tabAnti-HER2 (erbB-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO)
© Menendez et al; licensee BioMed Central Ltd. 2008
Received: 09 June 2008
Accepted: 18 December 2008
Published: 18 December 2008
The effects of the olive oil-rich Mediterranean diet on breast cancer risk might be underestimated when HER2 (ERBB2) 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.
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 in vitro 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.
Among the fractions mainly containing the single phenols hydroxytyrosol and tyrosol, the polyphenol acid elenolic acid, the lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, and the secoiridoids deacetoxy oleuropein aglycone, ligstroside aglycone, and oleuropein aglycone, all the major EVOO polyphenols (i.e. 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 (i.e. naturally by gene amplification and ectopically driven by a viral promoter). Pre-treatment with the proteasome inhibitor MG132 prevented EVOO polyphenols-induced HER2 depletion.
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.
Case-control, cohort, and prospective epidemiological studies have generated conflicting results regarding a protective effect of an olive oil (OO)-rich Mediterranean diet against several malignancies, especially breast cancer [1–5]. It has been assumed that, if protective agents (e.g. monounsaturated fatty acids such as oleic acid and/or antioxidants from olive oil and raw vegetables) are largely present in the diet of a population with a low risk of acquiring solid tumors (i.e. dietary patterns found in olive-growing areas of the Mediterranean basin), these diet-based anti-cancer mechanisms should influence the occurrence of all or most types of cancer. Sant et al. , by analyzing the data of the ORDET prospective study on hormones, diet and breast cancer , have recently suggested that the effects of the Mediterranean diet on breast cancer risk might be underestimated when HER2 (ERBB2) oncogene-positive and HER2-negative breast carcinomas are considered together. The Type I receptor tyrosine kinase (RTKs) HER2 regulates biological functions as diverse as cellular proliferation, transformation, differentiation, motility and apoptosis. HER2 is therefore one of the most commonly analyzed proto-oncogenes in human cancer studies, as it plays a pivotal role in oncogenic transformation, tumorigenesis, and metastasis [8–12]. HER2 gene is amplified and/or overexpressed in ~20% to 30% of invasive breast carcinomas and is associated with unfavorable prognosis, shorter relapse time, and decreased overall survival [8–12].
Although one observational study is not sufficient and consistency of findings across multiple cohort and case-control studies is paramount to definitely establish whether the protective effect of a diet rich in raw vegetables and OO is largely restricted to HER2-positive breast carcinomas, when considering experimental and epidemiological evidence together it is reasonable to suggest that dietary factors influencing the occurrence of HER2-positive breast carcinomas may differ from those influencing the occurrence of HER2-negative cancers . Moreover, an OO, salad and vegetable-rich dietary pattern might specifically exert a protective effect against HER2-positive breast cancer because the (anti-cancer) mechanism of action of some of its components largely depends on their ability to suppress HER2 expression. We previously demonstrated that an experimental diet with a high content in Extra Virgin OO (i.e. the juice of the olive obtained solely by pressing and consumed without any further refining process) acted as a negative modulator on the promotion stage of dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats by conferring to the tumors a more benign clinical behavior and lower histopathological malignancy . Mechanistically, both in vivo and in vitro studies revealed that oleic acid (OA; 18:1n-9) – the main EVOO's monounsaturated fatty acid (MUFA) – transcriptionally suppressed the expression of HER2 gene [14–19]. We recently described that the polyphenol oleuropein aglycone, a non-glyceridic constituent of EVOO, was capable to reverse breast cancer acquired autoresistance to the anti-HER2 monoclonal antibody trastuzumab (Herceptin™) . However, the HER2-related anti-breast cancer activities of polyphenolic compounds present in the soluble fraction of EVOO other than oleuropein aglycone, which have been suggested to contribute the oxidative stability of EVOO, and as such are often associated with the health benefits of EVOO [21–24], remained to be fully evaluated.
