cancers Article Extra-Virgin Olive Oil and Its Minor Compounds Influence Apoptosis in Experimental Mammary Tumors and Human Breast Cancer Cell Lines Maite Garcia-Guasch 1,† , Mireia Medrano 1,† , Irmgard Costa 2 , Elena Vela 1 , Marta Grau 1 , Eduard Escrich 1 and Raquel Moral 1, * 1 Department of Cell Biology, Physiology and Immunology, Faculty of Medicine, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; mariateresa.garcia.guasch@uab.cat (M.G.-G.); Mireia.Medrano@uab.cat (M.M.); Elena.Vela@uab.cat (E.V.); gr.mecm@uab.cat (M.G.); Eduard.Escrich@uab.cat (E.E.) 2 Department of Pathology, Corporació Parc Taulí-UDIAT, 08208 Sabadell, Spain; icosta@tauli.cat * Correspondence: Raquel.Moral@uab.cat † These authors contributed equally to this work. Simple Summary: Breast cancer is a disease influenced by dietetic factors, such as the type and amount of lipids in a diet. In this work, we aimed to elucidate the different effects of two high-fat diets on the histopathological and molecular characteristics of mammary tumors in an experimental model. Animals fed with a diet high in extra-virgin olive oil (EVOO), compared to those fed with a diet high in seed oil, developed tumors with less aggressiveness and proliferation. Tumor molecular analyses  of several cell death pathways also suggested an effect of EVOO in this process. In vitro experiments  indicated the role of EVOO minor compounds on the effects of this oil. Obtaining insights into Citation: Garcia-Guasch, M.; the influence and the mechanisms of action of dietary compounds are necessary to understand the Medrano, M.; Costa, I.; Vela, E.; Grau, relevance that dietetic habits from childhood may have on health and the risk of disease. M.; Escrich, E.; Moral, R. Extra-Virgin Olive Oil and Its Minor Compounds Abstract: Breast cancer is the most common malignancy among women worldwide. Modifiable Influence Apoptosis in Experimental factors such as nutrition have a role in its etiology. In experimental tumors, we have observed Mammary Tumors and Human the differential influence of high-fat diets in metabolic pathways, suggesting a different balance in Breast Cancer Cell Lines. Cancers proliferation/apoptosis. In this work, we analyzed the effects of a diet high in n-6 polyunsaturated 2022, 14, 905. https://doi.org/ fatty acids (PUFA) and a diet high in extra-virgin olive oil (EVOO) on the histopathological features 10.3390/cancers14040905 and different cell death pathways in the dimethylbenz(a)anthracene-induced breast cancer model. Academic Editors: Anupam Bishayee The diet high in n-6 PUFA had a stimulating effect on the morphological aggressiveness of tumors and Sanjay Gupta and their proliferation, while no significant differences were found in groups fed the EVOO-enriched Received: 16 September 2021 diet in comparison to a low-fat control group. The high-EVOO diet induced modifications in proteins Accepted: 8 February 2022 involved in several cell death pathways. In vitro analysis in different human breast cancer cell Published: 11 February 2022 lines showed an effect of EVOO minor compounds (especially hydroxytyrosol), but not of fatty acids, decreasing viability while increasing apoptosis. The results suggest an effect of dietary lipids Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in on tumor molecular contexts that result in the modulation of different pathways, highlighting the published maps and institutional affil- importance of apoptosis in the interplay of survival processes and how dietary habits may have an iations. impact on breast cancer risk. Keywords: breast cancer; experimental mammary tumors; high-fat diets; apoptosis; cell death; olive oil; hydroxytyrosol; oleuropein; luteolin Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and 1. Introduction conditions of the Creative Commons Attribution (CC BY) license (https:// Breast cancer is the most common cancer in women worldwide, with high incidence, creativecommons.org/licenses/by/ prevalence, and mortality rates [1]. This neoplasia has a multifactorial etiology, in which 4.0/). diet, as an environmental factor, has an important role. Epidemiological and especially Cancers 2022, 14, 905. https://doi.org/10.3390/cancers14040905 https://www.mdpi.com/journal/cancers Cancers 2022, 14, 905 2 of 20 animal studies have reported an influence of diet and dietary fat on breast cancer [2,3]. In general, saturated fatty acids and n-6 polyunsaturated fatty acids (PUFA) have shown a promoting effect on breast cancer, whereas n-3 PUFA, as well as the ratio of n-3/n-6 PUFA intake, showed an inverse association with breast cancer risk [3]. Regarding monoun- saturated fatty acids (MUFA), evidence is still uncertain, and experimental studies have described effects ranging from a weak promoting effect to a protective effect on mammary carcinogenesis [4]. In this sense, the Mediterranean diet, of which a main source of fat is olive oil (rich in the n-9 MUFA oleic acid), is considered a healthy dietary pattern, with ben- eficial effects on the risks of cardiovascular diseases, obesity, and cancer. Epidemiological studies have demonstrated that adherence to this diet is associated to a reduction of overall and cancer mortality [5–7]. Obtaining insight into the effects that diet and nutrition have on cancer is inherently complex, as nutritional factors would exert a subtle and probably long-term influence in such a multifactorial disease. However, it is of great importance to determine these effects and the mechanisms involved, since exposure to these nutritional factors can occur from childhood and throughout life. Animal experimental models are essential in diet and cancer research, considering that nutrients and other dietary components may play a physiopathological role, interacting at many molecular and cellular levels. We have previously demonstrated in the 7,12-dimethylbenz[α]anthracene (DMBA)-induced model of mammary cancer in rats a differential effect of high-fat diets, with a diet high in n-6 PUFA having a clear tumorigenesis-stimulating effect, while there was a weak influence of the isocaloric diet high in extra-virgin olive oil (EVOO; rich in MUFA and phenolic bioactive compounds) [4,8,9]. These effects were associated with several mechanisms, such as changes in growth and sexual maturation, hormonal status, mammary gland biology, and tumor molecular modifications, including transcriptomic changes modulating the function of cell death-related genes [4,8–13]. We have also recently reported that this high-EVOO diet, in comparison to the high n-6 PUFA diet, induced modifications in tumor metabolic characteristics suggesting a higher glucose uptake, glycolysis rate, and oxidative phosphorylation [13]. Although such metabolic features have been associated with tumor progression [14–16], these tumors displayed a less aggressive phenotype, highlighting that those metabolic characteristics without the context of other pathways, such as proliferation or apoptosis, may not reflect tumor malignancy. Apoptosis is a programmed and ordered cell death process occurring in many phys- iological and pathological conditions. The mechanisms of programmed cell death are complex and driven by many pathways. Caspases are the main involved proteins, as they act as initiators and executioners, and can be activated by three pathways: the extrinsic, the intrinsic, and the endoplasmic reticulum pathway. Apoptosis can also be induced in a caspase-independent manner [17,18]. Reduced cell death, or its resistance, has a key role in carcinogenesis. Cancer cells can disrupt the balance of pro-apoptotic and anti-apoptotic proteins, reduce caspase function, and impair death receptor signaling in order to avoid programmed cell death and sustain uncontrolled growth [18]. Targeting apoptosis mecha- nisms is important in cancer treatment and the basis of many therapeutic approaches [19]. From the field of nutrition, several works are addressing the effect of some phytochemicals on apoptosis as potential prevention and treatment agents in cancer [20], although several factors, such as their bioavailability, are currently important limitations. On the other hand, few studies have addressed the effect of diet on tumor apoptosis in vivo. Understanding the mechanisms by which diet can influence cancer risk is needed to establish prevention and treatment strategies. Thus, the aim of this work is to elucidate the effect that the high-fat diets, high in a seed oil (rich in n-6 PUFA) or in extra-virgin olive oil (rich in oleic acid and bioactive compounds), and with different timings of dietary interventions, may have on the interplay among metabolism, proliferation, and apoptosis, with a special focus on the different apoptotic pathways (Figure S1). In vitro analysis in different human breast cancer cells with different fatty acids (oleic acid, linoleic acid) and minor compounds Cancers 2022, 14, 905 3 of 20 (hydroxytyrosol, oleuropein, and luteolin) were also carried out to acquire insight into the molecular basis of the influence of EVOO on breast cancer. 