The olive oil phenolic (−)-oleocanthal modulates estrogen receptor expression in luminal breast cancer in vitro and in vivo and synergizes with tamoxifen treatment

Abstract

Luminal breast cancer represents a therapeutic challenge in terms of aggressive disease and emerging resistance to targeted therapy. (−)-Oleocanthal has demonstrated anticancer activity in multiple human cancers. The goal of this study was to explore the effect of (−)-oleocanthal treatment on growth of luminal breast cancer cells and to examine the effect of combination of (−)-oleocanthal with tamoxifen. Results showed that (−)-oleocanthal inhibited growth of BT-474, MCF-7, and T-47D human breast cancer cells in mitogen-free media with IC₅₀ values of 32.7, 24.07, and 80.93 μM, respectively. Similarly, (−)-oleocanthal suppressed growth of BT-474, MCF-7, and T-47D cells in 17β-estradiol-supplemented media with IC₅₀ values of 22.28, 20.77, and 83.91 μM, respectively. Combined (−)-oleocanthal and tamoxifen treatments resulted in a synergistic growth inhibition of BT-474, MCF-7, and T-47D cells with combination index values of 0.65, 0.61, and 0.53 for each cell line, respectively. In-silico docking studies indicated high degree of overlapping for the binding of (−)-oleocanthal and 17β-estradiol to estrogen receptors, while (−)-oleocanthal and tamoxifen have distinguished binding modes. Treatment with 5 mg/kg or 10 mg/kg (−)-oleocanthal resulted in 97% inhibition of tumor growth in orthotopic athymic mice bearing BT-474 tumor xenografts compared to vehicle-treated animals. (−)-Oleocanthal treatment reduced total levels of estrogen receptors in BT-474 cells both in vitro and in vivo. Collectively, (−)-oleocanthal showed a potential beneficial effect in suppressing growth of hormone-dependent breast cancer and improving sensitivity to tamoxifen treatment. These findings provide rational for evaluating the effect of (−)-oleocanthal in combination with endocrine treatments in luminal breast cancer.

Graphical abstract

1. Introduction

Breast cancer is the most commonly diagnosed carcinoma and a leading cause of cancer-related fatality among women worldwide (Siegel et al., 2017). In 2012, 1.76 million breast cancer cases were diagnosed globally accounting for a quarter of all newly diagnosed cancer cases in women (Ferlay et al., 2015). Breast cancer is a heterogeneous disease that is further classified into different subtypes based on gene expression profiling (Li et al., 2016; Polyak, 2007; 2011). The molecular subtypes include luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-positive, and basal-like breast cancer (Li et al., 2016; Perou and Borresen-Dale, 2011; Polyak, 2011). The molecular subtypes have been associated with distinct pathological features and clinical outcomes (Li et al., 2016; Perou and Borresen-Dale, 2011; Polyak, 2011).

Luminal breast cancers account for about 60% of all cases (Holowatyj et al., 2016). Luminal tumors are positive for hormone receptor and are further classified based on HER2 receptor status (Holowatyj et al., 2016; Polyak, 2011). Luminal A breast cancers are positive to estrogen receptor (ER) and/or progesterone receptor (PR) but HER2-negative (Holowatyj et al., 2016). In contrast, luminal B tumors demonstrate HER2-enrichment and could be positive to one or both hormone receptors (Holowatyj et al., 2016). Furthermore, luminal A tumors are characterized by low expression of the proliferation marker Ki67 while luminal B cancers have been reported to have high expression of Ki67 (Holliday and Speirs, 2011; Li et al., 2016; Subik et al., 2010). Clinically, luminal B cancers are characterized by high histologic grade and aggressive clinical behavior compared to luminal A tumors (Tran and Bedard, 2011). Recent evidence indicated that luminal B breast cancer is associated with higher risk of local recurrence and metastasis similar to rates associated with the more aggressive basal-like and HER2-enriched subgroups (Li et al., 2016). Recommended treatment for luminal B tumors include chemotherapeutic agents, anti-HER2 treatments, and endocrine therapy (Li et al., 2016). Nevertheless, luminal B tumors are associated with reduced sensitivity to endocrine treatment and emerged resistance to anti-HER2 targeted therapies (Lonning, 2012; Tran and Bedard, 2011). Clinical and experimental evidence revealed that HER2 overexpression or amplification reduced response to hormonal treatments in patients with luminal B tumors (Lonning, 2012; Pritchard, 2013). However, understanding the molecular mechanisms for treatment resistance in luminal B breast cancer is challenging and could be related to the interplay of multiple factors including HER2 overexpression, high Ki67 expression, the presence of hormone receptors, and the greater expression of other growth factor receptors (Lonning, 2012; Thakkar and Mehta, 2011).

(−)-Oleocanthal is a naturally occurring secoiridoid (Fig. 1) from extra-virgin olive oil (EVOO) known for its anticancer effects both in vitro and in vivo (Akl et al., 2014; Mohyeldin et al., 2016a; Pei et al., 2016). Anticancer activity of (−)-oleocanthal has been demonstrated in different tumor types including breast carcinoma, prostate carcinoma, hepatocellular carcinoma, colon cancer, melanoma, and multiple myeloma (Akl et al., 2014; Elnagar et al., 2011; Fini et al., 2008; Fogli et al., 2016; Pei et al., 2016; Scotece et al., 2013). (−)-Oleocanthal possesses a wide range of anticancer activities including anti-proliferative, anti-migratory, anti-invasive, and anti-angiogenic effects reported among multiple cancer types (Akl et al., 2014; Elnagar et al., 2011; Fini et al., 2008; Fogli et al., 2016; Pei et al., 2016; Scotece et al., 2013). Multiple molecular targets have been explored to be mediating the anticancer effects of (−)-oleocanthal. Nevertheless, the impact of (−)-oleocanthal on ER as a potential molecular target in breast cancer has gained little attention. In 2014, Keiler et al. investigated the potential of (−)-oleocanthal binding to ER and its effect on bone loss induced by estrogen ablation in experimental animal model (Keiler et al., 2014). This study showed that (−)-oleocanthal can potentially bind to ER (Keiler et al., 2014). These preliminary findings were insightful to further investigate the effect of (−)-oleocanthal treatment on estrogen-dependent breast cancers.

Fig. 1.

Chemical structure of (−)-oleocanthal.