We report for the first time that all the fractions containing the major EVOO polyphenols (i.e. the secoiridoids deacetoxy oleuropein aglycone, ligstroside aglycone, oleuropein aglycone and the lignans [+]-pinoresinol and 1-[+]-acetoxypinoresinol) can efficiently inhibit HER2 protein kinase activity by depleting the HER2 protein kinase itself. We suggest that the stereochemistry of these phytochemicals might provide an excellent and safe platform for the design of new HER2-targeted anti-breast cancer drugs.
Semi-preparative HPLC was performed with a HP 1100 series (Agilent Technologies, Palo Alto, CA, USA), equipped with a binary pump delivery system, a degasser, an autosampler, a diode array UV-VIS detector (DAD). The semi-preparative HPLC column used was a Phenomenex Luna (C18) column, 10 μm i.d., 25 cm × 10 mm and the flow rate was 3 mL/min.
Capillary electrophoresis (CE) were performed using a in a P/ACE™ System MDQ (Beckman Instruments, Fullerton, CA, USA). Fused-silica capillaries of 85 cm in length and 50 μm inner diameter (360 μm outer diameter) were used. CE apparatus was coupled to the mass spectrometer detector by an orthogonal electrospray interface (ESI). Mass spectrometer was a microTOF™ (Bruker Daltonik, Bremen, Germany), an orthogonal-accelerated TOF mass spectrometer (oaTOF-MS). Good sensitivity at a reasonable resolution was obtained (5,000–10,000 at 250 m/z). The trigger time was set to 50 μs, corresponding to a mass range of 50–800 m/z. Spectra were acquired by summarizing 30,000 single spectra, defining the time resolution to 1.5 s.
Reagents, stock solutions and reference compounds
Methanol and n-hexane HPLC-grade were from Merck (Darmstadt, Germany). Distilled water with a conductivity of 18.2 MΩ was deionized by using a Milli-Q system (Millipore, Bedford, MA, USA). Oleuropein glycoside was obtained from Extrasynthèse (Genay, France). Trastuzumab (Herceptin™) – kindly provided by Hospital Universitari de Girona Dr. Josep Trueta Pharmacy (Girona, Spain) – was solubilized in bacteriostatic water for injection containing 1.1% benzyl alcohol (stock solution at 21 mg/ml), stored at 4°C and used within one month. Lapatinib (GW572016; Tykerb®) was gently provided by GlaxoSmithKline (GSK), Corporate Environment, Health & Safety (Brentford, Middlesex TW8 9GS UK). MG-132 was purchased from Calbiochem (Calbiochem, San Diego, CA, USA). N-acetylcysteine and Trolox were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lapatinib, MG-132, and Trolox were dissolved in DMSO and stored in the dark as stock solutions (10 mM) at -20°C until utilization. NAC was dissolved in Phosphate Buffered Saline (PBS) immediately before utilization. For experimental use, trastuzumab, lapatinib, MG-132, NAC and Trolox were prepared freshly from stock solutions and diluted with growth medium. Control cells were cultured in medium containing the same concentration (v/v) of vehicles as the experimental cultures with treatments. The vehicle solutions had no noticeable influence on the proliferation of experimental cells.
Sample, extraction, isolation and analysis of polyphenol fractions from EVOO
Composition of the isolated EVOO phenolic fractions using CE-ESI-MS
Deacetoxy oleuropein aglycone (DAOA)
Deacetoxy ligstroside aglycone
Deacetoxy ligstroside aglycone
Cell lines and culture conditions
MCF-7 and SKBR3 breast cancer cells were obtained from the American Type Culture Collection (ATCC) and they where routinely grown in Improved MEM (IMEM; Biosource International) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-Glutamine. Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were screened periodically for Mycoplasma contamination. EVOO polyphenols were prepared freshly from stock solutions and diluted with growth medium. Control cells were cultured in medium containing the same concentration (v/v) as the experimental cultures with treatments. The vehicle solutions had no noticeable influence on the proliferation of experimental cells.