2. Materials and Methods 2.1. Animals and Experimental Design All animals received humane care under an institutionally approved experimental animal protocol, following the legislation applicable in this country. Female Sprague– Dawley rats (stain Crl: OFA (SD), n = 100) were obtained from Charles River Lab (L’Arbresle Cedex, France) at 23 days of age. Three semisynthetic diets were designed: the low-fat diet (LF; 3% corn oil -w/w-), the high corn oil diet (HCO; 20% corn oil) and the high extra-virgin olive oil diet (HEVOO; 3% corn oil + 17% EVOO). The definition, preparation, and suitability of the experimental diets were previously described in studies [8,21,22]. Animals were distributed in five groups depending on the diet and the timing of dietary intervention (n = 20 each group): the LF diet from weaning (LF group), the HCO diet from weaning (HCO group) or after induction (LF-HCO group), and the HEVOO diet from weaning (HEVOO group) or after induction (LF-HEVOO group). At 53 days of age, mammary tumors were induced by oral gavage with one single dose of 5 mg of DMBA (Sigma-Aldrich, St. Louis, MO, USA) dissolved in corn oil (25 mg DMBA/kg body weight). From day 74 onwards, animals were monitored weekly for the appearance of mammary tumors. At 236–256 days of age, animals were euthanized by decapitation, selecting the rats that were in the diestrus phase of the estrous cycle, and between 10:00 h and 13:00 h to avoid circadian rhythm variability (Figure S2). Tumors were removed, a portion of each was fixed in 4% formalin for histopathological analysis, and the rest was flash-frozen and stored at −80 ◦ C for molecular analysis. Tumor histopathology characterization was determined by applying the Scarff–Bloom– Richardson grading method (SBR3; scoring 1–3), and also the method adapted to rat mammary carcinomas that we have previously described (SBR11; scoring 3–11) [23], with the highest categories indicating the most aggressive tumors. SBR scores include differ- entiation pattern, nuclear pleomorphism, and mitotic index. Number of mitoses were determined in 10 high power fields (HPF) and classified into five categories [23]. Only confirmed mammary adenocarcinomas were included in this study. 2.2. Protein Extraction Tumor samples were homogenized in an extraction 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 Na3 VO4 , 10 µL/mL protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), 10 mM β-mercaptoethanol, and 1% Triton X-100) for total protein extraction. For fractioned ex- tracts, samples were cold centrifugated at 105,000× g for one hour to separate the soluble fraction (corresponding to cytosol) from the particulate fraction, which was further re- suspended in extraction buffer with 0.1% Triton X-100 and centrifuged at 105,000× g (supernatant corresponding to the membranous fraction containing all membranes). Mi- tochondrial extract was obtained from homogenized samples by centrifuging at 1500× g to separate nuclei, followed by supernatant centrifuging at 10,000× g to obtain precipi- tated mitochondria. Protein quantification was determined by Lowry’s method using the commercial DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA) (cytosolic, mitochondrial, and membrane). 2.3. Western Blot The different protein extracts (10–20 µg) were subjected to SDS-PAGE electrophoresis on an acrylamide gel (7.5% or 12% Mini-Protean TGX Stain-Free Gels, Bio-Rad, Hercules, CA, USA) and transferred to a PVDF membrane with Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with 5% BSA or 5% skimmed milk in TBS-0.1% Tween for 1 h at room temperature and incubated with primary antibody overnight at +4 ◦ C. The primary antibodies and dilutions used were: AIF (1:2000), APAF-1 (1:2000), Bak (1:2000), Cancers 2022, 14, 905 4 of 20 Bax (1:2000), Caspase-3 (1:2000), Caspase-8 (1:1000), Caspase-9 (1:1000), Cytochrome C (1:500), p53 (1:500), TNFR1 (1:500), TRADD (1:1000), and XIAP (1:500) from Cell Signaling (Leiden, Holland); Bcl2 (1:1000), Bid (1:3000), EndoG (1:1000), FADD (1:1000), and Fas (1:500) from Santa Cruz Bioechnologies (Dallas, TX, USA); TRAFf2 (1:3000) from ThermoFisher Scientific (Waltham, MA, USA); and Caspase-12 (1:2000) from Abcam (Cambridge, UK). Membranes were incubated with the secondary antibody (anti-mouse or anti-rabbit IgG peroxidase-conjugated) for 1 h at room temperature and developed with Luminata Forte Western HRP Substrate (Merk Millipore, Burlington, MA, USA) luminogen for 3–5 min. Proteins bands were visualized using the Chemidoc XRS+ hardware associated with Image Lab Software 5.1-Beta (Bio-Rad). Densitometric values were normalized with the total protein loaded [24] and relativized to an internal control sample loaded in duplicates in all blots. 2.4. Cell Culture Treatment For in vitro analyses, we used the MCF-7 cell line, representing the molecular subtype of human breast cancer Luminal A, and MDA-MB-231 cells, representing the triple-negative subtype. Cell lines were obtained from the American Type Culture Collection (ATCC; LGC Standards, Middlesex, UK). MCF-7 cells were grown in EMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and supplemented with 10% fetal bovine serum (FBS; Gibco) and 0.01 mg/mL of insulin. MDA-MB-231 cells were grown in DMEM (Gibco) and supplemented with 10% FBS. Cells were maintained at 37 ◦ C in a humidified atmosphere of 95% air and 5% CO2 . The cell cultures were subjected to Mycoplasma screen tests periodically. For fatty acid treatments, oleic acid- or linoleic acid-albumin from bovine serum (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) were used at 0 µM (control), 1 µM, 10 µM, 100 µM, or 1 mM concentration. All the solutions contained 0.1 mg/mL of BSA (Sigma-Aldrich). For treatments with EVOO minor compounds, we used hydroxytyrosol (PHL80152, Sigma Aldrich) at 0 µM (control), 100 µM, 250 µM, and 400 µM; oleuropein (12,247, Sigma Aldrich) at 0 µM (control), 10 µM, 30 µM, and 50 µM; and lutein (L9283, Sigma-Aldrich) at 0 µM (control), 5 µM, 10 µM, and 30 µM [25–27]. All the solutions contained 0.1% of DMSO. 2.5. Viability Assays For analysis of cellular metabolic activity as an indicator of cell viability and prolifera- tion, we used MTT colorimetric assay based on the reduction of thiazolyl blue tetrazolium bromide (Sigma-Aldrich) to formazan. Briefly, MCF-7 cells were seeded in 96-multiwell plates at 6 × 103 cells/well in 100 µL of complete EMEM (Gibco) medium with 5% FBS. MDA-MB-231 cells were seeded in 96-multiwell plates at 5 × 103 cells/well in 100 µL of complete DMEM (Gibco) medium with 5% FBS. After 24 h, the medium was replaced with fresh one containing the specific treatment and incubated for 24 to 72 h. MTT reagent was added to each well (10 µL/well of MTT solution at 5 mg/mL in PBS) and incubated 2 h at +37 ◦ C. Then, formazan crystals were solubilized in 100 µL of 100% DMSO and absorbances were read at 570 nm. Relative cell viability was calculated as the ratio of absorbance of treated samples and control samples. 2.6. Determination of Apoptotic Cells by Flow Cytometry MCF-7 and MDA-MB-231 cells were seeded 24 h before treatment in 12-multiwell plates at 2 × 105 cells/well in 1 mL of complete EMEM and DMEM (Gibco) medium, respec- tively, with 5% FBS. Cells were treated with the specific concentration of oleic acid, linoleic acid, hydroxytyrosol, oleuropein, or lutein for 24, 48, and 72 h. Cells were trypsinized, washed in PBS, and labelled with Annexin-V-Fluorescein and Propidium iodide (PI) using an Annexin-VFLUOS staining kit (Inmunostep, Salamanca, Spain) according to the manu- facturer instructions. Then, data were acquired on a FACSCalibur flow cytometer at SCAC Facility, UAB. Cancers 2022, 14, 905 5 of 20 2.7. Mitochondrial Membrane Potential Assay by JC-1 Staining MCF-7 cells were seeded 48 h before treatment in 6-multiwell plates at 4 × 105 cells/well and treated for 24, 48, and 72 h with 0 or 400 µM of hydroxytyrosol (Sigma Aldrich). Briefly, cells were collected by trypsinization, stained with JC-1 dye (15 µg/mL), and incubated at room temperature for 15 min at 37 ◦ C in a humidified atmosphere of 95% air and 5% CO2 . Data were acquired on a FACSCalibur flow cytometer at SCAC Facility, UAB. 2.8. Cell Cycle Analysis MCF-7 cells were seeded 48 h before treatment in 6-multiwell plates at 3 × 105 cells/well, 2.5 × 105 cells/well and 2 × 105 cells/well and treated for 24, 48, and 72 h respectively with 0 or 400 µM of hydroxytyrosol (Sigma-Aldrich). Cells were collected by trypsinization, fixed with 70% ethanol, and stored at −20 ◦ C. On the analysis day, cells were washed with PBS, labelled with 0.5 mL/1 × 106 cells of FxCycle PI/RNse staining solution (ThermoFisher Scien- tific), and incubated at room temperature for 30 min. Data were acquired on a FACSCalibur flow cytometer at SCAC Facility, UAB. 2.9. Statistical Analysis Statistical analyses were performed using R Deducer. The statistical test to be used was determined depending on the distribution of each variable (Shapiro–Wilk test) and the equality of variances among groups (Levene’s test). Data from tumors usually do not follow a normal distribution, and the non-parametrical Kruskal–Wallis test—Wilcoxon method was used. Quantitative parametrical data were analyzed with one-way ANOVA test (t-test equal variance) and paired t-test. For qualitative data (distributions and percentages) the Pearson’s Chi-squared test was used. The level of significance was established at p < 0.05. For in vivo results, distribution of data is represented using box plots. Box indicates interquartiles (IQR; first quartile Q1, second quartile = median, and third quartile Q3); whiskers extend from maximum (Q3 + 1.5 × IQR) to minimum (Q1 − 1.5 × IQR); dots represent outliers; and cross indicates the mean value. 