Based on the established anticancer properties of (−)-oleocanthal in breast cancer together with evidence for potential interaction of (−)-oleocanthal with ER, the purpose of the current study was to characterize the effect of (−)-oleocanthal on 17β-estradiol-dependent growth of luminal breast cancer cells alone and in combination with the endocrine treatment tamoxifen. In addition, the current study examined the effects of (−)-oleocanthal on luminal B cancer growth in vivo and the effect of the compound on ER expression in this subtype of luminal breast cancers.

2. Material and methods

2.1. Chemicals, reagents, and antibodies

All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated. All antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA), unless otherwise stated.

2.1.1 Isolation of (−)-oleocanthal

1 L of commercial extra-virgin olive oil (EVOO, Daily Chef, batch number: L022RE-565, Italy) was shaken with methanol (3×0.5 L, VWR, Suwanee, GA) and the concentrated methanol layer was fractionated twice on Sphadex LH20 (Sigma Aldrich, bead size 25–100 μm) using isocratic methylene chloride elution and finally HPLC purified on a Phenomenex Cosmosil 5C18-AR-II column (250 mm × 4.6 mm, 5 μm; Phenomenex Inc., Torrance, CA) using isocratic elution was performed using Water-Acetonitrile (6:4) as a mobile phase.

2.1.2 HPLC analysis

(−)-Oleocanthal purity of >99% was established based on HPLC analysis on a Simadzu HPLC system equipped with UV/Visible variable wavelength detector. (−)-Oleocanthal was dissolved in 50% v/v acetonitrile in water as a mobile phase. Samples (20 μl) were then injected into the Eclipse YD5 C18-RP analytical column (4.6 mm × 15 cm) which has been pre-heated to 40°C. The flow rate of the mobile phase was 1.0 ml/min and the analytes were simultaneously detected using the UV detector at λ 230 and 254 nm with 2.8 min retention time. Data acquisition and analysis were performed using Lab Solution™ chromatography software.

2.1.3 Quantitive proton NMR (q¹H NMR) spectral analysis

A >99% (−)-oleocanthal purity was established using q¹H NMR in CDCl₃ using tetramethylsilane (TMS) as an internal standard, on a JEOL Eclipse-ECS NMR spectrometer operating at 400 MHz. Calibration curve was established using known concentrations of pure (−)-oleocanthal as an external standard and quantitation was based on the integration ratio of the (−)-oleocanthal’s key aldehydic proton signal at 9.23 ppm and the residual CHCl₃ peak in the CDCl₃ at 7.24 ppm.

2.2. Cell lines and culture conditions

The human breast cancer cell lines BT-474, MCF-7, and T-47D were purchased from American Tissue Culture Collection (ATCC) (Rockville, MD, USA). BT-474 breast cancer cell line represents luminal B subtype because of the expression of ER and amplification of HER2 receptor (Holliday and Speirs, 2011). BT-474 cells are Ki67 high, endocrine-responsive, and trastuzumab-responsive (Holliday and Speirs, 2011). Both MCF-7 and T-47D cancer cell lines represent luminal A subtype which are positive for hormone receptors and negative for HER2. These cells are characterized by low Ki67 and being endocrine-responsive (Holliday and Speirs, 2011). Cells were maintained in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), 10 μg/ml insulin, 100 U/ml penicillin G, and 0.1 mg/ml streptomycin at 37°C in an environment of 95% air and 5% CO₂ in humidified incubator. For subculturing, cells were rinsed in sterile Ca²⁺ and Mg²⁺-free phosphate-buffered saline (PBS) and incubated in 0.05% trypsin containing 0.025% EDTA in PBS for 3–5 min at 37°C. Released cells were centrifuged, resuspended in 10% FBS medium and counted using a hemocytometer.

2.3. Experimental treatments

(−)-Oleocanthal was dissolved in dimethyl sulfoxide (DMSO) to provide a final 25 mM stock solution. Tamoxifen was dissolved in ethanol to form a 10 mM stock solution. These stock solutions were used to prepare various concentrations of treatment media. 17β-Estradiol powder was dissolved in absolute ethanol to form a 37 mM stock solution which was added to working media to form a final concentration of 10 nM. The final concentration of DMSO and ethanol were maintained the same in all treatment groups within a given experiment and never exceeded 0.1% of each.

2.4. Measurement of viable cell number

Viable cell count was determined using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay (Riss et al., 2004). Briefly, control and treatment media in various treatment groups were replaced with fresh media containing 0.41 mg/ml MTT. After 4 h incubation at 37°C, media was removed and formazan crystals were dissolved in DMSO (100 μl/well for 96-well plates). Optical density was measured for each sample at 570 nm on a microplate reader (BioTek, Winooski, VT, USA). Number of cells/well was calculated against a standard curve prepared by plating various concentrations of cells, as determined using a hemocytometer at the start of each experiment.

2.5. Cell growth and viability studies

To evaluate the effect of (−)-oleocanthal, tamoxifen, and the combination of both compounds on viability of luminal breast cancer cells, growth studies were performed. Cancer cells were initially seeded at 1×10⁴ cells/well (6 replicates/group) in 96-well plates in RPMI-1640 media containing 10% FBS and allowed to attach overnight. Next day, cells were washed with PBS and divided into different treatment groups. Cells were exposed to respective experimental treatments containing various concentrations of (−)-oleocanthal, tamoxifen, or combination of both compounds in mitogen-free or 17β-estradiol-containing media (10 nM) for 48 h. Viable cell number was determined at the end of the experiments using the MTT assay.

2.6. Western blot analysis

To evaluate the effect of (−)-oleocanthal on expression levels of estrogen receptor-alpha (ERα), BT-474 breast cancer cells were initially plated at 1×10⁶ cells/100 mm culture plates in RPMI-1640 media supplemented with 10% FBS and allowed to adhere overnight. Cells were washed afterwards with PBS and treated with the respective control or treatment media containing various concentrations of (−)-oleocanthal in mitogen-free media or media containing 10 nM 17β-estradiol as the mitogen for 48 h. Cells were lysed in RIPA buffer (Qiagen Sciences Inc., Valencia, CA) and protein concentration was determined by the Pierce BCA Protein Assay (Thermo Fisher Scientific Inc., Rockford, IL) at the end of treatment period. Equal amount of protein (30 μg) of each sample was subjected to electrophoresis through SDS-polyacrylamide gels. The gels were then electroblotted onto Polyvinylidene difluoride (PVDF) membranes. These PVDF membranes were then blocked with 2% BSA in 10 mM Tris-HCl containing 50 mM NaCl and 0.1% Tween 20, pH 7.4 (TBST), for 2 h at room temperature. Membranes were then incubated with primary antibody against ERα at 1:1000 dilution in 2% BSA in TBST overnight at 4°C. At the end of incubation period, membranes were washed five times with TBST and then incubated with respective horseradish peroxide-conjugated secondary antibody at 1:3000 dilution in 2% BSA in TBST for 1 h at room temperature followed by five times rinsing with TBST. Blots were then visualized by chemiluminescence according to the manufacturer’s instructions (Pierce, Rockford, IL). Images of protein bands from all treatment groups within a given experiment were acquired using Kodak Gel Logic 1500 Imaging System (Carestream Health Inc, New Haven, CT, USA). Visualization of β-tubulin was used to ensure equal sample loading in each lane. All experiments were repeated at least three times. ImageJ software (http://imagej.nih.gov/ij/) was used to run densitometric analysis for Western blot bands. For quantification, values obtained from densitometry of Western blot images for the various treatment groups were normalized to their respective β-tubulin and control densitometric values to clearly visualize differences between treatment groups.

