet [29]. Moreover, terconnection of different pathways. In vitro analysis with EVOO in the same experimental model presented here we observed an minor compounds will also get insight into the effect of that oil increase in proliferation proteins by the effect of the high corn oil on tumor metabolism. In conclusion, our study provides new data diet (activated p21Ras, phospho-AKT, phospho-Erk or Myc), while on the link between dietary lipids and tumor biology, and high- we found higher levels of pro-apoptotic proteins by the effect of light the importance of dietary habits from early life in the risk of the EVOO diet (FADD, Bid, Bax or p53 -unpublished results-). this disease. We have also observed that the groups fed with the EVOO- enriched diet present differences in metabolic profiles depending Author Contributions on the time of the dietary intervention. The LF-HOO group showed higher activity of TCA cycle and UCP2 protein expression. We pre- Maite Garcia-Guasch: Investigation, Methodology, Analysis, Vi- viously reported an increase in hepatic UCP2 mRNA by the effect of sualization, Writing - Original Draft; Lourdes Navarro: Investi- this high EVOO diet [30], in accordance with other authors report- gation; Vanessa Rivero: Investigation; Irmgard Costa: Methodol- ing increased UCP2 by EVOO or oleic acid in different tissues [31]. ogy, Investigation; Eduard Escrich: Conceptualization, Methodology, Although UCP2 has been related to avoiding ROS-mediated apopto- Funding acquisition, Resources; Raquel Moral: Conceptualization, sis in cancer [32], our previous results showed higher levels of p53 Supervision, Analysis, Writing - Review & Editing. M. Garcia-Guasch, L. Navarro, V. Rivero et al. / Journal of Nutritional Biochemistry 99 (2022) 108833 9 Declaration of Competing Interests [17] Zaidi N, Lupien L, Kuemmerle NB, Kinlaw WB, Swinnen JV, Smans K. 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