3. Results 3.1. The High-Corn Oil Diet Influenced the Manifestation of the Disease Carcinogenesis parameters are shown in Table 1. The tumor latency period (days from induction to first palpable malignant tumor) was shorter in the HCO and LF-HCO groups, although differences did not reach statistical significance. On the other hand, the percentage of tumor-bearing animals and total number of tumors were higher in the groups fed the HCO diet in comparison to the LF and HEVOO groups. Tumor multiplicity (mean number of tumors per animal) was also increased in the HCO group in comparison to the LF group. Table 1. Effect of high-fat diets on DMBA-induced tumorigenesis in rats. Parameter LF HCO LF-HCO HEVOO LF-HEVOO Latency period (days, mean ± SE) 98.6 ± 11.9 81.8 ± 9.9 76.4 ± 9.3 86.4 ± 10.3 94.8 ± 6.8 Tumor incidence (%) 80 100 a,b 100 a,b 75 85 (Tumor-bearing rats/total) 16/20 20/20 20/20 15/20 17/20 Total number of tumors 46 100 a,b 87 a 57 83 a Tumor multiplicity 2.3 ± 0.5 5.0 ± 0.7 a 4.4 ± 0.9 2.9 ± 0.8 4.2 ± 1.0 (no. tumors/animal, mean ± SE) a : p < 0.05 in comparison to control LF group; b : p < 0.05 in comparison to HEVOO group. Continuous variables (latency time, tumor multiplicity): Kruskal–Wallis test. Categorical variables (tumor incidence, total number of tumors): Pearson’s Chi-squared test. Tumor multiplicity(no. 2.3 ± 0.5 5.0 ± 0.7 a 4.4 ± 0.9 2.9 ± 0.8 4.2 ± 1.0 tumors/animal, mean ± SE) a:p < 0.05 in comparison to control LF group; b: p < 0.05 in comparison to HEVOO group. Continuous variables (latency time, tumor multiplicity): Kruskal–Wallis test. Categorical varia Cancers 2022, 14, 905 (tumor incidence, total number of tumors): Pearson’s Chi-squared test. 6 of 20 3.2. The High-Corn Oil Diet, but Not the High-EVOO Diet, Had a Clear Effect on Histopathological Malignancy and Proliferation of Tumors 3.2. The High-Corn Oil Diet, but Not the High-EVOO Diet, Had a Clear Effect on Results forand Histopathological Malignancy theProliferation morphological degree of tumor aggressiveness and proliferation of Tumors Results for the morphological degree of tumoranalyses shown in Figure 1. Histopathological indicated aggressiveness and a proliferation higher degreeare of maligna (both in SBR3 and SBR11 score) in the groups fed the HCO shown in Figure 1. Histopathological analyses indicated a higher degree of malignancy diet in comparison to (both in SBR3 groups and SBR11fed score) the control in theand high-EVOO groups fed the HCOdiets. A in diet histological comparison quantitation to the groupsof prolifera fed the control and high-EVOO diets. A histological quantitation of proliferation indicated espec indicated a higher mitosis score in the high-corn oil diet groups versus control, in score a higher mitosis LF-HCO,in theand a greatoil high-corn number of mitoses diet groups versusincontrol, both HCO-fed especiallygroups in relation to in LF-HCO, control. LF-HCO also showed a great number of mitoses in comparison and a great number of mitoses in both HCO-fed groups in relation to the control. LF-HCO to the high-EV diet groups. also showed a great number of mitoses in comparison to the high-EVOO diet groups. Figure 1. Effect of high-fat diets on the degree of aggressiveness and proliferation of experimental mammary tumors. (A) Histopathological classification of tumors. Images show representative DMBA- induced mammary adenocarcinomas with different degree of morphological aggressiveness. Stacked bar chart depicts the distribution of mammary adenocarcinomas of each group according to the Scarff– Bloom–Richardson index, scoring tumors in three categories (SBR3), with the highest representing the most aggressive. Box and whisker plot depicts data of the modified Scarff–Bloom–Richardson index (SBR11) of each tumor. (B) Mitosis study. Images show representative determination of mitoses in tumors with different mitosis score. Stacked bar chart depicts the distribution of mammary adenocarcinomas of each group according to the mitosis score. Box and whisker plot depicts data of the number of mitoses in 10 high power field (HPF, 400× magnification) for each tumor. Arrows connecting groups indicate differences statistically significant (p < 0.05), Chi-squared test (distribution data: SBR3 and mitosis score), non-parametric Mann–Whitney U test (quantitative data: SBR11 and number of mitoses). LF, n = 46; HCO, n = 100; LF-HCO, n= 87; HEVOO, n = 57; LF-HEVOO, n = 82. Scale bar. representative determination of mitoses in tumors with different mitosis score. Stacked bar chart depicts the distribution of mammary adenocarcinomas of each group according to the mitosis score. Box and whisker plot depicts data of the number of mitoses in 10 high power field (HPF, 400× magnification) for each tumor. Arrows connecting groups indicate differences statistically significant (p < 0.05), Chi-squared test (distribution data: SBR3 and mitosis score), non-parametric Cancers 2022, 14, 905 Mann–Whitney U test (quantitative data: SBR11 and number of mitoses). LF, n = 46; HCO, n =7100; of 20 LF-HCO, n= 87; HEVOO, n = 57; LF-HEVOO, n = 82. Scale bar. 3.3. The EVOO-Enriched Diet Promoted the Expression of Extrinsic Apoptosis Pathway Proteins 3.3. The EVOO-Enriched Diet Promoted the Expression of Extrinsic Apoptosis Pathway Proteins The protein expression levels of extrinsic pathway-related proteins (Fas, FADD total The protein expression levels of extrinsic pathway-related proteins (Fas, FADD total and membrane fraction, TNFR1, TRADD membrane fraction, TRAF2, pro-Caspase-8, and and membrane fraction, TNFR1, TRADD membrane fraction, TRAF2, pro-Caspase-8, and cleaved-Caspase-8) were studied by Western blot and the results are shown in Figure 2 cleaved-Caspase-8) were studied by Western blot and the results are shown in Figure 2 (Original Western blots in Supplementary material). Changes in protein expression levels (Original Western blots in Supplementary material). Changes in protein expression levels were observed in FADD, TNFR1, and pro-Caspase-8 with higher levels in the LF-HEVOO were observed in FADD, TNFR1, and pro-Caspase-8 with higher levels in the LF-HEVOO group groupwhen whencompared compared to to LF LF group. When group. comparing When between comparing high-fat between diets, FADD high-fat (total diets, FADD and (total and membrane fraction), TNFR1, and pro-Caspase-8 showed higher levels inLF- membrane fraction), TNFR1, and pro-Caspase-8 showed higher levels in the the HEVOO group versus LF-HCO group, while the HEVOO group showed LF-HEVOO group versus LF-HCO group, while the HEVOO group showed higher FADD higher FADD and andTRADD TRADDinincomparison comparisontotothetheHCO HCOgroup. group.Higher Higherexpression expressionlevels levelsofofTNFR1 TNFR1and and pro-Caspase-8 were also found in LF-HEVOO versus pro-Caspase-8 were also found in LF-HEVOO versus HEVOO.HEVOO. Figure 2. Effect of high-fat diets on the expression of apoptotic extrinsic pathway proteins in ex- perimental tumors. (A) Detection of proteins by Western blot in the different groups. (B) Box plots represent distribution data for Fas, FADD (total and membrane), TNFR1, TRADD (membrane), TRAF2, and Caspase-8 (pro- and cleaved-Casp8). FADD (membrane) and TRADD (membrane) were detected in the membrane fraction, the other proteins were detected in total protein extract. Box: 25th percentile, median, 75th percentile; whiskers: maximum to minimum; dots: outliers; cross: mean value. Solid arrows connecting groups indicate statistically significant differences (p < 0.05), dashed lines indicate differences close to significance (p < 0.1), Kruskal–Wallis test. 3.4. The EVOO-Enriched Diet Increased the Expression of Intrinsic Apoptosis Pathway Proteins We have also studied proteins involved in the apoptotic intrinsic pathway: the pro- apoptotic Bid and the active tBid, Bak, Bax, Cytochrome C in mitochondrial and cytoplasm fraction, APAF, pro-Caspase-9, cleaved-Caspase-9, and the anti-apoptotic Bcl2 (Figure 3 and Original Western blots in Supplementary material). Relative protein expression levels of the total Bid, tBid, and Cytochrome C (membrane fraction) were higher in the LF-HEVOO 3.4. The EVOO-Enriched Diet Increased the Expression of Intrinsic Apoptosis Pathway Proteins We have also studied proteins involved in the apoptotic intrinsic pathway: the pro- apoptotic Bid and the active tBid, Bak, Bax, Cytochrome C in mitochondrial and Cancers 2022, 14, 905 cytoplasm fraction, APAF, pro-Caspase-9, cleaved-Caspase-9, and the anti-apoptotic 8 of 20Bcl2 (Figure 3 and Original Western blots in Supplementary material). Relative protein expression levels of the total Bid, tBid, and Cytochrome C (membrane fraction) were higher in theinLF-HEVOO group than the LF group.group This than in the LF LF-HEVOO group. group alsoThis LF-HEVOO showed group higher levels of also totalshowed Bid, higher tBid, Bak, and Bax when compared to the HCO-fed groups, and those of Bid and Bak whenand levels of total Bid, tBid, Bak, and Bax when compared to the HCO-fed groups, those of Bid compared toand Bak when the HEVOO compared to the HEVOO group. group. 