2.7. Immunocytochemical fluorescent staining

To evaluate the effect of (−)-oleocanthal on expression and localization of ERα, BT-474 cells were seeded on 8-chamber culture slides (Becton Dickinson and Company, NJ, USA) at 4×10⁴ cells/chamber (2 replicates/group) and allowed to attach overnight in medium supplemented with 10% FBS. Cells were then washed in PBS and incubated with vehicle control or treatment medium containing various concentrations of (−)-oleocanthal in mitogen-free or 10 nM 17β-estradiol-supplemented media for 24 h. At the end of treatment, cells were washed in pre-cooled PBS, and fixed with 1:1 volume of methanol: acetone precooled to − 20°C for 2 min. Fixed cells were washed in PBS and blocked with 2% BSA-TBST for 1 h at room temperature. Afterwards, BT-474 cells were incubated in primary antibody to ERα (1:3000) in 2% BSA-TBST overnight at 4°C. Thereafter, cells were washed in pre-cooled PBS followed by incubation with goat anti-rabbit Alexa Fluor 594-conjugated secondary antibody (1:3000) in 2% BSA-TBST for 1 h at room temperature. After final washing, cells were embedded in Vectashield mounting medium with DAPI (Vector Laboratories IN, Burlingame, CA, USA). Fluorescent images were captured using Nikon Eclipse Ti–S inverted fluorescence microscope (Norcross, GA, USA) at a magnification of 200×.

2.8. Molecular modeling

The in silico experiments were carried out using the Schrödinger molecular modeling software package installed on an iMac 27-inch Z0PG workstation with a 3.5 GHz Quad-core Intel Core i7, Turbo Boost up to 3.9 GHz, processor and 16 GB RAM (Apple, Cupertino, CA, USA).

2.8.1. Protein structure preparation

The X-ray crystal structure of the human estrogen receptor-alpha ligand binding domain (hERα LBD); residues 297–554, (PDB code: 1A52) (Tanenbaum et al., 1998) was retrieved from the Protein Data Bank (www.rcsb.org). The Protein Preparation Wizard of the Schrödinger suite was implemented to prepare the binding domain (Olsson et al., 2011). The protein was reprocessed by assigning bond orders, adding hydrogens, creating disulfide bonds and optimizing H-bonding networks using PROPKA (Jensen Research Group, Copenhagen, Denmark). Finally, energy minimization with a root mean square deviation (RMSD) value of 0.30 Å was applied using an Optimized Potentials for Liquid Simulation (OPLS_2005, Schrödinger, New York, USA) force field.

2.8.2. Ligand structure preparation

The structures of (−)-oleocanthal and tamoxifen were sketched in the Maestro 9.3 panel (Maestro, version 9.3, 2012, Schrödinger, New York, USA). The Lig Prep 2.3 module (LigPrep, version 2.3, 2012, Schrödinger, New York, USA) of the Schrödinger suite was used to generate the 3D structures and to search for different conformers. The Optimized Potentials for Liquid Simulation (OPLS_2005, Schrödinger, New York, USA) force field was applied to geometrically optimize the structures of (−)-oleocanthal and tamoxifen and compute partial atomic charges. Finally, at most, 32 poses were generated with different steric features for subsequent docking studies.

2.8.3. Molecular docking

The prepared X-ray crystal structure of the human estrogen receptor ligand binding domain (hERα LBD) was employed to generate receptor energy grids using the default value of the protein atomic scale (1.0 Å) within the cubic box centered on the co-crystallized 17β-estradiol. After receptor energy grids generation, the prepared structures of (−)-oleocanthal and tamoxifen were docked using the Glide 5.8 module (Glide, version 5.8, 2012, Schrödinger, New York, USA) (Friesner et al., 2006).

2.9. Xenograft studies

In order to evaluate the effect of (−)-oleocanthal administration on luminal B tumor growth in vivo, efficacy studies in athymic mice bearing BT-474 tumor xenografts were conducted. Female athymic nude mice (Foxn1^nu/Foxn¹⁺, 4–5 weeks old) were purchased from Harlan (Indianapolis, IN, USA). The mice had free access to drinking water and pelleted rodent chow (no. 7012, Harlan/Teklad, Madison, WI, USA). The animals were acclimated to the animal housing facility and maintained under clean room conditions in sterile filter top cages with Alpha-Dri bedding and housed on high efficiency particulate air-filtered ventilated racks at a temperature of 18–25°C, with a relative humidity of 55% to 65% and a 12 h light/dark cycle, for at least one week before the study. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC), University of Louisiana at Monroe, and were handled in strict accordance with good animal practice as defined by the NIH guidelines.