Construction of pBABE/HER2 retroviruses and retroviral infection of MCF-7 cells
A full-length human HER2 cDNA construct in the pCMV-SPORT6 plasmid was purchased from RZPD (Berlin, Germany). The insert was excised from pCMV-SPORT6 using EcoRV and NotI sites and blunt end ligated into the pBABE-puro retroviral vector (Addgene) at the EcoRI site. Retroviruses were generated by co-transfection of 293T-derived phoenix cells with the retroviral constructs (pBABE, pBABE-HER2) and the packaging vector pCL-Eco by using FuGene transfection reagent (Roche Diagnostics, Barcelona, Spain) and 5 μg of each plasmid per 0.5 × 106 cells. 293T cells were cultured at 5% CO2, 37°C in DMEM containing 10% (v/v) heat-inactivated FBS. After 48 h, medium conditioned by transfected 293T cells was filtered and immediately added to MCF-7 cells in the presence of 4 μg/ml polybrene (Sigma-Chemicals, St. Louis, MO, USA). At 48 h following infection, MCF-7/pBABE and MCF-7/HER2 cells were selected by using 2.5 μg/ml puromycin for 72 h. Expression of virally encoded HER2 protein was confirmed by HER2-specific ELISA analyses (see below).
Metabolic status assessment (MTT-based cell viability assays)
Breast cancer cell viability was determined using a standard colorimetric MTT (3-4, 5-dimethylthiazol-2-yl-2, 5-diphenyl-tetrazolium bromide) reduction assay. Cells in exponential growth were harvested by trypsinization and seeded at a concentration of ~2.5 × 103 cells/200 μl/well into 96-well plates, and allowed an overnight period for attachment. Then the medium was removed and fresh medium along with various concentrations of EVOO phenolic fractions, trastuzumab, lapatinib, NAC, Trolox, and/or MG-132 were (concurrently or sequentially) added to cultures as specified. Control cells without agents were cultured in parallel using the same conditions with comparable media changes. Compounds were not renewed during the entire period of cell exposure. Following treatment (5 days), the medium was removed and replaced by fresh drug-free medium (100 μl/well), and MTT (5 mg/ml in PBS) was added to each well at a 1/10 volume. After incubation for 2–3 hr at 37°C, the supernatants were carefully aspirated, 100 μl of DMSO were added to each well, and the plates agitated to dissolve the crystal product. Optical Density (OD) was measured at 570 nm using a multi-well plate reader (Model Anthos Labtec 2010 1.7 reader). The cell viability effects from exposure of cells to each polyphenol fraction alone were analyzed as percentages of the control cell absorbances, which were obtained from control wells treated with appropriate concentrations of the compounds vehicles that were processed simultaneously. For each treatment, cell viability was evaluated as a percentage using the following equation:
>(OD570 of treated sample/OD570 of untreated sample) × 100.
Breast cancer cell sensitivity to agents was expressed in terms of the concentration of drug required to decrease by 50% cell viability (IC50 value). Since the percentage of control absorbance was considered to be the surviving fraction of cells, the IC50 values were defined as the concentration of agents that produced 50% reduction in control absorbance (by interpolation), respectively.
Cell proliferation (crystal violet staining)
The ability of EVOO polyphenol compounds to affect breast cancer cells proliferation was determined using a crystal violet cell staining assay. Crystal violet is an intense stain binding to the cell nuclei and gives an OD595 reading that is proportional to cell number. Cells were plated and treated as described above (MTT assays). Following treatments, cells were fixed by replacing the growth medium with 100 μl/well of 4% formaldehyde in PBS (20 minutes, RT). Formaldehyde solution was removed and cells were washed twice with 200 μl/well Wash Buffer (0.1% Triton X-100 in PBS) and 2 times with 200 μl/well 1 × PBS. 100 μl of crystal violet solution was added to each well and incubated 30 minutes at RT. Cell were then washed 3 times with 200 μl/well 1 × PBS, 100 μl of 1% SDS solution was added to each well, and plates were incubated on a shaker for 1 hour at RT. Optical Density (OD) was measured at 595 nm using a multi-well plate reader (Model Anthos Labtec 2010 1.7 reader), and the cell proliferation effects from exposure of cells to each fraction were analyzed as percentages of the control cell absorbances, which were obtained from control wells treated with appropriate concentrations of the compounds vehicles that were processed simultaneously. For each treatment, cell proliferation was evaluated as a percentage using the following equation:
(OD595 of treated sample/OD595 of untreated sample) × 100.
