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(−)‑Oleocanthal inhibits proliferation and migration by modulating Ca2+ entry through TRPC6 in breast cancer cells
Accepted Manuscript (−)-Oleocanthal inhibits proliferation and migration by modulating Ca2+ entry through TRPC6 in breast cancer cells
R. Diez-Bello, I. Jardin, J.J. Lopez, M. El Haouari, J. OrtegaVidal, J. Altarejos, G.M. Salido, S. Salido, J.A. Rosado PII: DOI: Reference:
S0167-4889(18)30451-8 doi:10.1016/j.bbamcr.2018.10.010 BBAMCR 18376
To appear in:
BBA - Molecular Cell Research
Received date: Revised date: Accepted date:
18 May 2018 8 October 2018 11 October 2018
Please cite this article as: R. Diez-Bello, I. Jardin, J.J. Lopez, M. El Haouari, J. OrtegaVidal, J. Altarejos, G.M. Salido, S. Salido, J.A. Rosado , (−)-Oleocanthal inhibits proliferation and migration by modulating Ca2+ entry through TRPC6 in breast cancer cells. Bbamcr (2018), doi:10.1016/j.bbamcr.2018.10.010
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(–)-Oleocanthal inhibits proliferation and migration by modulating Ca2+ entry through TRPC6 in breast cancer cells. Diez-Bello R1†, Jardin I1†, Lopez JJ1, El Haouari M2, Ortega-Vidal J3, Altarejos J3 , Salido
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GM1, Salido S3, Rosado JA1
Department of Physiology (Cellular Physiology Research Group), Institute of Molecular
Pathology Biomarkers, University of Extremadura, 10003-Caceres, Spain, 2Centre Régional des Métiers de l’Education et de la Formation de Taza, 35000-Taza and Laboratoire
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Matériaux, Substances Naturelles, Environnement & Modélisation (LMSNEM), Faculté Polydisciplinaire de Taza, Université Sidi Mohamed Ben Abdellah, Fès, Morocco, and Department of Inorganic and Organic Chemistry, University of Jaen, Campus de
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Excelencia Internacional Agroalimentario (ceiA3), 23071-Jaen, Spain These authors contributed equally to this work.
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†
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Running Title: Anticancer properties of (–)-Oleocanthal
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Address correspondence to: Dr. Salido S, Department of Inorganic and Organic Chemistry, University of Jaen, 23071-Jaen, Spain, Tel: +34 953 212746/213089, Fax: +34 953 211876;
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E-mail: [email protected] and Dr. Rosado JA, Department of Physiology, University of Extremadura, 10003-Caceres, Spain, Tel: +34 927257100, Fax: +34 927257110; Email: [email protected].
Key words. Oleocanthal, olive oil phenolic, calcium entry, countercurrent chromatography, proliferation, migration, MDA-MB-231 breast cancer cells, MCF7, TRPC6.
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ACCEPTED MANUSCRIPT Abstract. Triple negative breast cancer is an aggressive type of cancer that does not respond to hormonal therapy and current therapeutic strategies are accompanied by side effects due to cytotoxic actions on normal tissues. Therefore, there is a need for the identification of anti-cancer compounds with negligible effects on non-tumoral cells. Here we show that (–)-oleocanthal (OLCT), a phenolic compound isolated from olive oil, selectively
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impairs MDA-MB-231 cell proliferation and viability without affecting the ability of non-tumoral
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MCF10A cells to proliferate or their viability. Similarly, OLCT selectively impairs the ability of MDA-MB-231 cells to migrate while the ability of MCF10A to migrate was unaffected. The effect of OLCT was not exclusive for triple negative breast cancer cells as we found that OLCT also attenuate cell viability and proliferation of MCF7 cells. Our results indicate that
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OLCT is unable to induce Ca2+ mobilization in non-tumoral cells. By contrast, OLCT induces
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Ca2+ entry in MCF7 and MDA-MB-231 cells, which is impaired by TRPC6 expression silencing. We have found that MDA-MB-231 and MCF7 cells overexpress the channel TRPC6 as compared to non-tumoral MCF10A and treatment with OLCT for 24-72 h
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downregulates TRPC6 expression in MDA-MB-231 cells. These findings indicate that OLCT
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impairs the ability of breast cancer cells to proliferate and migrate via downregulation of
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TRPC6 channel expression while having no effect on the biology of non-tumoral breast cells.
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1. Introduction Triple negative breast cancer is an aggressive type of cancer that represents a therapeutic challenge mostly due to the absence of estrogen and progesterone receptors and lack of HER2 overexpression [1]. Current therapeutic strategies, based on the use of
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chemotherapeutics, are usually accompanied by a number of side effects due to cytotoxic
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actions on normal tissues, which considerably reduce the quality of life. Hence, there is a need for the identification of more selective compounds with anti-cancer properties and negligible effects on non-tumoral cells.
