Uptake and metabolism of olive oil polyphenols in human breast cancer cells using nano-liquid chromatography coupled to electrospray ionization–time of flight-mass spectrometry

Polyphenols from extra virgin olive oil (EVOO), a main component of the Mediterranean diet, have demonstrated repeatedly anti-tumor activity in several in vitro and in vivo studies. However, little is known about the efficiency of the absorption process and metabolic conversion of these compounds at cellular level. In this study, a nano liquid chromatography–electrospray ionization–time of flight mass spectrometry (nanoLC–ESI–TOF MS) method was developed to study the cellular uptake and metabolism of olive oil phenols in JIMT-1 human breast cancer cells. After incubation for different time periods with EVOO-derived phenolic extracts, culture media, cytosolic fraction and solid particles fraction were separated and analyzed. Most of the free phenols, mainly hydroxytyrosol, its secoiridoid derivatives, and the flavonoid luteolin, disappeared in the culture media in different ways and at different times. Besides, several metabolites were detected in the culture media, fact that may indicate absorption and intracellular metabolism followed by rapid cellular export. Low intracellular accumulation was observed with only traces of some compounds detected in the cytosolic and solid particles fractions. Methylated conjugates were the major metabolites detected, suggesting a catalytic action of catechol-O-methyl transferase (COMT) in cancer cells.The authors are very grateful to Ministry of Education and Science (FPU, AP2005-4356) and Junta de Andalucia (project P09-FQM-5469, project P07-AGR-02619 and AGL 2008-05108- CO3-03/ALI). Besides, this work was supported in part by Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, Fondo de Investigación Sanitaria – FIS, Spain, Grants CP05-00090, PI06-0778 and RD06-0020-0028)

Exploratory analysis of human urine by LC–ESI-TOF MS after high intake of olive oil: understanding the metabolism of polyphenols

Olive oil polyphenols have important biological properties which closely depend on their bioavailability, therefore it is essential to understand how polyphenols are absorbed, metabolized and eliminated from the body. An analytical methodology based on rapid resolution liquid chromatography (RRLC) coupled to mass spectrometry detection with a time of flight analyzer (RRLC-ESI-TOF MS) was developed for the determination of the main olive oil phenolic compounds and their metabolites in human urine. Urine samples from ten healthy volunteers were collected before and 2, 4 and 6 h after the intake of 50 mL of extra-virgin olive oil. The proposed method includes liquid-liquid extraction with ethyl acetate that provides extraction recoveries of the phenolic compounds studied between 35 and 75% from spiked urine samples. Good repeatability was obtained, since the relative standard deviations (RSDs) of peak areas in the intra- and inter-day studies were 4.3 and 6.5%, respectively. Statistical studies allowed us to discriminate between the urine samples before and after the intake, and facilitated to find out the m/z values responsible of this discrimination. Based on the very accurate mass information and the isotopic pattern provided by TOF-MS analyzer, together with other available information, ten of these biomarkers and more than 50 metabolites, obtained through phase I and phase II biotransformation reactions, were tentatively identified. Additionally, kinetic studies of the metabolites identified as possible biomarkers were developed, obtaining maximal values in the first two hours for most compounds.The authors are very grateful to Ministry of Education and Science (FPU, AP2005-4356 and Project AGL 2008- 05108-613 CO3-03/ALI), and Junta de Andalucía (P07-AGR-02629 and P09-FQM-5469)

tabAnti-HER2 (erbB-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO)