3. Effect Figure 3. Figure Effectofofhigh-fat diets high-fat on the diets on expression of apoptotic the expression intrinsicintrinsic of apoptotic pathwaypathway proteins inproteins exper- in experimental imental tumors.tumors. (A) Detection (A) Detection of proteins of proteins by Western by Western blot blot in theindifferent the different groups. groups. (B) Box (B) Box plotsplots represent distributiondata represent distribution dataforfor Bid Bid (total (total andand 22 kDa, 22 kDa, andactive and the the active truncated truncated 18 kDa 18 kDaBak, tBid), tBid), Bax,Bak, Bax, Bcl2,Bcl2, Cytochrome Cytochrome C (cyt.C) C (cyt.C) in membrane in membrane and cytosol, and cytosol, APAF in APAF in cytosol, cytosol, and Caspase-9 and Caspase-9 (pro- and(pro- and cleaved-Casp9). cleaved-Casp9). Cyt.cCyt.c (membrane) (membrane) was detected was detected in membrane in membrane fraction, fraction, Cyt.C and Cyt.C (cytosol) (cytosol) APAF and (cytosol) were detected in cytosol fraction, the other proteins were detected in total protein extract. Box: 25th percentile, median, 75th percentile; whiskers: maximum to minimum; dots: outliers; cross: mean value. Solid arrows connecting groups indicate statistically significant differences (p < 0.05), dashed lines indicate differences close to significance (p < 0.1), Kruskal–Wallis test. 3.5. Effect of High-Fat Diets on Endoplasmic Reticulum (ER) Stress-Induced Cell Death and in Apoptosis Pathways Convergence Results for the Caspase-12 protein, which is involved in ER stress-induced cell death, are shown in Figure 4A and in Original Western blots in Supplementary material. There was a trend in the HCO group to have lower levels of pro-Caspase-12, while a trend to have higher levels of the active cleaved form was observed in the LF-HCO group. was a trend in the HCO group to have lower levels of pro-Caspase-12, while a trend to have higher levels of the active cleaved form was observed in the LF-HCO group. On the other hand, the protein expression levels of pro-Caspase-3 and active Caspase-3 are shown in Figure 4B and in Supplementary material. There was a trend to have increased levels of 17 kDa cleaved-Caspase-3 in the LF-HEVOO group compared to Cancers 2022, 14, 905 the LF-HCO and HEVOO groups. The Caspase-3 inhibitor, XIAP, was increased in the 9 of 20 LF- HEVOO group in comparison to LF-HCO group. Figure 4. Effect of high-fat diets on the expression of apoptotic proteins in experimental tumors. (A) Detection of proteins by Western blot in the different groups. (B) ER stress-induced apoptosis protein Caspase-12 (pro- and cleaved-Casp12). (C) The key protein in confluence of apoptotic pathways Caspase-3 (pro-Casp3 and the 19 KDa and 17 Da cleaved forms), and its inhibitor XIAP. (D) Caspase- independent cell death proteins AIF (total in mitochondria and active cytosolic AIF levels) and EndoG (mitochondria and cytosolic). (E) Total protein levels of p53. AIF and EndoG were detected in mitochondria fraction and total protein extract, the other proteins were detected in total protein extract. Box: 25th percentile, median, 75th percentile; whiskers: maximum to minimum; dots: outliers; cross: mean value. Solid arrows connecting groups indicate statistically significant differences (p < 0.05), dashed lines indicate differences close to significance (p < 0.1), Kruskal–Wallis test. On the other hand, the protein expression levels of pro-Caspase-3 and active Caspase-3 are shown in Figure 4B and in Supplementary material. There was a trend to have increased levels of 17 kDa cleaved-Caspase-3 in the LF-HEVOO group compared to the LF-HCO and HEVOO groups. The Caspase-3 inhibitor, XIAP, was increased in the LF-HEVOO group in comparison to LF-HCO group. Cancers 2022, 14, 905 10 of 20 3.6. Effect of High-Fat Diets on Caspase Independent Cell Death and on p53 We studied AIF and EndoG as proteins related to caspase-independent cell death (Figure 4C and Original Western blots in Supplementary material). The total AIF (67 KDa) in mitochondria was decreased in the HCO group in comparison to the LF group. Active AIF (57 KDa) in cytosol was detected in a few samples, with a higher frequency (Pearson’s Chi-squared test, p < 0.05) in the LF-HEVOO group. EndoG levels in mitochondria were significantly higher in the LF-HEVOO and HEVOO groups compared to the LF-HCO group, while there was a trend toward higher levels of cytosolic EndoG in the LF-HEVOO group. In relation to p53, total levels were significantly higher in the LF-HEVOO group in comparison to all other high-fat diet groups (Figure 4D and Supplementary material). 3.7. Effect of Oleic and Linoleic Acids on Viability and Apoptosis in Breast Cancer Cells MDA-MB-231 and MCF-7 cells were treated with oleic and linoleic acid at different concentrations (1, 10, 100, and 1000 µM) for 24 h, 48 h, and 72 h. As shown in Figure 5A, oleic and linoleic acids had no effects on MCF-7 cell viability. In MDA-MB-231 cells, both fatty acids had a slight stimulating effect compared to the control (increased viability with oleic acid at 10 µM and 1 mM for 72 h, and with linoleic acid at 100 µM after 24 h Cancers 2022, 14, 905 of treatment). Regarding apoptosis (Figure 5B), no effects on MCF-7 were observed. 11 In of 21 MDA-MB-231, both oleic and linoleic acids decreased the percentage of apoptotic cells after 72 h of treatment with the highest doses (Figure 5B). Figure 5. Cont. Cancers 2022, 14, 905 11 of 20 Figure Figure 5. Effects 5. Effects of acid of fatty fatty acid treatments treatments on cell on cell viability viability andandapoptosis apoptosisin in human human breast breastcancer cancercell cell lines. lines. (A) Cell(A) Cell viability viability analysis analysis using using the the MTT MTT assay assay of of MDA-MB-231and MDA-MB-231 and MCF-7 MCF-7 cell celllines linestreated treated with different with different dosesdoses of oleic of oleic acid acid (OA) (OA) andand linoleic linoleic acid acid (LA)for (LA) for24 24h, h, 48 48 h, h, or or 72 72h.h.(B) (B)Apoptosis Apoptosis analyses by flow-cytometry assay of Annexin V/propidium iodide (PI) double staining.Picture analyses by flow-cytometry assay of Annexin V/propidium iodide (PI) double staining. Picture shows shows a representative a representative imageof image of dot dotplot diagrams plot diagramsidentifying cell population identifying stained withstained cell population Annexin-Vwith (AnxV, Annexin-V green)green) (AnxV, and PI (red). and PIFor MDA-MB-231 (red). and MCF-7, For MDA-MB-231 graphics and MCF-7, show the results graphics show forthe cellsresults in earlyfor cells inapoptosis (AnxV+/PI early apoptosis −), late apoptosis (AnxV+/PI−), (AnxV+/PI+), late apoptosis and total apoptosis (AnxV+/PI+), apoptosis±(AnxV+/PI±), and total(AnxV+/PI ), relative to control cells. Mean + standard deviation of three–five independent experiments. *: p < 0.05 compared to control cells (0.1 mg/mL of BSA). 3.8. EVOO Main Polyphenols Affected Cell Viability in Breast Cancer Cells MDA-MB-231 and MCF-7 cells were treated with different doses of hydroxytyrosol (HT), oleuropein (OLE), and luteolin (LUT) for 24, 48, and 72 h. Figure 6 depicts the comparison of treated cells versus control cells (treated only with 0.1% DMSO). HT had a time- and dose-dependent effect, decreasing cell viability both in MDA-MB-231 and MCF-7 cells. At the highest dose (400 µM), HT decreased viability at 48 h and 72 h in both cell lines. At 72 h, viability was also decreased with 100 µM (MDA-MB-231) and 250 µM (MDA-MB-231 and MCF-7). By 72 h, OLE at 50 µM decreased viability in both cell lines, and in MCF-7 there was also a significant effect at 30 µM after 48 h and 72 h. LUT did not decrease viability at the doses used in these cell lines; we only observed an increase in MDA-MB-231 with 5 µM by 24 h. A comparison among different doses and times of treatment are indicated in Figure S3. In both lines, cells treated with 400 µM of HT for 48 h and 72 h showed a significant decrease in the cell viability in comparison to cells treated with 100 µM and 250 µM. At 400 µM there were also a time-dependent effect, especially in MDA-MB-231 cells (lower viability at higher times of treatment). MDA-MB-231 cells treated with OLE 50 µM for 72 h had a decreased viability in comparison to cells treated with OLE at 30 µM. The highest concentration of LUT (30 µM) for 72 h also decreased cell viability in comparison to LUT at 5 µM and 10 µM. Cancers 2022, 14, 905 13 of 21 Cancers 2022, 14, 905 12 of 20 A 200 MDA-MB-231 MCF-7 250 24h cell viability (%) 150 200 48h * 150 72h 100 * 100 50 * * 50 * * 0 * 0 * 0 100 250 400 0 100 250 400 HT (µM) HT (µM) B 140 MDA-MB-231 120 MCF-7 24h 48h cell viability (%) 120 100 72h 100 80 * * * 80 * 60 60 40 0 10 30 50 0 10 30 50 OLE (µM) OLE (µM) C 140 MDA-MB-231 200 MCF-7 24h 48h cell viability (%) 150 120 * 72h 100 100 80 50 60 0 0 5 10 30 0 5 10 30 LUT (µM) LUT (µM) Figure Figure6.6.Effect EffectofofEVOO EVOOminor minorcompounds compoundson oncell cellviability viabilityininMDA-MB-231 MDA-MB-231and andMCF-7 MCF-7cell celllines lines treated treatedwith withdifferent differentdoses dosesfor for24 24h,h,48 48h,h,or or72 72h,h,relative relativetotocontrol controlcells cells(0(0μM). µM).