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The olive oil phenolic (–)-oleocanthal (OLCT), a natural product present in extra-virgin olive oil, has recently been reported to exert anti-cancer activity in a variety of human cancer
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types. For instance, OLCT has been reported to attenuate cell growth and to induce apoptosis in colorectal and hepatocellular carcinoma, probably due to the production of
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reactive oxygen species (ROS) [2]. Furthermore, OLCT attenuates tumor growth and metastasis of melanoma in a mouse subcutaneous xenograft model by inhibition of signal
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transducer and activator of transcription 3 (STAT3) phosphorylation, nuclear location and transcriptional activity [3]. OLCT also attenuates cell viability in PC3 prostate and BXPC3
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pancreatic adenocarcinomas [4] and inhibits multiple myeloma cells proliferation by down-
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regulation of the ERK1/2 and AKT signal transduction pathways [5]. OLCT inhibits growth of a number of breast cancer cell lines, including the estrogen receptor-positive (ER+) MCF7 and BT474 or the triple negative MDA-MB-231 cell line through the inhibition of c-Met receptor signaling [6], a receptor tyrosine kinase that is overexpressed in malignant cancer. Moreover, in ER+ breast cancer cells, OLCT has been found to reduce the expression of estrogen receptors and to improve the sensitivity to tamoxifen [7]. Treatment with OLCT has been reported to reduce cell viability in MDA-MB-231 cells [4]. Furthermore, OLCT induces a marked downregulation of phosphorylated mTOR in the
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ACCEPTED MANUSCRIPT MDA-MB-231 cell line, which has been associated with its ability to attenuate cell growth [8]. In the present study we have investigated the role of OLCT in the development of different cancer hallmarks, including proliferation and migration in triple negative MDA-MB-231 cells, as well as in the regulation of intracellular Ca2+ homeostasis. We have also extended our studies to the luminal MCF7 cell line. Our results indicate that OLCT induces a
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concentration-dependent effect on Ca2+ influx via TRPC6 channels. Interestingly, here we
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show that, at micromolar concentrations, OLCT is able to reduce the ability of MCF7 and MDA-MB-231 cells to migrate. Furthermore, OLCT decreases MDA-MB-231 cell proliferation in a concentration-dependent manner, without having any effect in non-tumoral MCF10A cells. The anti-proliferative and anti-migrative effects of OLCT parallel the attenuation of
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TRPC6 expression. These findings shed new light on the mechanisms underlying the anti-
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tumoral effects of OLCT in triple negative breast cancer cells and provide evidence for the
2. Material and methods
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2.1. Materials
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chemotherapeutic potential of this compound in breast cancer.
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Fura-2 acetoxymethyl ester (fura-2/AM), was from Molecular Probes (Leiden, The Netherlands). Thapsigargin (TG), rabbit polyclonal anti-β-actin antibody (catalog number
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A2066, epitope: amino acids 365-375 of human β-actin), OAG (oleoyl-2-acetyl-sn-glycerol) and bovine serum albumin (BSA) were from Sigma (Madrid, Spain). Rabbit polyclonal antiTRPC6 antibody (catalog number: ACC-120, epitope corresponding to amino acid residues 573–586) was from Alomone (Jerusalem, Israel). Horseradish peroxidase-conjugated antirabbit IgG antibody was from Abcam (Cambridge, U.K.). shRNA control vector was from Origene (Rockville, MD, U.S.A.). Enhanced chemiluminescence detection reagents were from
Pierce
(Cheshire, U. K.). Turbofect transfection reagent
and Live/Dead®
viability/cytotoxicity kit were from Thermo Fisher (Madrid, Spain). BrdU Cell proliferation
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ACCEPTED MANUSCRIPT assay kit was from BioVision (Milpitas, CA, USA). The solvents used for extractions, analytical thin-layer chromatography (TLC) and fast centrifugal partition chromatography (FCPC) separations, such as methanol (MeOH), dichloromethane (DCM), ethanol (EtOH), n-hexane (Hex) and ethyl acetate (EtOAc), were of analytical grade and were purchased from VWR Chemicals (Prolabo, Fontenay-sous-Bois, France). Methanol (MeOH), and
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acetonitrile (ACN) used for high-performance liquid chromatography (HPLC) analyses and
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semi-preparative HPLC purifications were of HPLC grade and were purchased from VWR (Madrid, Spain). Acetic acid (AcOH) used for TLC and HPLC analyses and deuterated chloroform (CDCl3) used to prepare solutions of the purified compound for nuclear magnetic resonance (NMR) analysis were purchased from VWR (Madrid, Spain). Water (H2O) used
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for extractions and chromatographic separations was of ultrapure grade produced by a MilliQ water (1.8 MΩ) equipment (Merck KGaA, Darmstadt, Germany). All other reagents were
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of analytical grade.
The olive oil sample used to isolate OLCT was obtained from ripe olives (Olea europaea
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L. cv. Picual) and was supplied by Drs. Francisco Espínola and Manuel Moya (Department
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of Chemical Engineering, University of Jaén, Spain).
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2.2. Extraction of phenolic compounds from olive oil The phenolic extract was obtained following a procedure based on that described by the
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International Olive Council [9]: an olive oil sample (300 g) was extracted in a separatory funnel with a mixture of MeOH/H2O 8:2 (v/v) (3 250 mL); the upper phases and the oily phase were combined again and sonicated at room temperature for 15 min. The upper phases were decanted from the rest and were centrifuged at 3000 g for 25 min with a centrifuge model Mixtasel-BL Selecta (JP Selecta, Barcelona, Spain). The resulting supernatant phases were combined and evaporated under vacuum at temperatures not higher than 40 ºC. The dry extract (1.50 g) was stored under argon at –20 ºC until use.
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ACCEPTED MANUSCRIPT 2.3. Isolation and identification of (–)-oleocanthal (OLCT) OLCT was isolated by a combination of FCPC and semi-preparative HPLC separation techniques. FCPC separations were performed on a FCPC-200® instrument (Kromaton Technologies, Angers, France) with a total column volume capacity of 200 mL and equipped with an Alltech 627 isocratic pump (Alltech Associates, Deerfield, IL, USA) and an UV-Vis
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Linear UVIS 200 detector (Linear Instrument Co., Reno, NV, USA) set at 280 nm. HPLC
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separations were performed with a semi-preparative C18 reversed-phase Spherisorb ODS2 column, 250 mm 10 mm i.d., 5 µm (Waters), on a Waters 600E instrument (Waters) equipped with a diode array detector (DAD) (Waters CapLC 2996 photodiode array
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detector).