The ability of EVOO-derived phenolic compounds to induce apoptosis was assessed using the Cell Death Detection ELISAPLUS kit obtained from Roche Diagnostics (Barcelona, Spain). Briefly, cells (5 to 10 × 103/well) were grown in 96-well plates and treated, in duplicates, for 72 h with the indicated doses of EVOO polyphenols, as specified. After treatment, the 96-well plates were centrifuged (200 × g) for 10 min. The supernatant was discharged, lysis buffer was added, and samples were incubated at room temperature (RT) for 30 min following the manufacturer's instructions. Anti-histone biotin and anti-DNA peroxidase antibodies were added to each well and incubated at RT for 2 h. After three washes, the peroxidase substrate was added to each well, and the plates were read at 405 nm at multiple time intervals. The enrichment of histone-DNA fragments in treated cells was expressed as fold increase in absorbance as compared with control (vehicle-treated) cells.
Transient transfection of small interference RNAs
The small interfering RNA sequences used for targeted silencing of human HER2 were supplied by Santa Cruz Biotech (Santa Cruz, CA, USA) as double-stranded small interference RNA [Neu siRNA (h) sc-29405]. siRNA A (sc-37007), which consists of a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA, was employed as negative control for experiments using HER2-targeted siRNA transfection. Transfections were performed as described in Santa Cruz technical bulletin. Briefly, cells at a confluence of 60 to 80% were transfected with the selected small interference RNAs using Santa Cruz Biotechnology's siRNA Transfection Reagent (sc-29528) and siRNA Transfection Medium (sc-36868) following the manufacturer's instructions.
HER2-specific Enzyme-Linked Immunosorbent Assay
Determination of HER2 protein content was performed with a commercially available quantitative ELISA (Oncogene Science, Bayer Diagnostics) according to the manufacturer's protocol. To assess the effects of EVOO phenols, HER2 siRNA, NAC, and Trolox on HER2 protein concentrations, breast cancer cells, after a 24 h starvation period in media without serum, were incubated with graded concentrations of EVOO phenolics, HER2 siRNA, siRNA A, NAC or Trolox as specified. After treatment, cells were washed twice with cold-PBS and then lysed in buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride) for 30 minutes on ice. The lysates were cleared by centrifugation in an Eppendorff tube (15 minutes at 14,000 × g, 4°C). Protein content was determined against a standardized control using the Pierce Protein Assay Kit (Rockford, IL, USA).
1:50, 1:500; 1: 5,000 and 1:10,000 dilutions of total cell lysates from EVOO phenols-treated, HER2 siRNA-transfected and control untreated cells were used to quantitate HER2 protein expression in cell cultures. A standard curve was generated by using standard solutions as per manufacturer's instructions. The concentrations of HER2 in test samples (in nanograms of HER2 per milligram of total protein) were determined by interpolation of the sample absorbances from the standard curve. Each experiment was performed in duplicate wells.
Activation status of HER2
Testing for the phosphorylation (activation) status of HER2 was performed by immunoblotting procedures using the monoclonal c-erbB2/HER2 (phosphor-specific) antibody Ab-18 (NeoMarkers, Fremont, CA, USA). Briefly, EVOO polyphenols-treated and untreated control cells were washed twice with cold PBS and then lysed as described above. Equal amounts of protein (i.e. 50 μg) were resuspended in 5× Laemli sample buffer (10 min at 70°C), resolved by electrophoresis on 3–8% NuPAGE Tris-Acetate and transferred onto nitrocellulose membranes. Non-specific binding on the nitrocellulose filter paper was minimized by blocking for 1 h at RT with TBS-T buffer [25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20] containing 5% (w/v) nonfat dry milk. The treated filters were washed in TBS-T and then incubated with the phospho-c-erbB2/HER2 (clone PN2A) antibody in 5% w/v BSA, 1 × TBS-T buffer, 0.1% Tween-20 at 4°C with gentle shaking, overnight. The membranes were washed in TBS-T, horseradish peroxidase-conjugated secondary anti-mouse IgG in TBS-T was added for 1 h, and immunoreactive bands were detected by chemiluminiscence reagent (Pierce, Rockford, IL). Blots were re-probed with an antibody for β-actin to control for protein loading and transfer (data not shown). Densitometric values of proteins bands were quantified using the Scion Image software (Scion Corporation, Frederick, MD, USA).
