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.

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.

A 50/50 mixture of two commercial EVOO samples (i.e. Picual and Arbequina Spanish varieties) was used for this study. We have used solid phase extraction (SPE) with Diol-cartridges followed by semi-preparative reverse phase-HPLC to isolate different fractions of phenolic compounds from the EVOO. Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) was then employed to establish the composition of the isolated fractions because the principles of separation are completely different to semi-preparative chromatographic employed to isolate the EVOO fractions 3031323334353637. The identification of the phenolic compounds was obtained from the accurate mass and the isotopic pattern of the peaks applying ESI-TOF-MS analyzer. The actual composition of the isolated phenolic fractions, including experimental m/z, the average molecular weight and, when available, the names of the phenolic compounds, is detailed in Table 1. The fractions 1, 2, 3 and 8 mainly included a single compound (i.e. hydroxytyrosol, tyrosol, elenolic acid and ligstroside aglycone, respectively), while the fractions 4, 5, 6 and 7 included a mixture of several polyphenolic compounds (Figure 2). In order to simplify both the results and the discussion sections, the total composition of fractions 4, 5, 6 and 7 was expressed as the main compound contributing to each fraction (i.e. deacetoxyoleuropein aglycone – DAOA-, pinoresinol, acetoxypynoresinol and oleuropein aglycone, respectively). Retention times, absorption maxima, the MS data using accurate mass and analytical parameter of the HPLC-MS method are detailed in Tables 1 and 2 (Additional file 1).

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.

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).

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.

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 ELISA^PLUS 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.

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.

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.

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.

We initially assessed the metabolic status of HER2-overexpressing SKBR3 breast cancer cells following treatments with graded micromolar concentrations of EVOO polyphenols (6.25 → 100 μM). After 5 days of treatment, cell viability was measured using a tetrazolium salt-based (MTT) assay, and the IC₅₀ value for each EVOO polyphenol was calculated as described in "Materials and methods". EVOO single phenols failed to significantly decrease SKBR3 cell viability. Thus, concentrations higher than 100 μM were needed to achieve cytotoxic responses in the presence of hydroxytyrosol, tyrosol, and elenolic acid (fractions 1, 2 and 3 respectively) (Figure 2). EVOO lignans exhibited significant cytotoxic activities against SKBR3 cells. Fraction 6, which mainly contained 1-(+)-acetoxypinoresinol was more effective than fraction 5, which mainly contained (+)-pinoresinol (IC_50s = 51 ± 2 μM and 72 ± 5 μM, respectively) (Figure 2). Among the EVOO phenolic fractions tested, those containing the secoiridoids DAOA, oleuropein aglycone, and ligstroside aglycone were the most potent at decreasing breast cancer cell viability, with all of them exhibiting IC₅₀ values lower than 50 μM (e.g. as low as 26 ± 6 μM for the fraction 8 mainly containing the secoiridoid ligstroside aglycone) (Figure 2). Equivalent pictures emerged when the dose-response effects of EVOO polyphenols on the proliferation rate of SKBR3 were assessed by photometry after crystal violet staining. As shown in Figure 3 (left panel), EVOO single phenols and polyphenol acids produced weak growth inhibitory effects in SKBR3 cells. By contrast, the fractions 5 and 6 containing EVOO lignans (Figure 3, middle panel) and the fractions 4, 7 and 8 containing EVOO secoiridoids (Figure 3, right panel) significantly inhibited SKBR3 cell proliferation in a concentration-dependent manner.