The dry extract previously obtained (1.50 g) was first fractionated with the FCPC-200®
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instrument using a quaternary biphasic solvent system composed of Hex/EtOAc/EtOH/H2O (2:3:2:3, v/v/v/v) in ascending mode, at a flow rate of 9 mL/min and a rotation speed of 1200
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rpm. Seventeen fractions of 12 mL each were collected and monitored by TLC and analytical HPLC. For TLC, a mixture of DCM/EtOH/AcOH (95:5:0.2, v/v/v) was used as
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developing solvent, and a UV lamp (254 nm) to visualize spots. For analytical HPLC, the separation was performed with a C18 reversed-phase Spherisorb ODS-2 column, 250 mm
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× 3 mm i.d., 5 μm (Waters Chromatography Division, Mildford, MA, USA) and carried out by a step gradient with mixtures of MeOH/ACN (1:1, v/v, solvent A) and H2O/AcOH (99.8:0.2,
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v/v, solvent B) at a flow rate of 0.7 mL/min. The gradient program consisted in (a) a linear gradient from 4 to 50% A in 40 min, (b) a linear gradient from 50 to 60% A in 5 min, (c) a linear gradient from 60 to 100% A in 15 min, (d) 100% A for 10 min, (e) and other 12 min to return to the initial conditions. As result of the FCPC separation, a sample of 180 mg of OLCT, with a purity of 73% (according to HPLC; see below), was obtained. Then, this sample was re-purified by semi-preparative HPLC with solvents ACN/AcOH (99.8:0.2, v/v, solvent A) and H2O/AcOH (99.8:0.2, v/v, solvent B) and flow rate of 5 mL/min. A linear gradient from 20 to 25% A in 50 min afforded pure (–)-oleocanthal (25 mg). This pure OLCT
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ACCEPTED MANUSCRIPT was used for structural identification (see next paragraph) and also to quantify the OLCT content of the 180-mg sample obtained from the FCPC separation (see above). To determine the OLCT content of that sample, an external-standard method was followed using the HPLC peak area of OLCT at 280 nm. For that, a standard calibration curve was previously constructed with 6 concentrations of pure OLCT (10–500 mg/L in MeOH) using
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the same conditions described above for the analytical HPLC analyses. The equation
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formulated and the coefficient of determination (R 2) were: y = 3124.6 x – 5755.1; and R2 = 0.998.
The structure of the purified OLCT was elucidated by proton nuclear magnetic resonance (1H NMR) and carbon nuclear magnetic resonance ( 13C NMR) spectra recorded on a Bruker
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Avance DPX 400 spectrometer (Bruker Daltonik GmbH, Rheinstetten, Germany), operating
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at 400 MHz and 100 MHz, respectively, and using CDCl 3 as solvent (containing tetramethylsilane (TMS) as internal reference). The NMR data (see supplementary information) of this compound agreed with those reported in the literature for oleocanthal
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[10].
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2.4. Cell culture and transfection
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MCF10A, MCF7 and MDA-MB-231 cell lines were obtained from ATCC (Manassas, VA, USA), and cultured at 37 ºC with a 5% CO 2 in DMEM-F12 (MCF10A) or DMEM (MCF7 and
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MDA-MB-231), supplemented with 20% (v/v) horse serum and 10% (v/v) fetal bovine serum, respectively, and 100 U/mL penicillin and streptomycin. Cells were transfected with expression plasmids for the shTRPC6 or scramble plasmids as described previously [11-12] using Turbofect transfection reagent and were used 48 h after transfection. Plasmids were used for silencing experiments at 1 µg/mL. 2.5. Measurement of cytosolic free-calcium concentration ([Ca2+]c) Cells were loaded with fura-2 by incubation with either 2 μM fura 2/AM for 30 min at 37 ºC
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ACCEPTED MANUSCRIPT for MCF7 and MDA-MB-231 cells or 5 μM fura 2/AM for 45 min at 37 ºC for MCF10A cells. Coverslips with cultured cells were mounted on a perfusion chamber and placed on the stage of an epifluorescence inverted microscope (Nikon Eclipse Ti-2, Amsterdam, The Netherlands) with image acquisition and analysis system for videomicroscopy (Nikon NISElements AR). Cells were continuously superfused with HEPES-buffered saline (HBS)
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containing (in mM): 125 NaCl, 5 KCl, 1 MgCl2, 5 glucose, 25 HEPES, and pH 7.4,
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supplemented with 0.1% (w/v) BSA. Cells were alternatively excited with light from a xenon lamp passed through a high-speed monochromator (Optoscan ELE 450, Cairn Research, Faversham, U.K.) at 340/380 nm. Fluorescence emission at 505 nm was detected using a cooled digital sCMOS camera (Zyla 4.2, Andor, Belfast, U.K.) and recorded using NIS-
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Elements AR software (Nikon, Amsterdam, The Netherlands). Fluorescence ratio (F340/F380) was calculated pixel by pixel, and the data are presented as F/F0, where F is
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the experimental fura-2 340/380 nm fluorescence ratio and F0 is the mean basal fura-2 340/380 fluorescence ratio [13]. TG- or OLCT-evoked Ca2+ release and influx was
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measured as the integral of the rise in ΔF/F 0 for 2½ min after the addition of the agent in the
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absence or presence of extracellular Ca2+, respectively.