Two-group comparisons were performed by the Student t test for paired and unpaired values. Comparisons of means of ≥ 3 groups were performed by ANOVA, and the existence of individual differences, in case of significant F values at ANOVA, tested by Scheffé's multiple contrasts.
Effects of EVOO phenolic compounds on breast cancer cell viability, proliferation, and apoptosis in HER2-overexpressing SKBR3 breast cancer cells
EVOO phenolics preferentially inhibit the proliferation of HER2-overexpressing breast cancer cells
EVOO polyphenols deplete HER2 oncoprotein
EVOO polyphenols inhibit HER2 tyrosine phosphorylation
EVOO polyphenols-induced depletion of HER2 protein depends on proteasomal degradation but does not relate to anti-oxidant effects
The finding that all the major complex phenols present in EVOO (i.e. lignans and secoiridoids) exhibit the ability to drastically down-regulate HER2 protein regardless of the molecular mechanism contributing to HER2 overexpression (i.e. naturally by gene amplification in SKBR3 cells and ectopically driven by a viral promoter in MCF-7 cells transduced with the human HER2 cDNA) suggested that the anti-HER2 effects of EVOO polyphenols did not relate to factors controlling the rate of HER2 gene promoter-regulated HER2 de novo synthesis [44, 45]. Accordingly, HER2 mRNA levels did not significantly decline even after 24 hours after treatment with EVOO polyphenols (data not shown). To better delineate the mechanism of EVOO polyphenols-mediated HER2 down-regulation, we tested the following hypotheses: 1.) Since all the main polyphenols that occur at high levels in EVOO have demonstrated antioxidant activity and antioxidants are believed to be responsible for a number of EVOO's biological activities, we envisioned that EVOO polyphenols-induced depletion of HER2 might represent a general response of HER2-overexpressing cancer cells growing upon anti-oxidant conditions. To test this hypothesis MCF-7/HER2 cells and MCF-7/pBABE matched control cells were treated with graded concentrations of the well-established anti-oxidants 6-hydroxy-N-acetylcysteine (NAC, a glutathione precursor and scavenger of reactive oxygen species) and 2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble vitamin E analogue). Interestingly, MCF-7/HER2 cells were more sensitive to the anti-proliferative effects of NAC (Figure 3; Additional file 1) and Trolox (Figure 4; Additional file 1) as they exhibited IC50 values significantly lower (3 to 4-times) than those found in MCF-7/pBABE cells. However, treatment with either Trolox or NAC failed to modulate HER2 protein levels in MCF-7/HER2 cells (Figures 3 and 4; Additional file 1). Overall, these results suggest that anti-oxidant agents preferentially suppress the growth of HER2-overexpressing cancer cells, whereas promoting an antioxidant status in HER2-positive breast cancer cells is not sufficient to down-regulate HER2 expression in human breast cancer cells.