To evaluate whether the inhibitory effects of EVOO-derived lignans and secoiridoids on breast cancer cell viability and proliferation might actually be due to changes in apoptotic cell death, SKBR3 cells were exposed to increasing concentrations of EVOO phenolics, apoptotic cell death was measured by a Cell Death ELISA detecting apoptosis-induced DNA-histone fragmentation, and the x-fold increase in apoptosis was calculated by comparing the ELISA optical density readings of treated samples (with the values obtained in untreated cells as 1.0). In agreement with the results obtained in cell viability and cell proliferation assays, the lowest degree of apoptotic cell death was achieved upon treatment with hydroxytyrosol and elenolic acid (Figure 4). Treatment with the lignans-rich fractions 5 and 6 notably increased (up to ~10-fold at 100 μM 1-(+)-acetoxypinoresinol) the apoptotic cell death of SKBR3 cells (Figure 4). SKBR3 cells were likewise exquisitely sensitive to secoiridoids-rich fractions 4, 7 and 8 as they exhibited the highest degree of apoptotic cell death following exposure to fraction 4 (DAOA, > 5-fold increase), fraction 8 containing (ligstroside aglycone, > 25-fold increase), and fraction 7 (oleuropein aglycone, > 10-fold increase) (Figure 4). Interestingly, the precursor form oleuropein glycoside was significantly less effective than its oleuropein aglycone derivative, thus revealing that the glucose moiety in oleuropein structure strongly affects its anti-breast cancer activity (Figure 4).

SKBR3 cells represent a widely used breast cancer in vitro model characterized by exhibiting natural HER2 gene amplification, HER2 receptor protein overexpression and HER2-dependency for cell proliferation and survival 3839. To evaluate whether EVOO polyphenols preferentially exhibit tumoricidal effects against HER2-overexpressing breast cancer cells, we further explored both the apoptotic/anti-proliferative effects of EVOO polyphenols in MCF-7 breast cancer cells – which express physiological levels of HER2 (i.e. one single copy of HER2 gene) and in MCF-7 cells stably transduced with pBABE-HER2 or pBABE (empty control) retroviral vectors. The level of HER2 overexpression achieved in MCF-7/(pBABE)HER2 cells (~70-fold increase when compared to MCF-7/pBABE matched control and MCF-7 parental cells) was comparable to that reported in MCF-7/Her2-18 cells, a well-characterized MCF-7-derived HER2-overexpressing clone engineered to stably express the full-length human HER2 cDNA controlled by a SV40 viral promoter 40. Forced expression of HER2 in MCF-7 cells increased by > 4-fold the apoptotic effects of the fraction 6 containing the EVOO lignan 1-(+)-acetoxypinoresinol. High levels of HER2 promoted also exacerbated apoptotic responses to fractions 4, 7 and 8 containing mainly EVOO secoiridoids DAOA, ligstroside aglycone, and oleuropein aglycone, respectively. For example, treatment with the DAOA-rich fraction 4 increased apoptotic cell death by 17.5-times and 7.1-times in HER2-overexpressing MCF-7/HER2 transfectants and in HER2-negative MCF-7/pBABE matched control cells, respectively (Figure 5). The notion that HER2 overexpression in breast cancer cells significantly decreases the capability of human breast epithelial cells to overcome EVOO polyphenols-induced cell injuries was further supported when analyzing the anti-proliferative effects of EVOO polyphenols in MCF-7/pBABE and MCF-7/HER2 cells. As shown in Figure 6 (left panel), EVOO single phenols and EVOO polyphenol acids were ineffective against HER2-negative MCF-7/pBABE cells while they exhibited weak growth-inhibitory effects against HER2-overexpressing MCF-7/HER2 cells. Remarkably, the fraction 6 containing mainly the EVOO lignan 1-(+)-acetoxypinoresinol reduced the proliferation rate of MCF-7/HER2 cells by 63%, whereas this reduction was as low as 26% in MCF-7/pBABE matched control cells (Figure 6, middle panel). All the EVOO secoiridoids exhibited slight anti-proliferative effects in HER2-negative MCF-7/pBABE cells. Conversely, HER2-overexpressing MCF-7/HER2 cells were exquisitely sensitive to the growth-inhibitory effects of DAOA-rich fraction 4 (~80% inhibition of cell proliferation at 100 μM), ligstroside aglycone-rich fraction 8 (~50% inhibition of cell proliferation at 100 μM) and oleuropein aglycone-rich fraction 7 (~55% inhibition of cell proliferation at 100 μM) (Figure 6, right panel). Overall, these results suggest that EVOO polyphenols preferentially suppress the growth of HER2-overexpressing breast cancer cells.