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2.6. Determination of cell proliferation
Cells were seeded at a concentration of 5 103/well (MCF7 and MDA-MB-231) or 1
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103 /well (MCF10A) into 96-well plates. Twenty four hours later cells were treated with OLCT and after 0, 24, 48 and 72 h, cell proliferation was assessed by a specific cell proliferation assay kit based on the measurement of BrdU incorporation during DNA synthesis according to the manufacturer’s instructions (BioVision, Milpitas, CA, USA) as described previously [14]. Data are presented as the percentage of proliferation relative to control. 2.7. Wound healing assay
For wound healing assay, MCF10A, MCF7 and MDA-MB-231 cells were seeded in 35-
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ACCEPTED MANUSCRIPT mm 6 well multidish to obtain confluence after 24 h. Next, cells were cultured in medium supplemented with 1% serum and a wound was created using a sterile 200-µL plastic pipette tip. Photographs were taken immediately or at the times indicated using an inverted microscope (Nikon Eclipse TS100, Japan). Migration of cells was quantitated using Fiji
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ImageJ (NIH, USA).
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2.8. Western blotting
The Western blotting was performed as described previously [15]. Briefly, cell lysates were resolved by 10% SDS-PAGE and separated proteins were electrophoretically
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transferred onto nitrocellulose membranes for subsequent probing. Blots were incubated overnight with 10% (w/v) BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) to block
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residual protein binding sites. Immunodetection of TRPC6 or β-actin was achieved by incubation overnight with the anti-TRPC6 antibody diluted 1:500 in TBST or by incubation for
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1 h with the anti-β-actin antibody diluted 1:2000 in TBST. The primary antibody was removed and blots were washed six times for 5 min each with TBST. To detect the primary antibody,
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blots were incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody diluted 1:10000 in TBST and then exposed to enhanced chemiluminescence
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reagents for 5 min. The density of bands was measured using C-DiGit Chemiluminescent
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Western Blot Scanner.
2.9. Cell viability
Cell viability was assessed using Live/Dead® viability/cytotoxicity kit. Cells were incubated for 45 min with 2 µM calcein-AM and 4 µM propidium iodide following the manufacturer´s instructions. Cells were washed and resuspended in fresh HBS. Coverslips with cultured cells were mounted on a perfusion chamber and placed on the stage of an epifluorescence inverted microscope (Nikon Eclipse Ti-2) with image acquisition and analysis system for
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ACCEPTED MANUSCRIPT videomicroscopy (Nikon NIS-Elements AR). Samples were excited at 430 nm and 555 nm for calcein and propidium iodide, respectively, and the resulting fluorescence was recorded at 542 nm (for viable cells) and 624 nm (for dead cells).
2.10. Statistical analysis
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Analysis of statistical significance was performed using one-way analysis of variance
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(ANOVA) combined with the Tukey test. P<0.05 was considered to be significant for a difference.
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3. Results
3.1. Isolation of (–)-oleocanthal (OLCT) from olive oil
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The isolation of OLCT from olive oil has been carried out in two steps: (a) a liquid-liquid extraction of an olive oil sample based on the method proposed by the International Olive
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Council (IOC) to make determinations of phenols in olive oil [9]; (b) a liquid-liquid chromatography of the resulting dry phenolic extract followed by a semi-preparative HPLC
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re-purification of the fractions enriched in OLCT (Fig. 1).
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OLCT has usually been isolated from olive oil by silica gel column chromatography and preparative thin-layer chromatography [16], by preparative HPLC [17] and by a combination
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of size-exclusion chromatography and HPLC [18]. Due to the known OLCT sensitivity to decomposition in contact with solid stationary phases [19] and upon exposure to oxygen and light [20] we have pre-purified OLCT using the fast centrifugal partition chromatography (FCPC) technique. Thus, an olive oil sample obtained from olives of cultivar Picual was extracted with a mixture of methanol/water 8:2 to finally give a dry extract enriched in phenolics (extraction yield: 0.5%), which was fractionated by FCPC. After several attempts with different solvent systems [21] we found that a biphasic solvent system composed of nhexane/ethyl acetate/ethanol/water (2:3:2:3, v/v/v/v) allowed us to get semi-pure OLCT
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ACCEPTED MANUSCRIPT fractions (purity: 73% according to HPLC) that could be further purified by semi-preparative HPLC on a C18 reversed-phase column to finally yield pure OLCT (>98%). The nuclear magnetic resonance (NMR) data for this pure compound agreed with those reported in the literature [10].
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3.2. Effect of OLCT in MCF7, MDA-MB-231 and MCF10A cell viability and proliferation
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OLCT has been reported to impair growth of different breast cancer cell lines, including MDA-MB-231 cells [6]. Hence, we have explored the effect of treatment with OLCT on cell viability by using the cell-permeant dye calcein and propidium iodide. Breast cancer MCF7 and MDA-MB-231 cells and non-tumoral MCF10A cells were treated with increasing
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concentrations of OLCT (1-20 µM) or the vehicle (Control) and 24 h later calcein and propidium iodide fluorescences were assessed. As shown in Fig. 2A, our results indicate
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that almost 100% of the untreated MCF10A cells show calcein fluorescence and calcein staining was found to be unaffected by treatment of MCF10A cells with OLCT, at least for 24
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h (p < 0.05; n = 4). However, treatment of MCF7 and MDA-MB-231 cells with OLCT significantly attenuated calcein staining in a concentration-dependent manner. Calcein
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staining was 99 ± 1, 83 ± 3 and 63 ± 8% of control in MCF7 cells and 97 ± 1, 81 ± 1 and 82 ±
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2% of control in MDA-MB-231 cells after treatment for 24 h with 1, 10 and 20 µM OLCT (Fig. 2A; p < 0.05; n = 4). Conversely, propidium iodide staining in MCF7 and MDA-MB-231 cells
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was 1 ± 1, 17 ± 3 and 37 ± 8% of control and 3 ± 1, 19 ± 1 and 18 ± 2% of control, respectively, after treatment for 24 h with 1, 10 and 20 µM OLCT (Fig. 2A; p < 0.05; n = 3). These findings indicate that OLCT reduces MCF7 and MDA-MB-231 cell viability without affecting the viability of MCF10A cells. We have further explored the effect of OLCT in MCF10A and MDA-MB-231 cells proliferation using the BrdU cell proliferation assay kit. As shown in Fig. 2B, cell treatment with 10 and 20 µM OLCT did not significantly modify MCF10A cell proliferation at least during 72 h (p < 0.05; n = 6). Interestingly, treatment of MDA-MB-231 cells with 10 µM OLCT
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ACCEPTED MANUSCRIPT abolished cell proliferation during the first 72 h as compared to MDA-MB-231 cells treated with the vehicle (Fig. 2B; p < 0.05; n = 6). Similar results were observed after treatment with 20 µM OLCT (Fig. 2B). Therefore, our observations reveal that OLCT selectively impairs MDA-MB-231 breast cancer cell proliferation compared to the effect on non-tumoral cells.