The ultimate molecular mechanism(s) determining how specific components of EVOO may influence the genetic program that drives breast cancer development and progression remain(s) largely obscure. Our experimental approach in the last few years has suggested that EVOO-related anti-breast cancer actions mainly affect the occurrence, the aggressive behavior and the therapeutic management of HER2 oncogene-driven breast carcinomas. First, when we used one of the most useful in vivo carcinogenesis systems of breast cancer – that of the DMBA-induced mammary carcinogenesis in female Sprague-Dawley rats, in which stepwise molecular analysis of the transformation process is conducted from the very early to the terminal stages of tumor development – a high EVOO diet was found to act as a negative modulator of the mammary carcinogenesis induced by genotoxic agents, conferring to the tumors mainly an indolent clinical behavior and a histopathological pattern compatible with a lower degree of malignancy [13, 14]. Mechanistically, we demonstrated that high EVOO diet influenced negatively experimental mammary carcinogenesis modifying the mRNA expression levels of neu (HER2) . Second, when using human breast cancer-derived in vitro models naturally exhibiting HER2 gene amplification and HER2 protein overexpression, exogenous supplementation with physiological concentrations of oleic acid (OA; 18:1n-9) – the main ω-9 monounsaturated fatty acid (MUFA) in EVOO – was found to drastically suppress the expression of HER2 and to synergistically enhance both the growth-inhibitory and the HER2 down-regulatory effects of the monoclonal antibody trastuzumab (Herceptin™) [15–17]. The above findings generated intense public interest, since no toxicities have been reported or suspected with OA, and suggested that supplementation with EVOO might represent a promising dietary intervention aimed to prevent and/or manage HER2-related carcinomas. However, it should be noted that EVOO consists primarily of triacylglycerols rich in the ω-9 MUFA OA and non-glyceridic constitutes comprising approximately 0.5% to 1% EVOO, which include at least 30 phenolic compounds. Although we recently presented evidence that the aglycone form of oleuropein – a member of the secoiridoid family of EVOO polyphenols that has mainly been implicated in the organoleptic characteristics of EVOO such as bitterness – significantly enhances the efficacy of anti-HER2 therapeutics in cultured HER2-overexpressing breast cancer cells when compared to simple phenols (e.g. tyrosol, hydroxytyrosol) , the anti-breast cancer effects of other EVOO-derived polyphenols such as the lignan (+)-1-acetoxypinoresinol or the secoiridoids deacetoxy oleuropein aglycone and ligstroside aglycone remained to be addressed.
We now provide new insights on the mechanisms by which good-quality OO, i.e. polyphenols rich-EVOO, may contribute to lower breast cancer risk in a HER2-dependent manner. First, EVOO phenolic compounds with a simple structure, involving only a single phenol ring might be incapable to exert strong anti-breast cancer actions. Indeed, a more complex (i.e. polyphenolic) structure appears to be required in order to exert these effects. Thus, both the EVOO-derived single phenols tyrosol and hydroxytyrosol and the EVOO-derived polyphenol acid elenolic acid exhibited significantly lower anti-proliferative and pro-apoptotic effects than those observed with the EVOO fractions rich in the complex polyphenols lignans and secoiridoids. Second, when we investigated the tumoricidal effects of EVOO polyphenols against breast epithelial cells bearing different endogenous levels of the HER2 oncogene, it became clear that high levels of HER2 oncoprotein constitute a molecular feature through which EVOO polyphenols exert, at least in part, their anti-breast cancer actions. EVOO polyphenols differentially induced breast cancer cell growth inhibition by promoting apoptotic cell death in HER2-positive breast cancer cells, with marginal tumoricidal effects occurring in HER2-negative breast cancer cells. Moreover, we found that EVOO polyphenols repressed the phosphor-Tyrosine levels of HER2 and also depleted HER2 protein levels. The definite mode of action underlying EVOO polyphenols-induced blockade of HER2 tyrosine kinase activity and down-regulation of HER2 protein expression was beyond the scope of this study. Nevertheless, when considering that the primary mechanism driving HER2 levels in human breast cancer cells is gene amplification and overexpression under the control of the endogenous promoter [44, 45], the fact that EVOO polyphenols drastically decreased HER2 protein content regardless the molecular mechanism contributing to HER2 overexpression strongly suggests that EVOO polyphenols do not significantly affect the cellular transcriptional machinery that controls the endogenous HER2 locus in breast cancer cells. Mechanistically, and unlike the EVOO ω-9 MUFA OA – which has been found to indirectly suppress the transcriptional activity of HER2 gene by up-regulating the transcriptional repressor PEA3 and inducing formation of inhibitory "PEA3 transcription factor-PEA3 DNA binding site" complexes at the HER2 promoter – [18, 19] the anti-HER2 effects of EVOO polyphenols appear to relate to the inhibition of HER2 tyrosine kinase activity as a result of HER2 protein depletion. This notion is supported by the following findings: 1.) EVOO polyphenols-induced inhibition of HER2 expression does not reflect a wider and unspecific inhibitory effect against other HER members closely related to HER2 in terms of structure and activity ; 2.) siRNA-induced depletion of HER2 protein protects breast cancer cells against EVOO-induced cell growth inhibition, thus suggesting that EVOO polyphenols should interact necessarily with the HER2 protein itself to trigger their mechanism of action; 3.) blocking ATP from binding to the tyrosine kinase (TK) domain of HER2 upon treatment with a potent, reversible, selective dual-HER1/HER2 TK inhibitor lapatinib prevents EVOO polyphenols-induced inhibition of breast cancer cell growth, thus suggesting that EVOO lignans and secoiridoids may function as phosphotyrosine-receptor kinase blockers by competing with ATP; and 4.) pre-treatment of HER2-overexpressing breast cancer cells with the proteasome inhibitor MG-132 blocks the depletion of HER2 protein induced by EVOO polyphenols, thus suggesting that EVOO-derived lignans and secoiridoids can efficiently inhibit HER2 protein kinase activity by depleting the HER2 protein kinase itself. Therefore, it is reasonable to suggest that EVOO polyphenols directly affect HER2 levels by promoting proteasomal degradation of HER2 as recently described for other structurally-related naturally-occurring polyphenols such as the flavonoids apigenin and luteolin [41–43].