Under anesthetic condition, sixty-day release pellets containing 17β-estradiol (0.72 mg) from Innovative Research of America (Sarasota, FL, USA) were implanted into the inter-scapular region of each nude mouse 24 h before implantation of BT-474 cells. BT-474 cells were harvested by trypsinization, pelleted by centrifugation at 850× g for 5 min, and re-suspended in sterile serum-free RPMI-1640 medium. BT-474 cells (1×10⁷ cells) were resuspended in 1:1 volume of matrigel/RPMI-1640 medium (30:30 μl) and were subcutaneously inoculated into the second mammary gland fat pad just beneath the nipple of each animal after anesthesia to generate orthotropic breast tumors. Two weeks post-inoculation, animals developed palpable tumors and were randomly divided into three groups: (i) Vehicle-treated control group (n=5); (ii) (−)-Oleocanthal-treated group at a dose of 5 mg/kg (n=5), and (iii) (−)-Oleocanthal-treated group at a dose of 10 mg/kg (n=5). Selection of (−)-oleocanthal doses used in animal experiments was primarily based on earlier in vivo studies on (−)-oleocanthal (Abuznait et al., 2013). (−)-Oleocanthal treatment was prepared by dissolving 5 mg of the compound in 250 μl of DMSO to prepare a stock solution, then dissolving 50 μl of this solution in 950 μl normal saline just prior to animal injection. Animal treatment started 14 days post-inoculation of BT-474 cells with intraperitoneal (i.p.) administration of vehicle control (DMSO/saline) or the respective (−)-oleocanthal treatments administered three times weekly for a duration of 43 days. Mice were monitored regularly by measuring tumor volume, body weight, and careful observation of their behavior. Tumor volume (V) was calculated using the formula V=(LxW²)/2, where L is the length and W is the width of tumors in millimeters as measured using a digital Vernier caliper every other day during the duration of the experiment. Similarly, body weight of each mouse was monitored every other day in grams. On the 60^th day post-inoculation, tumors were excised and weighed. Breast tumor tissues were stored at – 80°C until total protein extraction for Western blot analysis or stored in 70% ethanol at room temperature for immunohistochemistry studies.

2.10. Statistics

Data analysis was performed using IBM SPSS statistical package (IBM Corp. Version 21.0. Armonk, NY, USA). The results are presented as mean ± standard error of the mean (S.E.M.) for continuous variables. Differences between various treatment groups were determined by analysis of variance (ANOVA) followed by Tukey HSD test. All P-values were two-sided and differences were considered to be statistically significant at P<0.05.

IC₅₀ values (concentrations that induced 50% cell growth inhibition) were determined applying non-linear regression curve fit analysis using GraphPad Prism software version 5. Assessment for the effect of combination treatment of (−)-oleocanthal and tamoxifen was determined by combination index (CI), dose reduction index (DRI), and isobologram analysis. CI is a quantitative representation of pharmacological interaction between two compounds (Chou, 2006; 2010). CI values of less than 1 represents synergistic effects while CI values of 1 and greater than 1 indicate additive and antagonistic effects, respectively. CI value for the combination in this study was calculated as follows (Chou, 2006):

$$\mathrm{\text{CI}}=[\mathrm{\text{Oc}}/\mathrm{O}+\mathrm{\text{Tc}}/\mathrm{T}]$$

O and T stand for the IC₅₀ concentrations of individual (−)-oleocanthal and tamoxifen, respectively, which induced 50% cell growth inhibition; Oc and Tc are concentrations of (−)-oleocanthal and tamoxifen which inhibited cell growth by 50% when used in combination. DRI value represents fold decrease in dose of individual compounds when used in combination, compared to concentration of its single agent needed to achieve the same effect level (Chou, 2010). DRI values greater than 1 are favorable allowing reduction in drug toxicity, while maintaining therapeutic efficacy of compounds (Chou, 2006; 2010). DRI values for (−)-oleocanthal and tamoxifen were calculated as the following:

$$\mathrm{\text{DRI}}(\text{-})\mathrm{\text{-oleocanthal}}=\mathrm{O}/\mathrm{\text{Oc}}\mathrm{\text{and}}\mathrm{\text{DRI}}\mathrm{\text{tamoxifen}}=\mathrm{T}/\mathrm{\text{Tc}}$$

Isobologram analysis is a graphical method used to evaluate the effect of equally effective dose pairs for a single effect level (Tallarida, 2006). An isobologram is constructed on a coordinate system composed of individual drug doses and commonly contains a straight ‘line of additivity’ that is employed to distinguish additive from synergistic and antagonistic interactions (Tallarida, 2006). The straight line in each isobologram was formed by plotting IC₅₀ doses of tamoxifen and (−)-oleocanthal on x- and y-axes, respectively. The solid line connecting these points represents concentration for each compound that induced the same relative growth inhibition when used in combination, if the interaction between these compounds is additive. The data point in the isobologram corresponds to IC₅₀ dose of (−)-oleocanthal and tamoxifen given in combination. If a data point is on the line, this represents additive treatment effect, whereas a data point that lies below or above the line indicates synergism or antagonism, respectively (Tallarida, 2006). The tumor growth inhibition (TGI) is commonly used to quantify anti-tumor activity in drug screening tumor xenograft experiments (Wu, 2010). In this study, TGI was calculated for the entire dosing period on the last day of study using the following formula:

$$\%\mathrm{\text{TGI}}=(1-[(T_{\mathrm{t}}\text{/}{T}_0)/(C_{\mathrm{t}}/C_0)]\text{/}1-[C_0/C_{\mathrm{t}}])\times 100$$

where T_t = median tumor volume of treated animals at time t, T₀ = median tumor volume of treated animals at time 0, C_t = median tumor volume of control mice at time t, and C₀ = median tumor volume of control animals at time 0 (Ji et al., 2007). Tumor growth inhibition of more than 50% is considered meaningful for anti-tumor efficacy (Ji et al., 2007).

3. Results

3.1. Effect of (−)-oleocanthal treatment on growth of luminal breast cancer cell lines

Effects of various doses of (−)-oleocanthal on in vitro growth of multiple luminal breast cancer cell lines is indicated in Fig. 2. Luminal breast cancers are 17β-estradiol-responsive (Daniel et al., 2015; Holliday and Speirs, 2011). Therefore, effect of (−)-oleocanthal on viability of luminal breast cancer cells was examined in both mitogen-free and 17β-estradiol-supplemented culture media. Previous assessment for the concentration of 17β-estradiol in these experiments showed no significant difference in cancer cell growth over a range of 17β-estradiol concentrations (10–50 nM) over 48 h culture period (data not shown). Thus, a mitogenic concentration of 10 nM 17β-estradiol was considered for these experiments. (−)-Oleocanthal resulted in a dose-dependent reduction in cancer cell viability in both mitogen-free and 17β-estradiol-supplemented media (Fig. 2). Treatment with 20–60 μM (−)-oleocanthal significantly inhibited BT-474 cell growth compared to vehicle-treated control cells in 17β-estradiol-supplemented media. The IC₅₀ values for (−)-oleocanthal treatment in mitogen-free and 17β-estradiol-supplemented culture media were 32.7 μM and 22.28 μM in BT-474 cells, respectively. (−)-Oleocanthal treatment also resulted in a dose-dependent inhibition of MCF-7 and T-47D cell growth in mitogen-free media with IC₅₀ values of 24.07 μM and 80.93 μM, respectively. Similarly, (−)-oleocanthal suppressed growth of luminal A cells in media containing 17β-estradiol at IC₅₀ concentrations of 20.77 μM and 83.91 μM for MCF-7 and T-47D cells, respectively.