To evaluate whether, in terms of cell viability, the biological activity of EVOO polyphenols did require HER2 overexpression as a necessary molecular "hallmark", we used a siRNA HER2 depletion approach to determine the effect of "HER2 protein-dose" on the sensitivity of breast cancer cells to EVOO polyphenols. SKBR3 and MCF-7/HER2 cells were transfected with 80 pmols of HER2 siRNA or control siRNA (i.e. siRNA A). 72 h after HER2-targeted siRNA transfection, HER2 protein levels decreased up to ~65-70% of those present in SKBR3 and MCF-7/HER2 cells transfected with a control siRNA (Figure 1, Additional file 1). Interestingly, a combination of sequential HER2 siRNA followed by treatment with sub-optimal doses of the 1-[+]-acetoxypinoresinol-rich fraction 6 and of the DAOA-rich fraction 4 strikingly demonstrated an antagonistic to protective nature of the cytotoxic interaction (Interaction Indexes > 2.0; Figure 7).

The fact that depletion of endogenous HER2 significantly prevented the growth-inhibitory effects of EVOO polyphenols together with our earlier findings demonstrating that the down-regulatory effects of the secoiridoid oleuropein aglycone were specifically restricted to HER2 without affecting other key members of the oncogenic HER network such as HER1 (EGFR) 20, suggested that secoiridoids- and lignans-induced changes in cell viability might relate, at least in part, to changes in the expression of HER2 oncoprotein. To evaluate this hypothesis we examined the effects of long-term treatments (i.e. 48 hours) with graded concentrations of EVOO polyphenols on the naturally-occurring overexpression of HER2 protein in SKBR3 cells (Figure 8). At its highest concentration (i.e. 100 μM), the EVOO single phenol hydroxytyrosol slightly reduced HER2 protein expression by 35%. Remarkably, HER2 expression was drastically reduced by 68% and 86% in the presence of 100 μM of the fractions 5 and 6 containing mainly EVOO lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, respectively. Similarly to EVOO lignans, EVOO secoiridoids were very effective at inhibiting HER2 expression, with inhibitory percentages ranging from 50%–60% in the presence of DAOA-rich fraction 4 and ligstroside aglycone-rich fraction 8 to 87% inhibition upon treatment with the oleuropein aglycone-rich fraction 7. We then evaluated if the growth inhibitory effects of EVOO polyphenols against MCF-7/HER2 cells were also accompanied with changes in the expression levels of HER2 oncoprotein (Figure 9). The EVOO single phenol hydroxytyrosol notably decreased HER2 protein expression by 41%, while the fraction 6 containing mainly the EVOO lignan 1-(+)-acetoxypinoresinol drastically reduced HER2 expression by 83%. All the EVOO secoiridoids significantly reduced HER2 expression, from ~30% inhibition in the presence of DAOA-rich fraction 4 to 56% inhibition in the presence of the oleuropein aglycone-rich fraction 7. These findings reveal for the first time that all the major complex phenols present in EVOO (i.e. secoiridoids and lignans) drastically suppress HER2 oncoprotein overexpression in human breast cancer cells. Neither secoiridoids nor lignans treatments caused detectable changes in HER1 (EGFR) expression in breast cancer cells (data not shown).