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3.3. Effect of OLCT in MCF7, MDA-MB-231 and MCF10A cell migration
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Next, we assessed the effect of OLCT in the ability of non-tumoral MCF10A cells and both MCF7 and MDA-MB-231 breast cancer cells to migrate using the well-established wound healing assay. Cells were seeded, scratched, and cultured in medium supplemented with 1% serum to prevent further cell growth. Cell migration was estimated as described in
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Materials and Methods. MCF10A, MCF7 and MDA-MB-231 cells were treated with increasing concentrations of OLCT (1-20 µM) or the vehicle as control. As shown in Fig. 3, in the
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absence of OLCT all the cells significantly reduced the wound size during the first 48 h (p < 0.05; n = 5). Addition of OLCT (1-20 µM) to MCF10A cells did not significantly attenuate cell
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migration (Fig. 3A; p < 0.05; n = 5). Treatment with OLCT significantly attenuated MCF7 and MDA-MB-231 cell migration in a concentration-dependent manner, reaching the maximal
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effect at the concentration 20 µM but with significant effects also at 10 µM (Figs. 3B and C; p
cell migration.
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< 0.05; n = 5), which indicates that treatment with OLCT specifically impairs breast cancer
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3.4. OLCT induces Ca2+ entry in MCF7 and MDA-MB-231 breast cancer cells
Calcium signaling has long been reported to play a relevant role in cell proliferation [22]. Hence, we have assessed the ability of OLCT to mobilize Ca2+ in non-tumoral MCF10A cells and both MCF7 and MDA-MB-231 breast cancer cells. First of all, we have recorded Ca2+ release from the intracellular stores induced by the SERCA inhibitor TG and Ca 2+ entry via the activation of store-operated Ca2+ entry. As shown in Fig. 4A-C, treatment of fura-2loaded MCF10A, MCF7 and MDA-MB-231 cells with TG (1 µM) in a Ca2+-free medium
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ACCEPTED MANUSCRIPT results in a transient increase in the fura-2 340/380 nm fluorescence ratio due to passive Ca2+ efflux from the intracellular Ca2+ stores. Subsequent perfusion with HBS containing 1 mM Ca2+ resulted in a further rise in the fura-2 340/380 nm fluorescence ratio indicative of Ca2+ entry. In cells treated with DMSO instead of TG no significant Ca 2+ release or entry was detected (Fig. 4A-C). Treatment of MCF10A, MCF7 and MDA-MB-231 cells, perfused
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with a Ca2+-free medium, with increasing concentrations of OLCT (1-20 µM) induced a
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negligible effect, if any, in the fura-2 fluorescence ratio, thus suggesting that OLCT does not evoke Ca2+ release from the intracellular stores (Fig. 4D-L). In the presence of extracellular Ca2+, OLCT was unable to induce a detectable Ca2+ influx in non-tumoral MCF10A cells (Figs. 4D-F). By contrast, OLCT was found to stimulate Ca2+ entry in MCF7 and MDA-MB-
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231 cells in a concentration-dependent manner (Figs. 4G-L). Similar results were obtained when the cells were treated with OLCT in the presence of 1 mM extracellular Ca2+ (Fig. 5).
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These findings indicate that OLCT selectively stimulates Ca 2+ influx in MCF7 and MDA-MB231 breast cancer cells but not in MCF10A cells.
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Next, we explored the mechanism involved in the activation of Ca 2+ influx by OLCT.
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Several Ca2+-permeable channels have been reported in breast cancer cells, including Orai1 and 3, TRPC1, 3 and 6, TRPV4 and 6, TRPM7 and 8 [23]. Functional TRP channel
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expression has been reported in breast and prostate cancer cells, which play an important role in the development and progression of cancer [24-25] and TRPC6 expression has been
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found to be enhanced in breast carcinoma samples in comparison with normal tissue [26]. Furthermore, we have recently reported that TRPC6 plays a relevant role in Ca2+ influx as well as in proliferation and migration of MCF7 and MDA-MB-231 cells [27]. Hence, we have tested the expression of TRPC6 at the protein level in MDA-MB-231 cells by Western blotting and analyzed its involvement in Ca2+ influx stimulated by OLCT. As shown in Fig. 6A, our results indicate that TRPC6 is overexpressed in MCF7 and MDA-MB-231 cells at the protein level as compared to non-tumoral MCF10A cells (p < 0.05; n = 3). The functional expression of TRPC6 in MDA-MB-231 cells was confirmed by stimulation with the diacylglycerol analog,
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ACCEPTED MANUSCRIPT and TRPC6 activator, OAG. In the presence of 1 mM extracellular Ca2+, treatment of MCF10A, MCF7 and MDA-MB-231 cells with 100 µM OAG induces a transient increase in the fura-2 fluorescence ratio solely in breast cancer cells, indicative of Ca2+ influx via TRPC6 channels (Fig. 6C-E). Furthermore, treatment of MCF10A, MCF7 and MDA-MB-231 cells with 100 µM OAG for 24 h did not alter cell viability (supplemental Fig. 1). In order to confirm
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whether TRPC6 is involved in OLCT-evoked Ca2+ entry we have transfected MCF7 and
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MDA-MB-231 cells with shTRPC6 or control vector. As depicted in Fig. 6B, transfection with shTRPC6 significantly attenuated TRPC6 expression in these cells (p < 0.05; n = 3). Furthermore, TRPC6 expression silencing significantly inhibited Ca2+ entry evoked by 10 and 20 µM OLCT (Fig. 6F-M; p < 0.05), which indicates that OLCT-induced Ca2+ entry
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strongly depends on the activation of TRPC6 channels in MCF7 and MDA-MB-231 breast
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cancer cells.