Fig. 2.

Effects of (−)-oleocanthal treatment on viability of luminal breast cancer cells in vitro. (A) Effects of (−)-oleocanthal treatment on growth of luminal breast cancer cells in mitogen-free media after 48 h culture period. (B) Effects of (−)-oleocanthal treatment on growth of luminal breast cancer cells in 17β-estradiol-supplemented media (10 nM) after 48 h culture period. In these assays, cells were plated at a density of 1×10⁴ cells per well (six replicates/group) in 96-well plates in media supplemented with 10% FBS and allowed to adhere overnight. The next day, cells were washed with PBS, divided into different treatment groups and exposed to various concentrations of (−)-oleocanthal throughout two day culture period. At the end of treatment, viable cell number was determined by the MTT colorimetric assay. Each experiment was repeated three times. Vertical bars represent the mean cell count ± S.E.M. in each treatment group. *P<0.05 compared with respective vehicle-treated control group.

3.2. Effects of combined (−)-oleocanthal and tamoxifen treatment on growth of luminal breast cancer cell lines

Tamoxifen treatment caused a dose-dependent inhibition of growth of luminal breast cancer cells after 48 h of culture period in 17β-estradiol-supplemented media (Fig. 3A). The IC₅₀ values for tamoxifen treatment were 6.04 μM, 1.81 μM, and 16.46 μM for BT-474, MCF-7, and T-47D cells, respectively. The combination of subeffective concentrations of tamoxifen to a dose range of (−)-oleocanthal resulted in suppression of luminal breast cancer cell growth in a dose-dependent manner after 48 h of treatment in 17β-estradiol-containing culture media. Combined treatment of 0.5 μM tamoxifen with 20–40 μM (−)-oleocanthal significantly inhibited BT-474 cell growth compared to both vehicle-treated control cells and the respective (−)-oleocanthal-treated cells (Fig. 3B). Similarly, combination of subeffective dose of tamoxifen with (−)-oleocanthal suppressed both MCF-7 and T-47D cell growth. Isobologram analysis for the effect of combination treatment of (−)-oleocanthal and tamoxifen in all three cell lines indicated a synergistic inhibition of cell growth as the data points were located below the line of additivity in the isobolograms (Fig. 3C). The IC₅₀ value calculated for (−)-oleocanthal treatment in combination with tamoxifen was 12.63 μM for BT-474, 6.77 μM for MCF-7, and 31.46 μM for T-47D cells. Combination index (CI) calculated for growth suppressive effects of combined (−)-oleocanthal and tamoxifen treatment indicated high level of synergism with values less than 1 among all luminal cell lines (Table 1). In addition, DRI values calculated for combined (−)-oleocanthal and tamoxifen treatments showed multiple-fold reductions for both compounds when used in combination (Table 1).

Fig. 3. Effects of (−)-oleocanthal and tamoxifen combination treatment on viability of luminal breast cancer cell lines.

(A) Effects of tamoxifen treatment on 17β-estradiol-induced growth of luminal breast cancer cells after 48 h culture period. (B) Effects of combined (−)-oleocanthal and tamoxifen treatment on growth of luminal breast cancer cells in 10 nM 17β-estradiol-supplemented media after 48 h of treatment duration. In these assays, cells were plated at a density of 1×10⁴ cells per well (six replicates/group) in 96-well plates and maintained in media supplemented with 10% FBS and allowed to adhere overnight. The next day, cells were washed with PBS, divided into different treatment groups and exposed to various concentrations of (−)-oleocanthal and/or tamoxifen throughout two day culture period. At the end of treatment, viable cell number was determined by the MTT colorimetric assay. Each experiment was repeated three times. Vertical bars represent the mean cell count ± S.E.M. in each treatment group. *P<0.05 compared with respective vehicle-treated control group, and **P<0.05 compared to respective group with individual (−)-oleocanthal treatment. (C) Isobolograms of (−)-oleocanthal and tamoxifen anti-proliferative effect in luminal breast cancer cells. IC₅₀ concentrations for (−)-oleocanthal and tamoxifen were plotted on the y- and x-axis, respectively. The solid line connecting these points represents the concentration of each compound required to induce the same relative growth inhibition when used in combination if the interaction between the compounds is additive. The data point on each isobologram represents the actual concentrations of (−)-oleocanthal and tamoxifen which induced 50% inhibition of cell growth when used in combination. ND: Not detectable.

Table 1.

Combination index and dose reduction index values for combined treatment of (−)-oleocanthal and tamoxifen resulting in 50% reduction in 17β-estradiol-mediated growth of multiple luminal breast cancer cell lines.

Mammary cancer cell line	Classification	Receptor status	Ki67 expression level	CI	(−)-Oleocanthal DRI	Tamoxifen DRI
BT-474	Luminal Β	ER+, PR+/−, HER2+	High	0.65	1.8	12.1
MCF-7	Luminal A	ER+, PR+/−, HER2-	Low	0.61	3.1	3.62
T-47D	Luminal A	ER+, PR+/−, HER2-	Low	0.53	2.67	6.58

CI, combination index; DRI, dose reduction index; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; PR, progesterone receptor.

3.3. Effect of (−)-oleocanthal on expression of estrogen receptor in BT-474 breast cancer cells

Effects of (−)-oleocanthal treatment on the expression of estrogen receptor-α (ERα) in BT-474 luminal B cancer cells is shown in Fig. 4. Western blot analysis indicated that treatment with growth inhibitory concentrations of (−)-oleocanthal (20 and 40 μM) resulted in a reduction in the total intracellular levels of ERα compared to vehicle treated controls after 48 h of culture period (Fig. 4A). The effect of (−)-oleocanthal in reducing total levels of ERα was observed in both mitogen-free and 17β-estradiol-supplemented culture conditions in BT-474 cells. The effect of (−)-oleocanthal treatment on expression and localization of ERα was further analyzed using immunocytochemistry (Fig. 4B). Immunofluorescent staining of BT-474 cells indicated strong expression of ERα in both vehicle treated mitogen-free and 17β-estradiol-containing culture media. Estrogen receptors showed a prominent nuclear localization in BT-474 cells in both treatment groups. (−)-Oleocanthal treatment caused a dose-dependent reduction in total levels of ERα compared to cells in their respective control groups in both mitogen-free and 17β-estradiol supplemented treatment media (Fig. 4B).