(A) (A)Hydroxytyrosol Hydroxytyrosol (HT). (HT).(B) (B)Oleuropein Oleuropein(OLE). (OLE).(C) (C)Luteolin Luteolin(LUT). (LUT).MeanMean++standard standarddeviation deviationof ofthree threeindependent independent experiments. *: p < 0.05 compared to control cells (0.1% DMSO-treated). experiments. *: p < 0.05 compared to control cells (0.1% DMSO-treated). 3.9.Hydroxytyrosol 3.9. HydroxytyrosolPromoted PromotedApoptosis Apoptosisin inBreast BreastCancer CancerCell CellLines Lines AnnexinV/PI Annexin V/PIstaining stainingandandflow flowcytometry cytometrywere wereused usedtotoevaluate evaluatethetheeffect effectofofEVOO EVOO polyphenols in cell apoptosis. MDA-MB-231 and MCF-7 cells were polyphenols in cell apoptosis. MDA-MB-231 and MCF-7 cells were treated with increasing treated with increasing dosesof doses ofHT, HT,OLE,OLE,andandLUTLUTfor for24 24hhandand48 48h.h. Figure Figure77shows showsresults resultsfor forearly earlyapoptosis apoptosis (annexin positive/propidium iodide negative-labeled cells) and (annexin positive/propidium iodide negative-labeled cells) and late apoptosis (annexin late apoptosis (annexin positive/propidiumiodide positive/propidium iodidepositive-labeled positive-labeledcells),cells),and andaapercentage percentageofofliveliveand andapoptotic apoptotic cellsfor cells foreach eachtreatment treatmentin inMDA-MB-231 MDA-MB-231(Figure (Figure7A,C,E) 7A,C,E)andandMCF-7 MCF-7(Figure (Figure7B,D,F). 7B,D,F).HT HT increased early apoptosis in both cell lines (especially at 48 h in MDA-MB-231, increased early apoptosis in both cell lines (especially at 48 h in MDA-MB-231, Figure 7A, Figure 7A, and at the highest dose after 24 h and 48 h in MCF-7, Figure 7B). The and at the highest dose after 24 h and 48 h in MCF-7, Figure 7B). The highest dose (400 highest dose (400 µM) also also μM) increased late late increased apoptosis afterafter apoptosis 48 h48 in hMDA-MB-231 in MDA-MB-231 cells.cells. A comparison A comparison of the per- of the centage of live/apoptotic cells among doses showed a significant dose-effect percentage of live/apoptotic cells among doses showed a significant dose-effect after 48 h after 48 h in both cell lines, with a higher percentage of apoptotic cells in 400 µM-treated in both cell lines, with a higher percentage of apoptotic cells in 400 μM-treated cells cells compared to all other doses. Few differences were found with OLE and LUT treatments, with no clear trend. Cancers 2022, 14, 905 14 of 21 Cancers 2022, 14, 905 compared to all other doses. Few differences were found with OLE and LUT treatments, 13 of 20 with no clear trend A B Early apoptosis Late apoptosis Early apoptosis Late apoptosis 12 5 100 2 * 3 100 10 4 * * Relative to control Relative to control Relative to control Relative to control 8 % of cells % of cells 3 2 6 * 1 50 50 2 4 * 1 2 1 0 0 0 0 0 0 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h HT (µM) HT (µM) HT (µM) HT (µM) HT (µM) HT (µM) live cells live cells apoptotic cells apoptotic cells C Early apoptosis Late apoptosis D Early apoptosis Late apoptosis 14 2 2 2 100 100 12 Relative to control Relative to control Relative to control Relative to control 10 % of cells % of cells 8 1 50 1 1 50 6 * 4 2 0 0 0 0 0 0 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h OLE (µM) OLE (µM) OLE (µM) OLE (µM) OLE (µM) OLE (µM) live cells live cells apoptotic cells apoptotic cells E Early apoptosis Late apoptosis F Early apoptosis Late apoptosis 2 2 2 2 100 100 Relative to control Relative to control Relative to control Relative to control % of cells 1 1 * % of cells 1 1 * 50 50 0 0 0 0 0 0 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h LUT (µM) LUT (µM) LUT (µM) LUT (µM) LUT (µM) LUT (µM) live cells live cells apoptotic cells apoptotic cells Figure 7. Effect of treatment with HT, OLE, and LUT on apoptosis in MDA-MB-231 and MCF- Figure 7. Effect of treatment with HT, OLE, and LUT on apoptosis in MDA-MB-231 and MCF-7 cell 7 cell Apoptosis lines. lines. Apoptosis analysesanalyses by flow-cytometry by flow-cytometry assay ofassay of Annexin Annexin V/propidiumV/propidium iodide iodide (PI) (PI) double double staining staining in MDA-MB-231 in MDA-MB-231 (A,C,E) (A,C,E) and MCF-7 and MCF-7 (B,D,F) (B,D,F) cellscells treated treated withdifferent with differentdoses doses of of hydroxytyrosol (A,B), hydroxytyrosol (A,B), oleuropein oleuropein (C,D), (C,D), or luteolin luteolin (E,F) for 24 h or 48 h. For each treatment and cell line, results show values values of of early early apoptosis apoptosis compared compared toto control control cells cells (0.1% (0.1%DMSO-treated DMSO-treated cells), cells), values of late apoptosis compared control cells, compared to control cells, and percentage percentage ofof live/apoptotic cells. Histograms Histograms show show mean mean ++ standard standard deviation deviation of of three three independent experiments. *: independent experiments. *: pp << 0.05 0.05 compared compared toto control control cells (0.1% DMSO-treated). Solid lines connecting bars indicate statistically significant cells (0.1% DMSO-treated). Solid lines connecting bars indicate statistically significant differences differences between doses. between doses. 3.10. Hydroxytyrosol Did Not Significantly Affect Cell Cycle or Mitochondrial Membrane Potential in MCF-7 Further analysis on the effect of HT was carried out on MCF-7 cells. We studied by flow cytometry the cycle of cells treated with HT at 400 µM for 24, 48, and 72 h (Figures 8A and S4A). On the other hand, mitochondrial membrane potential was an- alyzed by staining with JC-1, a cationic dye indicator of potential-dependent accumulation in the mitochondria (Figures 8B and S4B). No statistically significant differences were observed in the HT treatment. Further analysis on the effect of HT was carried out on MCF-7 cells. We studied by flow cytometry the cycle of cells treated with HT at 400 μM for 24, 48, and 72 h (Figures 8A and S4A). On the other hand, mitochondrial membrane potential was analyzed by staining with JC-1, a cationic dye indicator of potential-dependent accumulation in the Cancers 2022, 14, 905 14 of 20 mitochondria (Figures 8B and S4B). No statistically significant differences were observed in the HT treatment. Effect Figure8.8.Effect Figure of of treatment treatment with with HTHT on cell on cell cycle cycle andand membrane membrane depolarization depolarization in MCF-7 in MCF-7 cells. cells. (A) (A) Flow Flow cytometry cytometry DNA DNA content content analysis analysis of cells of cells stained stained withpropidium with propidiumiodide iodide(G0/G1 (G0/G1and andG2/M G2/M phase phasepeaks peaksareareseparated separatedbybythe theS-phase). S-phase).Distribution Distributionof ofcells cells in in G0/G1, G0/G1,S,S,and andG2/M G2/Minincontrol control and and400 400μM µMHT-treated HT-treated MCF-7 MCF-7cells of three cells independent of three independent experiments. experiments.(B) Flow cytometry (B) Flow dot plot cytometry dot showing the gating of JC1 aggregates (live cells, red fluorescence) and JC1 monomer plot showing the gating of JC1 aggregates (live cells, red fluorescence) and JC1 monomer (low mito- (low mitochondrial membrane chondrial membrane potential, potential, apoptotic apoptotic cells, andcells, greenand green fluorescence) fluorescence) populations. populations. Percentage of Percentage of live/apoptotic cells in control and 400 μM HT-treated MCF-7 cells of three live/apoptotic cells in control and 400 µM HT-treated MCF-7 cells of three independent experiments. independent experiments. 