<p>Abstract</p> <p>Background</p> <p>The effects of the olive oil-rich Mediterranean diet on breast cancer risk might be underestimated when HER2 (<it>ERB</it>B2) oncogene-positive and HER2-negative breast carcinomas are considered together. We here investigated the anti-HER2 effects of phenolic fractions directly extracted from Extra Virgin Olive Oil (EVOO) in cultured human breast cancer cell lines.</p> <p>Methods</p> <p>Solid phase extraction followed by semi-preparative high-performance liquid chromatography (HPLC) was used to isolate phenolic fractions from commercial EVOO. Analytical capillary electrophoresis coupled to mass spectrometry was performed to check for the composition and to confirm the identity of the isolated fractions. EVOO polyphenolic fractions were tested on their tumoricidal ability against HER2-negative and HER2-positive breast cancer <it>in vitro </it>models using MTT, crystal violet staining, and Cell Death ELISA assays. The effects of EVOO polyphenolic fractions on the expression and activation status of HER2 oncoprotein were evaluated using HER2-specific ELISAs and immunoblotting procedures, respectively.</p> <p>Results</p> <p>Among the fractions mainly containing the <it>single phenols </it>hydroxytyrosol and tyrosol, the <it>polyphenol acid </it>elenolic acid, the <it>lignans </it>(+)-pinoresinol and 1-(+)-acetoxypinoresinol, and the <it>secoiridoids </it>deacetoxy oleuropein aglycone, ligstroside aglycone, and oleuropein aglycone, all the major EVOO polyphenols (<it>i.e. </it>secoiridoids and lignans) were found to induce strong tumoricidal effects within a micromolar range by selectively triggering high levels of apoptotic cell death in HER2-overexpressors. Small interfering RNA-induced depletion of HER2 protein and lapatinib-induced blockade of HER2 tyrosine kinase activity both significantly prevented EVOO polyphenols-induced cytotoxicity. EVOO polyphenols drastically depleted HER2 protein and reduced HER2 tyrosine autophosphorylation in a dose- and time-dependent manner. EVOO polyphenols-induced HER2 downregulation occurred regardless the molecular mechanism contributing to HER2 overexpression (<it>i.e</it>. naturally by gene amplification and ectopically driven by a viral promoter). Pre-treatment with the proteasome inhibitor MG132 prevented EVOO polyphenols-induced HER2 depletion.</p> <p>Conclusion</p> <p>The ability of EVOO-derived polyphenols to inhibit HER2 activity by promoting the proteasomal degradation of the HER2 protein itself, together with the fact that humans have safely been ingesting secoiridoids and lignans as long as they have been consuming olives and OO, support the notion that the stereochemistry of these phytochemicals might provide an excellent and safe platform for the design of new HER2-targeting agents.</p

Olive oil's bitter principle reverses acquired autoresistance to trastuzumab (Herceptin™) in HER2-overexpressing breast cancer cells

[Background] A low incidence of breast cancer in the Mediterranean basin suggests that a high consumption of Extra Virgin Olive Oil (EVOO) might confer this benefit. While the anti-HER2 oncogene effects of the main ω-9 fatty acid present in EVOO triacylglycerols (i.e., oleic acid) have been recently described, the anti-breast cancer activities of EVOO non-glyceridic constituents -which consist of at least 30 phenolic compounds-, remained to be evaluated. [Methods] Semi-preparative HPLC was used to isolate EVOO polyphenols (i.e., tyrosol, hydroxytyrosol, oleuropein). Both the anti-proliferative and the pro-apoptotic effects of EVOO phenolics were evaluated by using MTT-based quantification of metabolically viable cells and ELISA-based detection of histone-associated DNA fragments, respectively. The nature of the interaction between oleuropein aglycone and the anti-HER2 monoclonal antibody trastuzumab (Herceptin™) was mathematically evaluated by the dose-oriented isobologram technique. HER2-specific ELISAs were employed to quantitatively assess both the basal cleavage of the HER2 extracellular domain (ECD) and the expression level of total HER2. The activation status of HER2 was evaluated by immunoblotting procedures using a monoclonal antibody specifically recognizing the tyrosine phosphorylated (Phosphor-Tyr1248) form of HER2. [Results] Among EVOO polyphenols tested, oleuropein aglycone was the most potent EVOO phenolic in decreasing breast cancer cell viability. HER2 gene-amplified SKBR3 cells were ~5-times more sensitive to oleuropein aglycone than HER2-negative MCF-7 cells. Retroviral infection of the HER2 oncogene in MCF-7 cells resulted in a "SKBR3-assimilated" phenotype of hypersensitivity to oleuropein aglycone. An up to 50-fold increase in the efficacy of trastuzumab occurred in the presence of oleuropein aglycone. A preclinical model of acquired autoresistance to trastuzumab (SKBR3/Tzb100 cells) completely recovered trastuzumab sensitivity (> 1,000-fold sensitization) when co-cultured in the presence of oleuropein aglycone. Indeed, the nature of the interaction between oleuropein aglycone and trastuzumab was found to be strongly synergistic in Tzb-resistant SKBR3/Tzb100 cells. Mechanistically, oleuropein aglycone treatment significantly reduced HER2 ECD cleavage and subsequent HER2 auto-phosphorylation, while it dramatically enhanced Tzb-induced down-regulation of HER2 expression. [Conclusion] Olive oil's bitter principle (i.e., oleuropein aglycone) is among the first examples of how selected nutrients from an EVOO-rich "Mediterranean diet" directly regulate HER2-driven breast cancer disease.JAM is the recipient of a Basic, Clinical and Translational Research Award (BCTR0600894) from the Susan G. Komen Breast Cancer Foundation (Texas, USA). This work was also supported by the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, Fondo de Investigación Sanitaria -FIS-, Spain, Grants CP05-00090 and PI06-0778 to JAM, and Grant RD06-0020-0028 to JAM, RC and JB)