Fig. 4. Effects of (−)-oleocanthal treatment on estrogen receptor expression in BT-474 cells in vitro.

(A) Western blot analysis for the effect of (−)-oleocanthal treatment on ERα expression in BT-474 cancer cells. Cells were plated at 1×10⁶ cells/100 mm culture plates in RPMI-1640 media supplemented with 10% FBS and allowed to adhere overnight. Next day, cells were incubated with respective (−)-oleocanthal treatment in serum-free media or media containing 10 nM 17β-estradiol as the mitogen for two days. At the end of treatment, whole cell lysates were prepared then subjected to polyacrylamide gel electrophoresis and Western blot analysis for total intracellular levels of ERα. β-Tubulin was visualized to ensure equal sample loading in each lane. Scanning densitometric analysis was performed on all blots and the integrated optical density of each band was normalized with corresponding β-tubulin, as shown in bar graphs under their respective Western blot images. Vertical bars in the graph indicate the normalized integrated optical density of bands visualized in each lane. (B) Immunocytochemical fluorescence staining of total levels of ERα in BT-474 breast cancer cells treated with variable concentrations of (−)-oleocanthal. Cells were initially plated at density of 4×10⁴ cells/chamber (2 replicates/group) and allowed to attach overnight in media supplemented with 10% FBS. Cells were then washed with PBS and incubated with vehicle control or treatment media containing various concentrations of (−)-oleocanthal in mitogen-free or 10 nM 17β-estradiol-supplemented media for 24 h. Afterwards, cells were fixed in methanol: acetone system and subjected to immunofluorescence analysis for detection of total ERα levels. Red staining indicates positive immunofluorescence signal for ERα and blue staining indicates cell nuclei counter-stained with DAPI. Magnification of each photomicrograph is 200×. Scale is 50 μm.

3.4. In-silico binding of (−)-oleocanthal and tamoxifen with estrogen receptor

(−)-Oleocanthal was docked into the ligand-binding domain of the prepared human estrogen receptor-alpha (hERα LBD, PDB code: 1A52) (Tanenbaum et al., 1998). The virtual binding mode (Fig. 5A) showed that (−)-oleocanthal can orient, in its best energetically favored conformation, at the ligand-binding pocket by two types of interactions: hydrogen bonding and van der Waals. The C-6` phenolic hydroxy group of the tyrosol part and aldehydic functionality of elenolic acid anchored (−)-oleocanthal to the helix-3 of the ERα LBD, through hydrogen bonding interactions. (−)-Oleocanthal’s phenolic hydroxy group displayed a hydrogen bond donor interaction with the backbone carbonyl oxygen of Glu346, an identical interaction to the 17β-estradiol C-3 phenolic hydroxy group. The C-3 aldehydic oxygen of (−)-oleocanthal accepted a hydrogen bond with the side-chain hydroxyl of Hid524, an identical interaction for the C-17β hydroxy group in estradiol ring D. Interestingly, the phenolic ring of (−)-oleocanthal’s tyrosol part showed aromatic face-to-edge type π-π stacking with the phenyl side-chain of Phe404, the same interacting residue with the cognate ligand 17β-estradiol (Brzozowski et al., 1997). Moreover, the overlay of the 3D structures of (−)-oleocanthal and 17β-estradiol showed a high degree of overlapping (Fig. 5B). (−)-Oleocanthal’s aromatic ring was in a perfect π-π stacking interaction with the aromatic ring A of the 17β-estradiol steroidal scaffold and the hydrogen bonding acceptor C-3 aldehyde group was in a close proximity to the C-17 terminal β- hydroxyl group of the 17β-estradiol ring D, which accepted a hydrogen bond interaction with Hid524. The ester linkage in (−)-oleocanthal seems to properly direct its acid and alcohol ends in such a way to occupy the same binding pocket where the 17β-estradiol binds and thus enables (−)-oleocanthal’s pharmacophores to favorably interact with the hotspots residues of ERα. The Arg394 did not directly interact with 17β-estradiol but it supports the favorable locations of both Glu353 and Phe404 for proper interactions with 17β-estradiol functionalities. Docking of tamoxifen to the ERα LBD of the same crystal structure (PDB 1A52) indicated that while tamoxifen aromatic ring C is π-π-stacking with Phe404, it also has a unique hydrogen bond donor interaction between its quaternized tertiary amine and the carboxyl group of Asp351, unlike both 17β-estradiol and (−)-oleocanthal (Fig. 5C). Meanwhile, the 4-hydroxytamoxifen, the active human metabolite of tamoxifen, showed similar binding mode and interactions with Phe404 and Asp351 like tamoxifen, in addition to strong hydrogen bond donor interaction between its new ring C hydroxy group with Glu353 and hydrogen bond accepting interaction with Arg394, justifying its superior activity (30–100 fold) versus the parent compound tamoxifen (Fig. 5D). Comparison of binding modes (Figs. 5E–H) may justify the potential of (−)-oleocanthal to synergize the estrogen receptor modulatory activity of tamoxifen and 4-hydroxytamoxifen via its unique hydrogen bonding interaction with Hid524, which is analogous to the 17β-estradiol binding interaction and not targeted by tamoxifen or its active metabolite.

Fig. 5. In-silico binding mode of (−)-oleocanthal and tamoxifen to human estrogen receptor.

(A) The prepared X-ray crystal structure of the human estrogen receptor ligand binding domain (hERα LBD, PDB code 1A52) was employed to generate receptor energy grids using the default value of the protein atomic scale (1.0 Å) within the cubic box centered on the cocrystallized 17β-estradiol. The olive oil phenolic (−)-oleocanthal was docked into the ligand-binding domain of the prepared human estrogen receptor-alpha (hERα LBD). (B) The overlay of the 3D structures of (−)-oleocanthal and 17β-estradiol showing a high degree of overlapping. (C) The overlay of the 3D structures of tamoxifen and 4-hydroxytamoxifen (D) at the same hERα LBD crystal structure (PDB code 1A52). (E-H) Comparison of the 2D binding modes of oleocanthal, 17β-estradiol, tamoxifen, and 4-hydroxytamoxifen, respectively, at the hERα LBD crystal structure PDB code 1A52.