4. Discussion 4. Discussion In this work, we aimed to analyze the effects of two high-fat diets, rich in n-6 PUFA In this or rich work, we in EVOO, aimed on the to analyze the characteristics effects of the of two high-fat aggressiveness anddiets, rich in n-6 proliferation PUFA of DMBA- orinduced rich in EVOO, mammary on the characteristics tumors, of the and to obtain aggressiveness insight and proliferation into the molecular mechanisms of DMBA- of such induced effects. mammary tumors, anddemonstrated We have previously to obtain insight into the molecular a different influence of mechanisms these dietsofon suchthe effects. We have previously demonstrated a different influence of manifestation of experimental tumors, clearly stimulating in the case of the high n-6 these diets on the manifestation PUFA diet, and of experimental tumors, clearly with the high-EVOO stimulating diet having a weakin the case of the influence high n-6 [4,9–12]. PUFA Here, we diet, haveand with the observed thathigh-EVOO tumors from dietboth having groupsa weak influence fed with [4,9–12]. the high Here, diet, n-6 PUFA we havefrom observed weaningthat (HCOtumors group) from both induction or after groups fed(LF-HCO with the group), high n-6displayed PUFA diet, thefrom weaning highest tumor histopathological (HCO group) or grade, after indicating induction more aggressiveness (LF-HCO [23]. The number group), displayed of mitoses the highest tumor and the mitosis score grade, histopathological were also higher in indicating suchaggressiveness more groups. On the[23]. other Thehand, tumors number from groups of mitoses and fedmitosis the with the high-EVOO score diet were were also higher not groups. in such significantly On the different to those other hand, tumorsfromfromthegroups control low-fat group. Considering that high-fat intake is associated with fed with the high-EVOO diet were not significantly different to those from the controlan increase in breast cancer group. low-fat risk [28,29], and thatthat Considering bothhigh-fat experimental intake high-fat diets with is associated were anisocaloric, increaseour results in breast suggest cancer a specific risk [28,29],beneficial and that effect of the EVOO that both experimental can partially high-fat diets werecounteract theour isocaloric, detrimental results impact of suggest a an excessive specific intake of beneficial fat. of the EVOO that can partially counteract the effect detrimental impact of an excessive may The effects that dietary lipids intakeexert on mammary tumorigenesis are likely driven of fat. by multiple and complex molecular mechanisms acting simultaneously. We have recently demonstrated that these high-fat diets induced metabolic changes depending on the type and timing of dietary intervention [13]. The high-EVOO diet, in comparison to the high n-6 PUFA diet, induced modifications suggesting an increase in the glucose uptake and glycol- ysis, pentose phosphate pathway (PPP), tricarboxylic acid cycle, and energy dissipation by UCP2. Although these pathways have been reported as deregulated in cancer [14–16], the clinical and morphological characterization of tumors showed that such changes were not associated to aggressiveness. Our data also pointed out that the relevance of metabolic changes depend on the interplay with other signaling pathways, such as apoptosis [30]. In this sense, in a previous transcriptomic analysis, we have observed different effects of Cancers 2022, 14, 905 15 of 20 these high-fat diets on the expression profiles of genes related to metabolism, proliferation, and apoptosis pathways [9]. Thus, we have analyzed in the experimental tumors the effect of diets on the protein levels of several pathways leading to cell death: intrinsic, extrinsic, ER stress-induced, and caspase-independent (Figure S1). The results showed that several pro-apoptotic proteins increased in the tumors from animals fed the EVOO diet, especially in the LF-HEVOO group. In this group, we found higher levels of the proteins of the extrinsic apoptosis pathway TNFR1, FADD, and pro-Caspase-8, although there were no differences in active-Caspase-8 levels. On the other hand, several mitochondrial proteins with a role in the intrinsic apoptosis pathway were also increased (tBid, Bax, Bak, and Cytochrome C), and no differences were found in cytoplasmic Cytochrome C or active- Caspase-9. Caspase-12, involved in ER stress-induced apoptosis, showed a tendency to be increased in the LF-HEVOO group, and the active-Caspase-12 increased in the LF-HCO group. Caspase-3, the key protein in the confluence of the apoptotic pathways, showed no marked changes, which could be related to the increase in its inhibitor XIAP. Despite this, a statistical trend towards higher levels of 17 kDa cleaved-Caspase-3 was found in the LF-HEVOO group. Regarding the caspase-independent pathway, membrane EndoG levels were increased in such EVOO diet groups, also showing a trend towards higher levels of cytosolic EndoG. Although few samples showed active AIF, the percentage of tumors with detectable cytoplasmatic levels of active AIF was significantly higher in the LF-HEVOO group. Finally, we also observed significantly higher levels of the p53 protein in this group. Taken together, all these results raise several hypotheses. One is that, given the complexity and heterogeneity of the experimental tumors, the set of results is much more informative than the specific proteins, so these data suggest greater cell death due to the effect of the high-EVOO diet through several pathways. Another possibility is that despite observing increases in various pro-apoptotic proteins, anti-apoptotic proteins are also upmodulated, thus some effectors are not increased. Additionally, little changes in these proteins are enough to contribute to the aggressive phenotype in the context of other pathways, and methodological issues cannot be ruled out. In any case, the analysis of all these proteins and pathways suggests that cell death could be increased in the LF-HEVOO group (which displays a tendency towards greater levels of cleaved-Caspase-3, active AIF, EndoG, and p53), which is consistent with the histopathological characteristics of this group. The fact that the same results have not been found in the group fed this diet from weaning (HEVOO) is consistent with the greater volume of their tumors [9,12]. It is also concordant with metabolic changes observed in the HEVOO group, i.e., an increase in glycolysis and PPP, which has been associated with a decrease in apoptosis [13]. This metabolic profile favors NADPH generation, which may lead to caspase inhibition [31,32]. Further analyses were performed with the components of the oils in different cell lines characteristic of different molecular subtypes of human breast cancer: MDA-MB-231, representing the triple-negative molecular subtype, and MCF-7, representing the Luminal A subtype. MCF-7 is a non-aggressive, hormone receptor-positive, and non-invasive cell line, while the MDA-MD-231 cell line is an estrogen receptor-, progesterone receptor-, and HER2- negative, highly proliferative and invasive cell line [33]. In accordance, the proliferation rate was higher in MDA-MD-231 cells, while the percentage of cells in apoptosis was lower in comparison to MCF-7 cells. We analyzed cell viability and apoptosis in cells treated with the main fatty acids of the two oils used in vivo: oleic acid (main fatty acid of EVOO) and linoleic acid (main fatty acid of corn oil). In MDA-MB-231, but not in MCF-7, both fatty acids had a slight effect on viability, while the higher doses after 72 h decreased apoptosis. It is likely that the effect of oleic acid depends on time, dose, and the type of cell. It has been reported in other cell lines that oleic acid treatment induced apoptosis [34,35], but it has also been shown to increase proliferation, migration, or invasion in cancer cells [36,37]. It has been suggested that the heterogeneity in fatty acid metabolism may be at the basis of the different response of cell lines to fatty acid treatments [38]. On the other hand, we analyzed the effect of some of the main minor compounds of EVOO: the secoiridoid oleuropein (OLE), its metabolite hydroxytyrosol (HT), and the main flavonoid luteolin Cancers 2022, 14, 905 16 of 20 (LUT) [39]. HT and OLE had a dose- and time-dependent effect, decreasing cell viability on both MDA-MB-231 and MCF-7 cells. HT also stimulated apoptosis in both cell lines, while little effect was observed with OLE and LUT. The preliminary results with HT have not shown changes by this polyphenol in cell cycle or membrane potential, the latter suggesting that apoptosis would not be led from the intrinsic pathway, although further analyses are needed to elucidate the involvement of the different cell death pathways. The results with hydroxytyrosol and oleuropein agree with those described by other authors in relation to their anti-proliferative and apoptotic properties [40,41]. On the contrary, with the doses used, and in these cell lines, we did not find a significant effect of luteolin on viability and apoptosis, although other authors reported anti-inflammatory and antioxidant effects [42], and tumor suppressor activity in DMBA-induced tumors [43,44]. Recently, a mitogenic effect of oleic acid has also been observed in colon cancer cells, while the effect of several minor compounds of EVOO was the opposite [45], which highlights the importance of the specific composition of the oils consumed and the complexity of the interaction of nutritional factors in vivo. All this data suggest that dietary fats have a molecular effect on tumors that influences their histopathological degree and proliferation through various mechanisms, specific and nonspecific, that interact with each other, creating a myriad of responses. The results are consistent, for example, with changes in the membrane composition of tumors as a function of the type of lipid consumed [46,47], concomitantly with a specific effect of different minor compounds, which would have effects in different signaling pathways heterogeneously. Thus, we have observed increases in the EVOO groups in the proteins involved in the extrinsic apoptosis pathway, in which changes in microdomains can have a role, and in intrinsic and caspase-independent pathways, which can be also related to the effect of EVOO minor compounds. In this