Single-cultivar extra virgin olive oil classification using a potentiometric electronic tongue

Label authentication of monovarietal extra virgin olive oils is of great importance. A novel approach based on a potentiometric electronic tongue is proposed to classify oils obtained from single olive cultivars (Portuguese cvs. Cobrançosa, Madural, Verdeal Transmontana; Spanish cvs. Arbequina, Hojiblanca, Picual). A meta-heuristic simulated annealing algorithm was applied to select the most informative sets of sensors to establish predictive linear discriminant models. Olive oils were correctly classified according to olive cultivar (sensitivities greater than 97%) and each Spanish olive oil was satisfactorily discriminated from the Portuguese ones with the exception of cv. Arbequina (sensitivities from 61% to 98%). Also, the discriminant ability was related to the polar compounds contents of olive oils and so, indirectly, with organoleptic properties like bitterness, astringency or pungency. Therefore the proposed E-tongue can be foreseen as a useful auxiliary tool for trained sensory panels for the classification of monovarietal extra virgin olive oils.This work was co-financed by FCT and FEDER under Program COMPETE (Project PEst-C/EQB/LA0020/2013)

Monovarietal extra-virgin olive oil classification: a fusion of human sensory attributes and an electronic tongue

Olive oil quality grading is traditionally assessed by human sensory evaluation of positive and negative attributes (olfactory, gustatory, and final olfactorygustatory sensations). However, it is not guaranteed that trained panelist can correctly classify monovarietal extra-virgin olive oils according to olive cultivar. In this work, the potential application of human (sensory panelists) and artificial (electronic tongue) sensory evaluation of olive oils was studied aiming to discriminate eight single-cultivar extra-virgin olive oils. Linear discriminant, partial least square discriminant, and sparse partial least square discriminant analyses were evaluated. The best predictive classification was obtained using linear discriminant analysis with simulated annealing selection algorithm. A low-level data fusion approach (18 electronic tongue signals and nine sensory attributes) enabled 100 % leave-one-out cross-validation correct classification, improving the discrimination capability of the individual use of sensor profiles or sensory attributes (70 and 57 % leave-one-out correct classifications, respectively). So, human sensory evaluation and electronic tongue analysis may be used as complementary tools allowing successful monovarietal olive oil discrimination.This work was co-financed by FCT/MEC and FEDER under Program PT2020 (Project UID/EQU/50020/2013); by Fundacao para a Ciencia e Tecnologia under the strategic funding of UID/BIO/04469/2013 unit; and by Project POCTEP through Project RED/AGROTEC-Experimentation network and transfer for development of agricultural and agro industrial sectors between Spain and Portugal

The COMT Val158 Met polymorphism as an associated risk factor for Alzheimer disease and mild cognitive impairment in APOE 4 carriers