3.5. In vivo antitumor activity of (−)-oleocanthal in BT-474 xenograft animal model

Antitumor activity of (−)-oleocanthal was tested in an orthotopic athymic mice bearing BT-474 tumor xenografts. Animals were treated with (−)-oleocanthal in two different concentrations (5 mg/kg and 10 mg/kg) for approximately six consecutive weeks. At the end of treatment duration, mean tumor weight in vehicle-treated control group was 1515.32±273.10 grams. Mean tumor weight for (−)-oleocanthal-treated groups was 178.9±37.04 and 103.86±40.92 grams for the 5 mg/kg and 10 mg/kg treatments, respectively (Fig. 6A, left). In addition, mean tumor volume at the end of study was 2066.02±274.73, 156.67±48.86, and 44.19±9.47 mm³ for vehicle control, 5 mg/kg (−)-oleocanthal, and 10 mg/kg (−)-oleocanthal-treated groups, respectively (Fig. 6A, right). Post hoc analysis showed no significant difference in mean tumor weight or volume between both (−)-oleocanthal treatment groups. (−)-Oleocanthal treatment at 5 mg/kg resulted in 97.26% inhibition of BT-474 tumor growth, while 10 mg/kg treatment resulted in reduction of tumor growth by 97.06%, compared to vehicle-treated control animals (Fig. 6B). Effect of (−)-oleocanthal treatment on body weight of animals is shown in Fig. 6C. (−)-Oleocanthal treatment did not result in loss of mice body weight and considered to lack systemic toxicity in treated animals. In addition, Western blot analysis of isolated tumors showed relatively lower levels of total ERα in (−)-oleocanthal-treated groups when compared to the vehicle-treated control group (Fig. 6D).

Fig. 6. Antitumor effects of (−)-oleocanthal treatment in human xenograft model of BT-474 breast cancer cells.

Under anesthetic condition, sixty-day release pellets containing 17β-estradiol were implanted into the interscapular region of female nude mouse 24 h before implantation of cancer cells. BT-474 human breast cancer cells were cultured and resuspended in serum-free RPMI-1640 medium and inoculated (1×10⁷cells) subcutaneously into the second mammary gland fat pad just beneath the nipple of each animal under anesthetic condition to generate orthotopic breast tumors. Two weeks post-inoculation, animals developed palpable tumors and were randomly divided into three groups: (i) Vehicle-treated control group (n=5); (ii) (−)-Oleocanthal-treated group at a dose of 5 mg/kg (n=5), and (iii) (−)-Oleocanthal-treated group at a dose of 10 mg/kg (n=5). Animal treatment started 14 days post-inoculation of BT-474 cells with intraperitoneal (i.p.) administration of vehicle control (DMSO/saline) or respective (−)-oleocanthal treatments administered three times weekly for a duration of six weeks. (A) Left panel; Vertical bars represent mean tumor weight at the end of the experiment. Right panel; Vertical bars represent mean tumor volume at the end of the experiment. Tumor volume was calculated as V=(LxW²)/2, where L was the length and W was the width of tumors. Bars ± S.E.M. *P<0.05 as compared to vehicle-treated control group. (B) Left panel; Tumor volumes during the treatment period of the experiment. Points represent mean of tumor volume of several tumors (n=5) in each experimental group during the course of the treatment period. Error bars indicate S.E.M. for n=5. Right panel; Mice representing each experimental group. The first mouse harboring breast tumor from vehicle-treated control group, while the mice in the middle and left represent animals treated with (−)-oleocanthal 5 mg/kg and 10 mg/kg, respectively. (C) Body weight of animals during the duration of the experiment. Points represent mean of body weight for animals in each group (n=5) during the duration of experiment. Error bars indicate S.E.M. for n=5. (D) Protein expression of total levels of ERα in breast tumors detected by Western blot. Scanning densitometric analysis was performed on all blots done in triplicate and the integrated optical density of each band was normalized with corresponding β-tubulin, as shown in bar graphs beside their respective Western blot images. Vertical bars in the graph indicate the normalized integrated optical density of bands visualized in each lane.

4. Discussion

Luminal breast cancers are characterized by expression of ER and being endocrine responsive. Both luminal A and luminal B subtypes have expression patterns indicative of the luminal epithelial component of the breast demonstrated by expression of luminal cytokeratins and genes associated with ER activation (Tomaskovic-Crook et al., 2009; Tran and Bedard, 2011). However, luminal B tumors presented with increased expression of growth receptor signaling genes contributing to increased proliferation and poor prognosis of these tumors compared to luminal A subtype (Perou and Borresen-Dale, 2011; Subik et al., 2010; Tran and Bedard, 2011). Approximately 20% of luminal B tumors were HER2-positive by immunohistochemistry (Wirapati et al., 2008) and 30% of HER2-enriched breast cancers belong to the luminal B subtype (Subik et al., 2010; Tran and Bedard, 2011).

Results of the present study demonstrated that (−)-oleocanthal treatment suppressed growth of both luminal A and B breast cancer cell lines. (−)-Oleocanthal reduced the viability of BT-474, MCF-7, and T-47D cells in a dose-dependent manner. (−)-Oleocanthal retained its antiproliferative activity in luminal breast cancer cells in which cell growth was inhibited in media containing estradiol as well as in mitogen-free media. The inhibition of BT-474 cell growth was associated with the ability of (−)-oleocanthal treatment to reduce total levels of ERα in these cells. (−)-Oleocanthal treatment reduced nuclear expression of ERα, which was associated with suppressed cell growth in vitro. Earlier studies by Menendez et al. demonstrated that polyphenolic secoiridoids of EVOO reduced viability of HER2-overexpressing SKBR3 and MCF-7-HER2-overexpressing breast cancer cells by induction of apoptosis (Menendez et al., 2007). The catechol secoiridoid oleuropein aglycone was the most potent among EVOO phenolics tested by Menendez et al. in decreasing breast cancer cell viability (Menendez et al., 2007). Results from our study further indicates that (−)-oleocanthal from EVOO reduced viability of HER2-positive BT-474 cells by downregulation of total levels of ERα.