sense, we have previously observed changes in p21Ras location and activity, which has been related to an oleic acid-enrichment of membranes that can modify microdomains and thus several signaling pathways [12,46]. In addition, our in vitro results, both in hormone receptor-positive (representing Luminal A human breast cancer) and hormone receptor-negative (representing the triple-negative subtype) cells, suggest that HT and OLE are able to influence different breast cancer cell lines through estrogen receptor-independent pathways. In the case of the rodent DMBA model, the mammary tumors developed reflect the most common less-aggressive human breast cancer, being hormone-sensitive, low proliferative, and with no overexpression of HER2 [23,48]. In any case, data in the literature suggest several mechanisms of action of EVOO minor compounds, including hormone receptor-dependent and -independent effects [49]. Many EVOO components have shown anticancer activities by modulating gene expression, the direct activation/inhibition of proteins and enzymes, or antioxidant activity, which may in turn regulate apoptosis [49,50]. The results also showed the effect of the timing of dietary intervention. High-fat diets were administered from weaning (groups HCO and HEVOO), or after induction (groups LF-HCO and LF-EVOO). Both groups fed the high-EVOO diet had lower clinical and histopathological levels of malignancy than high corn oil diet groups. However, the LF-HCO group had the highest degree of clinical and morphological malignancy, while LF-EVOO is the group in which more differences in cell death proteins have been found. In previous works, we have observed that the high-fat diets, especially the high corn oil diet, advanced the puberty onset [10]. Thus, with an accelerated puberty maturation, the observed difference in carcinogenesis yield can be related to the different degree of differentiation that the mammary gland was at during the time of the carcinogenic insult. If the mammary gland of groups LF-HCO and LF-HEVOO were less differentiated at the time of induction, the gland could be more sensitive to the influence of the high-fat diets, clearly carcinogenesis-promoting in the case of the high corn oil diet (resulting in the highest degree of malignancy in the LF-HCO group), and with an effect of EVOO on cell death pathways (more evident in the LF-EVOO group). Moreover, we also previously observed an increase in body weight and mass if the high corn oil diet is administered from Cancers 2022, 14, 905 17 of 20 weaning [10]. Considering that advanced puberty onset and obesity are known risk factors of breast cancer, our results highlight the importance of acquiring healthy choices from an early age. Despite the variability of some results, the group of tumors from animals fed with the high-EVOO diet have different characteristics, such as low proliferation, modifications in several cell death pathways, and the reported metabolic changes [13]. However, neither of these pathways, without the interrelation of all networks, characterized by itself the clinical manifestation and the degree of tumor aggressiveness in vivo. In vitro analyses support an effect of EVOO on proliferation and apoptosis, with minor compounds such as hydroxytyrosol and oleuropein playing a role. In vivo, it is likely that the fatty acids also have a role as carriers of minor compounds and/or in the absorption of such com- pounds, in addition to the reported importance of the ratio between fatty acids on breast cancer risk [51,52]. Taking into account the tumor-promoting influence of a high intake of total fat [28,29], our investigations support the beneficial effect of the moderate intake of extra-virgin olive oil (which contains all the bioactive elements) on the risk of breast cancer. The complexity of this disease suggests that the effects of nutritional factors are subtle, heterogeneous, and probably in the long-term, but potentially relevant, especially considering that exposure, depending on dietary habits, can occur chronically throughout life. Thus, more investigation on the effects and mechanisms of these modifiable factors is needed, which can provide important scientific bases for dietary recommendations in health and disease. 5. Conclusions The results reported show the specific effect of the type of dietary lipid in breast carcinogenesis with the diet high in n-6 PUFA diet but not the high-EVOO diet, promoting mammary tumors with a high degree of histopathological malignancy and proliferation. This is the first study reporting that EVOO increased proteins leading to different cell death pathways in experimental mammary tumors. In vitro results support that the potential ben- eficial effect of EVOO is mainly elicited by its minor components, such as hydroxytyrosol (decreased tumor cell variability and increased cell death), and highlight the importance of specific food compositions (e.g., different effects of olive oil, extra-virgin olive oil, and oleic acid-enriched seed oil) in breast cancer prevention and treatment strategies. Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/cancers14040905/s1. Figure S1: cell death signaling pathways; Figure S2: experimental design; Figure S3: cell viability of MDA-MB-231 and MCF-7 cell lines treated with EVOO minor compounds; Figure S4: original plots of the in vitro analysis of cell cycle and apoptosis by flow cytometry; Original Western blot data: original blots and densitometry analyses for all the proteins studied. Author Contributions: Conceptualization, E.E. and R.M.; funding acquisition, E.E. and R.M.; in- vestigation, M.G.-G., M.M., I.C., E.V. and M.G.; methodology, M.G.-G., M.M., I.C., E.V., M.G., E.E. and R.M.; project administration, E.E. and R.M.; supervision, R.M.; writing—original draft, M.G.-G., M.M. and R.M.; writing—review and editing, all authors. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by “Plan Nacional de I + D + I” (AGL2006-07691); “Fundación Patrimonio Comunal Olivarero (FPCO)” (FPCO2008-165.396; FPCO2013-CF611.084); “Agencia para el Aceite de Oliva del Ministerio de Agricultura, Alimentación y Medio Ambiente” (AAO2008- 165.471); “Organización Interprofesional del Aceite de Oliva Español (OIAOE)” (OIP2009-CD165.646); “Departaments de Salut i d’Agricultura, Alimentació i Acció Rural de la Generalitat de Catalunya” (GC2010-165.000); and FPCO and OIAOE (FPCO-OIP2016-CF614.087). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethical Committee of Animal and Human Experimen- tation of Universitat Autonoma de Barcelona (CEEAH 566/3616). Informed Consent Statement: Not applicable. Cancers 2022, 14, 905 18 of 20 Data Availability Statement: Molecular data from Western blots generated in this study are included in Supplementary Materials. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. IARC Global Cancer Observatory. GLOBOCAN. 2021. Available online: https://gco.iarc.fr (accessed on 15 December 2021). 2. World Cancer Research Fund; American Institute for Cancer Research. Diet, Nutrition, Physical Activity and Cancer: A Global Perspective. Continuous Update Project Expert Report. 2018. Available online: https://www.wcrf.org/diet-and-cancer/ (accessed on 15 December 2021). 3. Escrich, E.; Solanas, M.; Moral, R. Olive oil, and other dietary lipids, in cancer: Experimental approaches. In Olive Oil and Health; Quiles, J.L., Ramírez-Tortosa, M.C., Yaqoob, P., Eds.; CABI Publishing: Oxford, UK, 2006; pp. 317–374. 4. Escrich, E.; Solanas, M.; Moral, R.; Escrich, R. Modulatory effects and molecular mechanisms of olive oil and other dietary lipids in breast cancer. Curr. Pharm. Des. 2011, 17, 813–830. [CrossRef] [PubMed] 5. Buckland, G.; Travier, N.; Cottet, V.; González, C.A.; Luján-Barroso, L.; Agudo, A.; Trichopoulou, A.; Lagiou, P.; Trichopoulos, D.; Peeters, P.H.; et al. Adherence to the mediterranean diet and risk of breast cancer in the European prospective investigation into cancer and nutrition cohort study. Int. J. Cancer 2013, 132, 2918–2927. [CrossRef] [PubMed] 6. Schwingshackl, L.; Schwedhelm, C.; Galbete, C.; Hoffmann, G. Adherence to Mediterranean Diet and Risk of Cancer: An Updated Systematic Review and Meta-Analysis. Nutrients 2017, 9, 1063. [CrossRef] [PubMed] 7. Di Daniele, N.; Noce, A.; Vidiri, M.F.; Moriconi, E.; Marrone, G.; Annicchiarico-Petruzzelli, M.; D’Urso, G.; Tesauro, M.; Rovella, V.; De Lorenzo, A. Impact of Mediterranean diet on metabolic syndrome, cancer and longevity. Oncotarget 2017, 8, 8947–8979. [CrossRef] [PubMed] 8. Solanas, M.; Hurtado, A.; Costa, I.; Moral, R.; Menéndez, J.A.; Colomer, R.; Escrich, E. Effects of a high olive oil diet on the clinical behavior and histopathological features of rat DMBA-induced mammary tumors compared with a high corn oil diet. Int. J. Oncol. 2002, 21, 745–753. [CrossRef] 9. Escrich, R.; Costa, I.; Moreno, M.; Cubedo, M.; Vela, E.; Escrich, E.; Moral, R. A high-corn-oil diet strongly stimulates mammary carcinogenesis, while a high-extra-virgin-olive-oil diet has a weak effect, through changes in metabolism, immune system function and proliferation/apoptosis pathways. J. Nutr. Biochem. 