<p>Abstract</p> <p>Background</p> <p>The aim of this study is to examine the influence of the <it>catechol-O-methyltranferase (COMT) </it>gene (polymorphism Val158 Met) as a risk factor for Alzheimer's disease (AD) and mild cognitive impairment of amnesic type (MCI), and its synergistic effect with the <it>apolipoprotein E gene (APOE)</it>.</p> <p>A total of 223 MCI patients, 345 AD and 253 healthy controls were analyzed. Clinical criteria and neuropsychological tests were used to establish diagnostic groups.</p> <p>The DNA Bank of the University of the Basque Country (UPV-EHU) (Spain) determined <it>COMT </it>Val158 Met and <it>APOE </it>genotypes using real time polymerase chain reaction (rtPCR) and polymerase chain reaction (PCR), and restriction fragment length polymorphism (RFLPs), respectively. Multinomial logistic regression models were used to determine the risk of AD and MCI.</p> <p>Results</p> <p>Neither <it>COMT </it>alleles nor genotypes were independent risk factors for AD or MCI. The high activity genotypes (GG and AG) showed a synergistic effect with <it>APOE ε4 </it>allele, increasing the risk of AD (OR = 5.96, 95%CI 2.74-12.94, p < 0.001 and OR = 6.71, 95%CI 3.36-13.41, p < 0.001 respectivily). In AD patients this effect was greater in women.</p> <p>In MCI patients such as synergistic effect was only found between AG and <it>APOE ε4 </it>allele (OR = 3.21 95%CI 1.56-6.63, p = 0.02) and was greater in men (OR = 5.88 95%CI 1.69-20.42, p < 0.01).</p> <p>Conclusion</p> <p><it>COMT </it>(Val158 Met) polymorphism is not an independent risk factor for AD or MCI, but shows a synergistic effect with <it>APOE ε4 </it>allele that proves greater in women with AD.</p

Application of an electronic tongue for Tunisian olive oils' classification according to olive cultivar or physicochemical parameters

Olive oil commercialization has a great impact on the economy of several countries, namely Tunisia, being prone to frauds. Therefore, it is important to establish analytical techniques to ensure labeling correctness concerning olive oil quality and olive cultivar. Traditional analytical techniques are quite expensive, time consuming and hardly applied in situ, considering the harsh environments of the olive industry. In this work, the feasibility of applying a potentiometric electronic tongue with cross-sensitivity lipid membranes to discriminate Tunisian olive oils according to their quality level (i.e., extra virgin, virgin or lampante olive oils) or autochthonous olive cultivar (i.e., cv Chétoui and cv Shali) was evaluated for the first time. Linear discrimination analysis coupled with the simulated annealing variable selection algorithm showed that the signal profiles of olive oils hydroethanolic extracts allowed olive oils discrimination according to physicochemical quality level (classification model based on 25 signals enabling 84 ± 9% correct classifications for repeated K-fold cross-validation), and olive cultivar (classification model based on 20 signals with an average sensitivity of 94 ± 6% for repeated K-fold cross-validation), regardless of the geographical origin and olive variety or the olive quality, respectively. The results confirmed, for the first time, the potential discrimination of the electronic tongue, attributed to the observed quantitative response (sensitivities ranging from 66.6 to +57.7 mV/decade) of the E-tongue multi-sensors towards standard solutions of polar compounds (aldehydes, esters and alcohols) usually found in olive oils and that are related to their sensory positive attributes like green and fruity.This work was financially supported by Project POCI-01–0145-FEDER-006984–Associate Laboratory LSRE-LCM and by Project UID/QUI/00616/2013–CQ-VR both funded by FEDER—Fundo Europeu de Desenvolvimento Regional through COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI)—and by national funds through FCTFundação para a Ciência e a Tecnologia, Portugal. Strategic funding of UID/BIO/04469/2013 unit is also acknowledged. Nuno Rodrigues thanks FCT, POPH-QREN and FSE for the Ph.D. Grant (SFRH/ BD/104038/2014).info:eu-repo/semantics/publishedVersio