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3.5. OLCT induces attenuation of TRPC6 expression in MDA-MB-231 cells
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ACCEPTED MANUSCRIPT TRPC6 has been previously reported to promote HGF-induced cell proliferation of human prostate cancer DU145 and PC3 cells [28]. In agreement with this study, we have found that HGF-mediated Ca2+ entry in MDA-MB-231 cells is entirely dependent on the expression of TRPC6 channels (supplemental Fig. 2). Hence, in order to ascertain the involvement of TRPC6 on OLCT-mediated anti-proliferative and anti-migrative effects we have assessed
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the effect of treatment with OLCT on TRPC6 channel expression. MDA-MB-231 cells were
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treated with 20 µM OLCT for 10 min, 24, 48 and 72 h or the vehicle (DMSO) and then subjected to Western blotting with specific anti-TRPC6 antibody. Interestingly, cell treatment with OLCT significantly attenuated TRPC6 channel expression in a time-dependent manner as compared to vehicle-treated cells where the expression of TRPC6 was unaffected during
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the time investigated (Fig. 7; p < 0.05; n = 3). These findings indicate that OLCT induces downregulation of TRPC6 expression, which might be responsible for the inhibition of cell
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proliferation and migration.
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4. Discussion
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Our results and previous reports [6] show that OLCT induces selective anti-proliferative and anti-migrative effects on the triple negative MDA-MB-231 and the luminal MCF7 cell
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lines, without having any effect on the non-tumoral MCF10A cells. In MDA-MB-231 cells this mechanism involves the downregulation of TRPC6 channel expression, a channel that plays
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a relevant functional role in MCF7 and MDA-MB-231 cells [27]. The lack of effect of OLCT in non-tumoral MCF10A cells might be attributed to the low TRPC6 expression in these cells; subsequently, OLCT was unable to evoke Ca2+ mobilization in MCF10A cells or to alter the ability of these cells to proliferate or migrate. In agreement with previous studies [4], we have found that OLCT also reduces the viability of MCF7 and MDA-MB-231 cells without having any significant effect on MCF10A cell viability. These findings make OLCT a promising agent for anti-cancer strategies in cells where the different cancer features require TRPC6 channel function, especially in triple negative breast cancer, which is not sensitive to
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ACCEPTED MANUSCRIPT hormonal therapies and the current chemotherapeutics are not free from serious side effects [29]. We have isolated OLCT from olive oil by a combination of liquid-liquid extraction and liquid-liquid chromatographies. Liquid-liquid extraction and solid-phase extraction are the most used techniques to extract phenolic compounds from olive oil [30], whereas silica gel
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column chromatography (CC) is the most applied one to isolate pure phenolics [31]. We
including olive tree extracts
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have also routinely used silica gel CC to isolate phenolic compounds from many plants [32], and also in combination with size-exclusion
chromatography (SEC) and semi-preparative high-performance liquid chromatography (HPLC) [33]. However, more recently we have turned our attention towards countercurrent
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chromatography (CCC), since this technique is becoming a good alternative to traditional
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solid support chromatographic techniques for natural products purifications [34]. The absence of a solid stationary phase in CCC gives to this liquid-liquid chromatography several advantages, such as better sample recovery, lesser solvent consumption, minimum
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peak tailing, shorter separation times, etc. [35]. During the course of our work an integrated
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process for the recovery of OLCT and other high added-value phenolics from olive oil was published [36]. This optimized procedure combined a centrifugal partition extraction of olive
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oil and a gradient elution FCPC purification of the resulting total phenolic fraction, using the same quaternary solvent system than us.
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Our results indicate for the first time that OLCT induces a transient Ca2+ entry in MCF7 and MDA-MB-231 cells by activation of TRPC6. This statement is based on the observation that Ca2+ influx evoked by OLCT is impaired by attenuation of TRPC6 expression using siRNA. Later on, exposure to OLCT results in downregulation of TRPC6 expression in MDAMB-231 cells, which might be responsible for the anti-proliferative and anti-migrative roles of OLCT in these cells, as previously reported for prostate cancer cells [28] Downregulation of TRPC6 expression by different agents has been reported to exert diverse effects in a number of cell types. For instance, a recent study has reported that TRPC6 expression is
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ACCEPTED MANUSCRIPT downregulated by astragaloside IV, a saponin isolated from Astragalus membranaceus, to prevent high glucose-induced podocyte apoptosis and diabetic nephropathy [37]. Losartan, a type 1 angiotensin II receptor antagonist, reduces proteinuria and podocyte injury induced by angiotensin II via downregulation of TRPC6 [38]. Furthermore, ATRA (all-trans-retinoic acid) attenuates proteinuria in a rat model of glomerulosclerosis by downregulation of TRPC6
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expression [39]. We have recently reported that silencing TRPC6 expression results in
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attenuation of the ability of MCF7 and MDA-MB-231 cells to proliferate and migrate due to the impairment of Ca2+ entry via Orai channels [27]. These findings, together with the role of TRPC6 in cell proliferation in different cell types [28, 40-41], might provide an explanation to
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the anti-proliferative effects of OLCT in MDA-MB-231 cells.