2019, 64, 218–227. [CrossRef] 10. Moral, R.; Escrich, R.; Solanas, M.; Vela, E.; Costa, I.; de Villa, M.C.R.; Escrich, E. Diets high in corn oil or extra-virgin olive oil provided from weaning advance sexual maturation and differentially modify susceptibility to mammary carcinogenesis in female rats. Nutr. Cancer 2011, 63, 410–420. [CrossRef] 11. Moral, R.; Escrich, R.; Solanas, M.; Vela, E.; de Villa, M.C.R.; Escrich, E. Diets high in corn oil or extra-virgin olive oil differentially modify the gene expression profile of the mammary gland and influence experimental breast cancer susceptibility. Eur. J. Nutr. 2016, 55, 1397–1409. [CrossRef] 12. Solanas, M.; Grau, L.; Moral, R.; Vela, E.; Escrich, R.; Escrich, E. Dietary olive oil and corn oil differentially affect experimental breast cancer through distinct modulation of the p21Ras signaling and the proliferation-apoptosis balance. Carcinogenesis 2009, 31, 871–879. [CrossRef] 13. Garcia-Guasch, M.; Navarro, L.; Rivero, V.; Costa, I.; Escrich, E.; Moral, R. A high extra-virgin olive oil diet induces changes in metabolic pathways of experimental mammary tumors. J. Nutr. Biochem. 2022, 99, 108833. [CrossRef] 14. Jin, L.; Zhou, Y. Crucial role of the pentose phosphate pathway in malignant tumors. Oncol. Lett. 2019, 17, 4213–4221. [CrossRef] [PubMed] 15. Vozza, A.; Parisi, G.; De Leonardis, F.; Lasorsa, F.M.; Castegna, A.; Amorese, D.; Marmo, R.; Calcagnile, V.M.; Palmieri, L.; Ricquier, D.; et al. UCP2 transports C4 metabolites out of mitochondria, regulating glucose and glutamine oxidation. Proc. Natl. Acad. Sci. USA 2014, 111, 960–965. [CrossRef] [PubMed] 16. Faubert, B.; Solmonson, A.; DeBerardinis, R.J. Metabolic reprogramming and cancer progression. Science 2020, 368, eaaw5473. [CrossRef] [PubMed] 17. Elmore, S. Apoptosis: A review of programmed cell death. Toxicol. Pathol. 2007, 35, 495–516. [CrossRef] 18. Wong, R.S. Apoptosis in cancer: From pathogenesis to treatment. J. Exp. Clin. Cancer Res. 2011, 30, 87. [CrossRef] 19. Carneiro, B.A.; El-Deiry, W.S. Targeting apoptosis in cancer therapy. Nat. Rev. Clin. Oncol. 2020, 17, 395–417. [CrossRef] 20. Rajabi, S.; Maresca, M.; Yumashev, A.V.; Choopani, R.; Hajimehdipoor, H. The Most Competent Plant-Derived Natural Products for Targeting Apoptosis in Cancer Therapy. Biomolecules 2021, 11, 534. [CrossRef] 21. Escrich, E.; Solanas, M.; Segura, R. Experimental diets for the study of lipid influence on induced mammary carcinoma in rats: I—Diet definition. In Vivo 1994, 8, 1099–1106. 22. Escrich, E.; Solanas, M.; de Villa, R.M.C.; Ribalta, T.; Muntane, J.; Segura, R. Experimental diets for the study of lipid influence on induced mammary carcinoma in rats: II—Suitability of the diets. In Vivo 1994, 8, 1107–1112. Cancers 2022, 14, 905 19 of 20 23. Costa, I.; Solanas, M.; Escrich, E. Histopathologic characterization of mammary neoplastic lesions induced with 7,12 dimethylbenz(α)anthracene in the rat: A comparative analysis with human breast tumors. Arch. Pathol. Lab. Med. 2002, 126, 915–927. [CrossRef] 24. Gürtler, A.; Kunz, N.; Gomolka, M.; Hornhardt, S.; Friedl, A.A.; McDonald, K.; Kohn, J.E.; Posch, A. Stain-Free technology as a normalization tool in Western blot analysis. Anal. Biochem. 2013, 433, 105–111. [CrossRef] 25. Calahorra, J.; Martínez-Lara, E.; Granadino-Roldán, J.M.; Martí, J.M.; Cañuelo, A.; Blanco, S.; Oliver, F.J.; Siles, E. Crosstalk between hydroxytyrosol, a major olive oil phenol, and HIF-1 in MCF-7 breast cancer cells. Sci. Rep. 2020, 10, 6361. [CrossRef] [PubMed] 26. Przychodzen, P.; Kuban-Jankowska, A.; Wyszkowska, R.; Barone, G.; Bosco, G.L.; Celso, F.L.; Kamm, A.; Daca, A.; Kostrzewa, T.; Gorska-Ponikowska, M. PTP1B phosphatase as a novel target of oleuropein activity in MCF-7 breast cancer model. Toxicol. In Vitro 2019, 61, 104624. [CrossRef] [PubMed] 27. Huang, L.; Jin, K.; Lan, H. Luteolin inhibits cell cycle progression and induces apoptosis of breast cancer cells through downregu- lation of human telomerase reverse transcriptase. Oncol. Lett. 2019, 17, 3842–3850. [CrossRef] [PubMed] 28. Guthrie, N.; Carroll, K.K. Specific versus non-specific effects of dietary fat on carcinogenesis. Prog. Lipid Res. 1999, 38, 261–271. [CrossRef] 29. Sieri, S.; Krogh, V.; Ferrari, P.; Berrino, F.; Pala, V.; Thiébaut, A.C.; Tjønneland, A.; Olsen, A.; Overvad, K.; Jakobsen, M.U.; et al. Dietary fat and breast cancer risk in the European Prospective Investigation into Cancer and Nutrition. Am. J. Clin. Nutr. 2008, 88, 1304–1312. [CrossRef] 30. Matsuura, K.; Canfield, K.; Feng, W.; Kurokawa, M. Metabolic Regulation of Apoptosis in Cancer. Int. Rev. Cell Mol. Biol. 2016, 327, 43–87. [CrossRef] 31. Yi, C.H.; Vakifahmetoglu-Norberg, H.; Yuan, J. Integration of apoptosis and metabolism. Cold Spring Harb. Symp. Quant. Biol. 2011, 76, 375–387. [CrossRef] 32. Majors, B.S.; Betenbaugh, M.J.; Chiang, G.G. Links between metabolism and apoptosis in mammalian cells: Applications for anti-apoptosis engineering. Metab. Eng. 2007, 9, 317–326. [CrossRef] 33. Holliday, D.L.; Speirs, V. Choosing the right cell line for breast cancer research. Breast Cancer Res. 2011, 13, 215. [CrossRef] 34. Yamagata, K.; Uzu, E.; Yoshigai, Y.; Kato, C.; Tagami, M. Oleic acid and oleoylethanolamide decrease interferon-gamma-induced expression of PD-L1 and induce apoptosis in human lung carcinoma cells. Eur. J. Pharmacol. 2021, 903, 174116. [CrossRef] [PubMed] 35. Carrillo, C.; Cavia, M.M.; Alonso-Torre, S.R. Antitumor effect of oleic acid; mechanisms of action: A review. Nutr. Hosp. 2012, 27, 1860–1865. [CrossRef] [PubMed] 36. Zhao, J.; Zhi, Z.; Wang, C.; Xing, H.; Song, G.; Yu, X.; Zhu, Y.; Wang, X.; Zhang, X.; Di, Y. Exogenous lipids promote the growth of breast cancer cells via CD36. Oncol. Rep. 2017, 38, 2105–2115. [CrossRef] [PubMed] 37. Marcial-Medina, C.; Ordoñez-Moreno, A.; Gonzalez-Reyes, C.; Cortes-Reynosa, P.; Salazar, E.P. Oleic acid induces migration through a FFAR1/4, EGFR and AKT-dependent pathway in breast cancer cells. Endocr. Connect. 2019, 8, 252–265. [CrossRef] 38. Balaban, S.; Lee, L.S.; Varney, B.; Aishah, A.; Gao, Q.; Shearer, R.F.; Saunders, D.N.; Grewal, T.; Hoy, A.J. Heterogeneity of fatty acid metabolism in breast cancer cells underlies differential sensitivity to palmitate-induced apoptosis. Mol. Oncol. 2018, 12, 1623–1638. [CrossRef] 39. Jimenez-Lopez, C.; Carpena, M.; Lourenço-Lopes, C.; Gallardo-Gomez, M.; Lorenzo, J.M.; Barba, F.J.; Prieto, M.A.; Simal-Gandara, J. Bioactive Compounds and Quality of Extra Virgin Olive Oil. Foods 2020, 9, 1014. [CrossRef] 40. Romani, A.; Ieri, F.; Urciuoli, S.; Noce, A.; Marrone, G.; Nediani, C.; Bernini, R. Health Effects of Phenolic Compounds Found in Extra-Virgin Olive Oil, By-Products, and Leaf of Olea europaea L. Nutrients 2019, 11, 1776. [CrossRef] 41. Imran, M.; Nadeem, M.; Gilani, S.A.; Khan, S.; Sajid, M.W.; Amir, R.M. Antitumor Perspectives of Oleuropein and Its Metabolite Hydroxytyrosol: Recent Updates. J. Food Sci. 2018, 83, 1781–1791. [CrossRef] 42. Antoniou, C.; Hull, J. The Anti-cancer Effect of Olea europaea L. Products: A Review. Curr. Nutr. Rep. 2021, 10, 99–124. [CrossRef] 43. Cook, M.T.; Mafuvadze, B.; Besch-Williford, C.; Ellersieck, M.R.; Goyette, S.; Hyder, S.M. Luteolin suppresses development of medroxyprogesterone acetate-accelerated 7,12-dimethylbenz(a)anthracene-induced mammary tumors in Sprague-Dawley rats. Oncol. Rep. 2016, 35, 825–832. [CrossRef] 44. Samy, R.P.; Gopalakrishnakone, P.; Ignacimuthu, S. Anti-tumor promoting potential of luteolin against 7,12-dimethylbenz(a)anthracene- induced mammary tumors in rats. Chem. Biol. Interact. 2006, 64, 1–14. [CrossRef] [PubMed] 45. Storniolo, C.E.; Martínez-Hovelman, N.; Martínez-Huélamo, M.; Lamuela-Raventos, R.M.; Moreno, J.J. Extra Virgin Olive Oil Minor Compounds Modulate Mitogenic Action of Oleic Acid on Colon Cancer Cell Line. J. Agric. Food Chem. 2019, 67, 11420–11427. [CrossRef] [PubMed] 46. Escrich, E.; Solanas, M.; Soler, M.; de Villa, M.C.R.; Sanchez, J.A.; Segura, R. Dietary polyunsaturated n-6 lipids effects on the growth and fatty acid composition of rat mammary tumors. J. Nutr. Biochem. 2001, 12, 536–549. [CrossRef] 47. Hulbert, A.J.; Turner, N.; Storlien, L.H.; Else, P.L. Dietary fats and membrane function: Implications for metabolism and disease. Biol. Rev. Camb. Philos. Soc. 2005, 80, 155–169. [CrossRef] [PubMed] 48. Clarke, R.; Jones, B.C.; Sevigny, C.M.; Hilakivi-Clarke, L.A.; Sengupta, S. Experimental models of endocrine responsive breast cancer: Strengths, limitations, and use. Cancer Drug Resist. 2021, 4, 762–783. [CrossRef] Cancers 2022, 14, 905 20 of 20 49. Moral, R.; Escrich, E. Influence of olive oil and its components on breast cancer: Molecular mechanisms. Molecules 2022, 27, 477. [CrossRef] 50. Chen, Y.; Zhang, H.; Zhou, H.J.; Ji, W.; Min, W. Mitochondrial Redox Signaling and Tumor Progression. Cancers 2016, 8, 40. [CrossRef] 51. Simopoulos, A.P. The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomed. Pharmacother. 2002, 56, 365–379. [CrossRef] 52. Sczaniecka, A.K.; Brasky, T.M.; Lampe, J.W.; Patterson, R.E.; White, E. Dietary intake of specific fatty acids and breast cancer risk among postmenopausal women in the VITAL cohort. Nutr. Cancer 2012, 64, 1131–1142. [CrossRef]