Resistance to hormonal therapy in breast cancer management is emerging (Milosevic et al., 2013). Despite clinical advances of ER-targeted therapy, resistance to all forms of endocrine therapy remains a great obstacle. Earlier reports indicated that almost half of ER-positive breast cancer patients exhibit de novo or acquired resistance to tamoxifen (Welsh et al., 2012). The cross-talk between HER2 and ER signaling pathways in breast cancer has been shown to promote resistance to ER-targeted therapy (Li et al., 2016; Welsh et al., 2012). In line with this, increased relapse rates among ER-positive tumors treated with hormonal therapies has been shown to be associated with HER2 overexpression in luminal B breast cancer (Lonning, 2012). Tamoxifen is a corner-stone anti-estrogen therapy in clinical practice for ER-positive breast cancer (Jordan, 1997). Results in this study indicated a synergistic interaction between (−)-oleocanthal and tamoxifen in multiple luminal breast cancer cell lines. Combined (−)-oleocanthal and tamoxifen treatment significantly enhanced sensitivity to both compounds in BT-474, MCF-7, and T-47D cancer cells. In addition, DRI analysis showed multifold reduction of growth inhibitory concentrations for combined (−)-oleocanthal and tamoxifen treatments, compared to each compound alone. (−)-Oleocanthal downregulated ERα expression, an effect which could partially sensitized tamoxifen activity on inhibition of breast cancer cell growth. In this regard, (−)-oleocanthal can be an appealing option to improve sensitivity to endocrine treatments in luminal breast cancer and reduce the emergence of resistance to hormonal therapies. This is particularly interesting taking into consideration that the antiproliferative effects of (−)-oleocanthal in cancer cell lines are achieved at concentrations that have no or little effect on the viability of non-tumorigenic cells and its exceptionally potent in vivo activities as indicated by multiple previous studies (Akl et al., 2014; Fogli et al., 2016; Pei et al., 2016).

Steroid/nuclear hormones exert their molecular pharmacological effects on target cells by binding to their cognate steroid/nuclear receptor superfamily of transcription factors. Structurally, members of these receptors family are modular and arranged in domains, including discrete DNA binding and ligand binding domains (LBDs) (Katzenellenbogen and Katzenellenbogen, 1996; Mangelsdorf et al., 1995). Docking studies in this report indicated the potential for direct binding of (−)-oleocanthal at the ERα LBD in a pattern comparable to the natural ligand, 17β-estradiol. Based on the virtual results in this study, (−)-oleocanthal is hypothesized to directly interact with the ERα receptor and thus can modulate pharmacological signaling of such important nuclear hormone receptor. The high degree of overlapping between the binding of (−)-oleocanthal and 17β-estradiol to ERα could infer some degree of competitiveness for the binding to the receptor, which could explain, in part, the synergistic activity between (−)-oleocanthal and tamoxifen treatment. In addition, docking studies indicated different binding modes for (−)-oleocanthal and tamoxifen at the ERα LBD suggesting that synergy of (−)-oleocanthal and tamoxifen might be due to different binding modes and target amino acids at the receptor site. In the same context, earlier studies by Keiler et al. revealed (−)-oleocanthal preferentially binds to ERα, compared to ERβ, however at weak binding affinities compared to 17β-estradiol (Keiler et al., 2014). Results from our study showed (−)-oleocanthal to reduce total levels of ERα in BT-474 cells, both in vitro and in vivo. In agreement to these findings, Keiler et al. demonstrated anti-estrogenic activity of (−)-oleocanthal in terms of inhibition of 17β-estradiol-induced reporter gene activity independent of ER or tissue subtype (Keiler et al., 2015). Interestingly, weak ERα transactivation was observed in bone-derived osteosarcoma cells exposed to (−)-oleocanthal (Keiler et al., 2015). It is worth to mention that the effect of (−)-oleocanthal on estrogenic signaling could be a complex issue to address. Noticeably, the type of tissue and ER expressed could modify the ultimate effect of (−)-oleocanthal in target cells.

Multiple studies evaluated the underlying mechanisms and potential molecular targets for the anticancer activity of (−)-oleocanthal. Earlier studies indicated that (−)-oleocanthal is a potential inhibitor of the hepatocyte growth factor receptor (c-Met) pathway in breast cancer cells (Akl et al., 2014; Elnagar et al., 2011; Mohyeldin et al., 2016b). The antiproliferative effects of (−)-oleocanthal are mediated by inhibiting multiple c-Met downstream signaling molecules including protein kinase B (Akt), mitogen-activated protein kinase (MAPK), signal transducers and activators of transcription-3 (STAT-3), and mammalian target of rapamycin (mTOR) in multiple cancer cells (Akl et al., 2014; Fogli et al., 2016; Khanal et al., 2011; Khanfar et al., 2015; Pei et al., 2016; Scotece et al., 2013). In addition, (−)-oleocanthal induced cancer cell arrest by modulating the expression of cyclins, cyclin-dependent kinases, and arrest proteins (Akl et al., 2014). (−)-Oleocanthal also induced programmed cell death resulting in cytotoxic activity in breast cancer cells characterized by activation of caspases-8 and -3, elevation in cleaved poly (ADP-ribose) polymerase (PARP) levels, and downregulation of Bcl-2 expression (Akl et al., 2014; Fogli et al., 2016; Pei et al., 2016; Scotece et al., 2013). Interestingly, LeGendre et al. indicated that (−)-oleocanthal promoted cancer cell death by destabilization of lysosomal membranes leading to cancer cell necrosis and/or apoptosis (LeGendre et al., 2015). Furthermore, (−)-oleocanthal is known for its anti-oxidant and anti-inflammatory activities (Galvano et al., 2007; Scotece et al., 2012). Reported anti-inflammatory activity of (−)-oleocanthal has been shown to be mediated by inhibition of macrophage inflammatory protein 1-alpha (MIP-1α), interleukin-6 (IL-6) expression and secretion, 5-lipoxygenase, and heat shock protein 90 (Hsp90) (Margarucci et al., 2013; Scotece et al., 2012; 2013; Vougogiannopoulou et al., 2014).

Results from this study revealed that (−)-oleocanthal administration resulted in a powerful inhibition of luminal B tumor growth in a xenograft animal model. (−)-Oleocanthal treatment remarkably reduced 17β-estradiol-mediated growth of BT-474 xenografts in treated animals compared to vehicle-treated control group. This inhibition was associated with reduced total levels of ERα in isolated tumors. Nevertheless, the remarkable antitumor activity of (−)-oleocanthal in vivo could be also attributed to above described molecular effects of the compound on cancer cell growth and proliferation. To the best of our knowledge, this study is the first to show the growth inhibitory effects of (−)-oleocanthal treatment in animal models representing luminal B breast cancer.

In conclusion, (−)-oleocanthal suppressed growth of luminal breast cancer cells, in part, by reducing total levels of ERα in cell culture and animal studies. The combination of (−)-oleocanthal with the standard anti-estrogen drug, tamoxifen, showed synergistic activity in both luminal A and B breast cancer cells. These findings support further evaluation of (−)-oleocanthal as a potential therapeutic option in combination with endocrine treatments in hormone-dependent breast cancer, particularly the luminal B subtype which represents a therapeutic challenge to treat because of disease aggressiveness and emergence of drug resistance.