TRPC6 channels have been reported to be involved in receptor-operated Ca2+ entry, both
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as a STIM1-regulated store-operated channel [42] and as a second messenger-operated channel [43]. The different expression of TRPC6 in non-tumoral and tumoral cells might
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provide an explanation to the different effect of OLCT in both cell types, as non-tumoral cells show a low TRPC6 expression as compared to MCF7 and MDA-MB-231 cells.
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Summarizing, our results demonstrate for the first time selective activation of TRPC6-
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dependent Ca2+ influx and TRPC6 downregulation by olive oil-derived OLCT at low micromolar concentrations in breast cancer cell lines, which might be responsible for the
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inhibitory effects of OLCT on cell proliferation and migration. Interestingly, OLCT was without effect on cell proliferation and migration of the non-tumoral breast cell line MCF10A, most likely due to the low expression, and, thus, low dependence of TRPC6. The selective effect of OLCT on breast cancer cells over non-tumoral cells indicate that OLCT might be taken into consideration as a potential anti-cancer agent with low side effects for the treatment of triple negative breast cancer.
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ACKNOWLEDGEMENTS This work is supported by MINECO (Grant BFU2016-74932-C2-1-P) and Junta de
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Extremadura-FEDER (Fondo Europeo de Desarrollo Regional Grants IB16046 and
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GR18061). I.J. is supported by contract Juan de la Cierva (Ministerio Economia y Competitividad, Spain; IJCI-2015-25665). R.D.-B. is supported by MINECO. Part of the work was supported by the Centro de Instrumentación Científico-Técnica of the University of
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Jaén.
Author contributions: J.A.R. conceived the project and wrote the manuscript. R.D.-B., M.E.,
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J.J.L. and I.J. performed the experiments and analyzed the data. J.O-V, S.S. and J.A. isolated OLCT. G.M.S., S.S. and J.A. helped write the manuscript and were involved in data
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discussion. All authors reviewed and approved the manuscript.
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DISCLOSURES
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The authors declare no conflict of interests.
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ACCEPTED MANUSCRIPT FIGURE LEGENDS
Figure 1. Schematic procedure for the extraction of olive oil, fractionation of the phenolic extract and purification of (–)-oleocanthal (OLCT).
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Figure 2. OLCT impairs cell viability and proliferation in MDA-MB-231 cells.
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(A) MCF10A, MCF7 and MDA-MB-231 cells were treated with OLCT (1-20 µM) or the vehicle. Twenty four hours later cells were loaded with calcein and propidium iodide and cell staining was visualized using an inverted microscope as described in Material and Methods. (B) MCF10A and MDA-MB-231 cells were treated with 10 and 20 µM OLCT or the vehicle
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(Control) and cell proliferation was assessed for a further 24, 48 and 72 h using the BrdU cell proliferation assay kit, as described in Material and Methods. Bar graphs represent
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MCF10A and MDA-MB-231 cell proliferation after 24, 48 and 72 h presented as BrdU uptake rate in a.u. and expressed as mean ± SEM. *p < 0.05 compared to the corresponding
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control.
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Figure 3. OLCT attenuates MCF7 and MDA-MB-231 cell migration. MCF10A (A), MCF7 (B) and MDA-MB-231 (C) cells were treated with OLCT (1-20 µM) or the
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vehicle (Control) and were subjected to wound healing assay as described in Methods. Images were acquired at 0, 24 and 48 h from the beginning of the assay. The dotted lines
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define the areas lacking cells. The bar graphs represent the wound size, in micrometers, at the different conditions, expressed as the mean ± SEM of three independent experiments. * p < 0.05 compared to the time = 0 h.
§
p < 0.05 compared to the corresponding time in
vehicle-treated cells. Figure 4. OLCT evokes Ca2+ entry but not Ca2+ release in MCF7 and MDA-MB-231 breast cancer cells.
(A-C) Fura-2-loaded MCF10A (A), MCF7 (B) and MDA-MB-231 (C) cells were perfused with
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ACCEPTED MANUSCRIPT a Ca2+-free medium (100 µM EGTA added) and then stimulated with TG (1 µM) or the vehicle (DMSO) followed by reintroduction of external Ca2+ (final concentration 1 mM) to initiate Ca2+ entry. Data are mean ± SEM of 50 cells/day/5-7 days. MCF10A (D-F), MCF7 (GI) and MDA-MB-231 cells (J-L) were perfused with a Ca2+-free medium (100 µM EGTA added) and then treated with increasing concentrations (1-20 µM) of OLCT followed by
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reintroduction of external Ca2+ (final concentration 1 mM) to visualize Ca2+ entry. Data are
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mean ± SEM of 50 cells/day/5-7 days. Dashed lines represent Ca2+ mobilization in vehicletreated cells.
Figure 5. Ca2+ mobilization by OLCT in non-tumoral MCF10A cells and MCF7 and
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MDA-MB-231 breast cancer cells.
Fura-2-loaded MCF10A (A-C), MCF7 (D-F) and MDA-MB-231 cells (G-I) were perfused with
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a medium containing 1 mM Ca2+ and then stimulated with increasing concentrations of OLCT (1-20 µM) or the vehicle (DMSO; dashed lines). Data are mean ± SEM of 50
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cells/day/5 days.