Since apigenin and luteolin, two naturally occurring flavonoids structurally related to EVOO polyphenols, have been found to inhibit tyrosine phosphorylation and deplete HER2 protein through HER2 protein degradation 414243, we envisioned that an analogous posttranslational mechanism may also contribute to the anti-HER2 actions of EVOO polyphenols. First, to evaluate whether the biological activity of EVOO polyphenols in terms of cell viability not only related to HER2 protein overexpression but further required an enhanced tyrosine kinase activity of HER2 as a molecular "hallmark" in EVOO polyphenols-sensitive breast cancer cells, we used a pharmacological approach to determine the effect of "HER2 activity-dose" on the tumoricidal effects of EVOO polyphenols. Similarly to siRNA HER2-induced depletion of HER2 protein, specific blockade of HER2 constitutive hyperactivation (autophosphorylation at Tyr1248; Figure 2, Additional file 1) using the dual-HER1/HER2 tyrosine kinase inhibitor lapatinib (Tykerb™) was sufficient to preclude any further cytotoxic action of EVOO polyphenols. Thus, a combination of sequential lapatinib (0.1 μM) followed by treatment with sub-optimal doses of the 1-[+]-acetoxypinoresinol-rich fraction 6 and DAOA-rich fraction 4 demonstrated an antagonistic to protective nature in their cytotoxic interactions (Interaction Indexes > 2.0; Figure 10). To investigate the kinetics of inhibition/depletion of HER2 activity/expression, we treated SKBR3 and MCF-7/HER2 cells with EVOO polyphenols for different time periods and harvested them for ELISA-based analysis of HER2 protein expression. HER2 tyrosine autophosphorylation levels (i.e. activation status of HER2 Tyr1248) were measured by immunoblotting procedures. The HER2 protein levels decreased in a time-dependent manner following exposure to EVOO polyphenols, with significant HER2 depleting effects occurring as early as 6 hr after treatment with the fraction 6 containing mainly 1-[+]-acetoxypinoresinol (Figure 11). Fraction 6-decreased HER2 protein led to a coordinate decrease in HER2 autophosphorylation, which significantly declined after 6 hr and was barely detectable after 24 hr (Figure 11). Although less markedly than fraction 6, the fraction 4 containing mainly the EVOO secoiridoid DAOA also decreased HER2 protein expression and HER2 tyrosine kinase activity in a time-dependent manner, with the most significant anti-HER2 expression/activity effects occurring after 48 hr (Figure 11). These findings, altogether, strongly suggest that EVOO polyphenols could inhibit HER2 protein kinase activity by depleting the HER2 protein kinase itself.

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 4445. 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.

Since the ability of EVOO polyphenols to deplete HER2 protein did not appear to relate to their impact on the antioxidant status of breast cancer cells, we then speculated that EVOO secoiridoids and lignans might deplete HER2 through proteolysis, the molecular mechanism through which other structurally related plant polyphenols such as the flavonoids apigenin and luteolin suppress HER2 protein expression in breast cancer cells 414243. A role of proteasomal degradation in EVOO polyphenols-mediated HER2 down-regulation was likewise supported by the fact that pre-treatment (2 hr) with the proteasome inhibitor MG-132 fully prevented the reduction of HER2 protein levels induced by EVOO polyphenols (Figure 12). To further corroborate the suggestion that EVOO polyphenols induce cell growth inhibition by depleting HER2 protein in HER2-overexpressing breast cancer cells via proteasomal degradation, we analyzed whether supra-additive or synergistic interactions occurred upon concurrent treatment with EVOO polyphenols and anti-HER2 agents that promote HER2 down-regulation through proteasome-independent mechanisms 124647. Simultaneous exposure of SKBR3 cells to the anti-HER2 monoclonal antibody trastuzumab with sub-optimal doses of the 1-(+)-acetoxypinoresinol-rich fraction 6 or the DAOA-rich fraction 4 likewise demonstrated greater decreases in cell viability that did each agent alone (Interaction Indexes < 1.0; Figure 13). This synergistic interaction occurring between EVOO-derived lignans and secoiridoids further extends our previous findings that demonstrated a supra-additive cytotoxic effect when combining trastuzumab with the EVOO secoiridoid oleuropein aglycone 20.