Although our current findings provide new insights on the mechanisms by which good-quality OO, i.e. polyphenols rich-EVOO, might contribute to lower breast cancer risk in a HER2-dependent manner, extreme caution must be applied when extrapolating in vitro results into clinical practice. One obvious limitation of our current results is that the phenolics that were active (i.e. lignans and secoiridoids) exhibited tumoricidal effects against cultured breast cancer cells at concentrations (> 50 μM) that are unlikely to be achieved in vivo [46–48]. Indeed, it has to be clarified if these compounds will be accessible in the breast tumor tissue in vivo. In this regard, an important step in the body metabolism might be that EVOO polyphenols rapidly split into inactive compounds. In this regard, the secoiridoid oleuropein aglycone is split into hydroxytyrosol or tyrosol and elenolic acid [46–50], both of them notably less effective than the parental secoiridoid in terms of cytotoxicity and HER2 down-regulation. Nevertheless, EVOO-derived lignans may represent a different molecular scenario when compared to EVOO-derived secoiridoids. Saarinen et al. recently evaluated the accessibility and accumulation of lignans to breast cancer tissue after their oral administration to athymic mice bearing MCF-7 human breast cancer tumors . Importantly, tumor tissue accumulated up to 92% of the lignans levels found in serum, thus suggesting that the anticancer activity of lignans may be due to their direct local effects on the breast cancer tissues. Of note, a randomized double-blind placebo-controlled clinical trial recently evaluated the effects of dietary flaxseed, which has an exceptionally high concentration of lignans on tumor biological markers in postmenopausal patients with newly diagnosed breast cancer . The results of this clinical trial demonstrated that daily intake of 25 g flaxseed can significantly reduce cell proliferation, increase apoptosis, and affect cell signaling by reducing HER2 expression of breast tumors. A 71.0% reduction in HER2 expression and an increase in apoptosis (30.7%) were observed in the flaxseed, but not in the placebo group. In fact, the total intake of flaxseed correlated with changes in HER2 expression and apoptotic index .
American Type Culture Collection
Extra Virgin Olive Oil
Capillary Electrophoresis coupled to Mass Spectrometry
Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry
Enzyme-Linked ImmunoSorbent Assay
Epidermal Growth Factor Receptor
Fetal Bovine Serum
Human Epidermal growth factor Receptor 1
Human Epidermal growth factor Receptor 2
High-Performance Liquid Chromatography
Iscove's modified Eagle's medium
3-4, 5-dimethylthiazol-2-yl-2, 5-diphenyl-tetrazolium bromide
Monounsaturated Fatty Acid
Polyomavirus enhancer activator protein 3
small interfering RNA
Time of Flight.
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 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 to JAM). ASC and JAM were also supported by a Grant from the Fundación Científica de la Asociación Española Contra el Cáncer (AECC, Spain) and by the Ministerio de Educación y Ciencia CTQ2005-01914/BQU and Junta de Andalucía (Proyecto de Excelencia AGR-02619).
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