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Figure 6. TRPC6 mediates Ca2+ entry induced by OLCT.
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(A) MCF10A, MCF7 and MDA-MB-231 cells were lysed and whole cell lysates were subjected to 10% SDS-PAGE and Western blotting with the anti-TRPC6 and anti-β-actin
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antibodies, as described in Material and Methods. Blots are representative of 3 separate experiments. Bar graphs represent TRPC6 expression presented as the percentage of MCF10A cells. (B) MCF7 (left panel) and MDA-MB-231 cells (right panel) were transfected with shTRPC6 or scramble (shRNAcv) plasmids. Forty eight hours later cells were lysed and subjected to Western blotting with the anti-TRPC6 antibody, followed by reprobing with anti-β-actin antibody for protein loading control. Blots are representative of 3 separate experiments. Bar graphs represent TRPC6 expression presented as the percentage of Control (cells treated with shRNAcv). (C-E) Fura-2-loaded MCF10A (C), MCF7 (D) and
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ACCEPTED MANUSCRIPT MDA-MB-231 cells (E) were perfused with a medium containing 1 mM Ca2+ and then stimulated with OAG (100 µM) or the vehicle (DMSO). Data are mean ± SEM of 50 cells/day/5 days. (F-K) MCF7 (F-H) and MDA-MB-231 (I-K) cells were transfected with shTRPC6 or scramble (shRNAcv) plasmids, as indicated. After 48 h cells were loaded with fura-2 and perfused with Ca2+-free HBS. Cells were treated with increasing concentrations
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of OLCT (1-20 µM) followed by perfusion with HBS containing 1 mM Ca 2+ to estimate Ca2+
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entry. Data are mean ± SEM of 50 cells/day/5-7 days. (L and M) Data represent Ca2+ entry evoked by OLCT (1-20 µM) in MCF7 (L) and MDA-MB-231 (M) cells transfected with shTRPC6 or the corresponding control plasmid.
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Figure 7. OLCT downregulates TRPC6 expression.
MDA-MB-231 cells were treated for 10 min, 24, 48 or 72 h with OLCT (20 µM; B) or the
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vehicle (DMSO; A) and lysed. Whole cell lysates were subjected to 10% SDS-PAGE and Western blotting with the anti-TRPC6 and anti-β-actin antibodies, as described in Material
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and Methods. Blots are representative of 3 separate experiments. Bar graphs represent
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compared to control.
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TRPC6 expression presented as the percentage of control (untreated cells). * p < 0.05
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ACCEPTED MANUSCRIPT Highlights 2+ • (–)-Oleocanthal (OLCT) induces Ca entry in MCF7 and MDA-MB-231 cells via TRPC6 channel. • OLCT attenuates viability and impairs proliferation and migration in MDA-MB-231 cells. • OLCT downregulates TRPC6 expression in MDA-MB-231 cells.
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• The effects of OLCT are selective for breast cancer cells which show high TRPC6 expression.
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Figure 1
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R. Diez-Bello, I. Jardin, J.J. Lopez, M. El Haouari, J. OrtegaVidal, J. Altarejos, G.M. Salido, S. Salido, J.A. Rosado PII: DOI: Reference:
S0167-4889(18)30451-8 doi:10.1016/j.bbamcr.2018.10.010 BBAMCR 18376
To appear in:
BBA - Molecular Cell Research
Received date: Revised date: Accepted date:
18 May 2018 8 October 2018 11 October 2018
Please cite this article as: R. Diez-Bello, I. Jardin, J.J. Lopez, M. El Haouari, J. OrtegaVidal, J. Altarejos, G.M. Salido, S. Salido, J.A. Rosado , (−)-Oleocanthal inhibits proliferation and migration by modulating Ca2+ entry through TRPC6 in breast cancer cells. Bbamcr (2018), doi:10.1016/j.bbamcr.2018.10.010
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(–)-Oleocanthal inhibits proliferation and migration by modulating Ca2+ entry through TRPC6 in breast cancer cells. Diez-Bello R1†, Jardin I1†, Lopez JJ1, El Haouari M2, Ortega-Vidal J3, Altarejos J3 , Salido
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GM1, Salido S3, Rosado JA1
Department of Physiology (Cellular Physiology Research Group), Institute of Molecular
Pathology Biomarkers, University of Extremadura, 10003-Caceres, Spain, 2Centre Régional des Métiers de l’Education et de la Formation de Taza, 35000-Taza and Laboratoire
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Matériaux, Substances Naturelles, Environnement & Modélisation (LMSNEM), Faculté Polydisciplinaire de Taza, Université Sidi Mohamed Ben Abdellah, Fès, Morocco, and Department of Inorganic and Organic Chemistry, University of Jaen, Campus de
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Excelencia Internacional Agroalimentario (ceiA3), 23071-Jaen, Spain These authors contributed equally to this work.
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†
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Running Title: Anticancer properties of (–)-Oleocanthal
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Address correspondence to: Dr. Salido S, Department of Inorganic and Organic Chemistry, University of Jaen, 23071-Jaen, Spain, Tel: +34 953 212746/213089, Fax: +34 953 211876;
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E-mail: [email protected] and Dr. Rosado JA, Department of Physiology, University of Extremadura, 10003-Caceres, Spain, Tel: +34 927257100, Fax: +34 927257110; Email: [email protected].
Key words. Oleocanthal, olive oil phenolic, calcium entry, countercurrent chromatography, proliferation, migration, MDA-MB-231 breast cancer cells, MCF7, TRPC6.
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ACCEPTED MANUSCRIPT Abstract. Triple negative breast cancer is an aggressive type of cancer that does not respond to hormonal therapy and current therapeutic strategies are accompanied by side effects due to cytotoxic actions on normal tissues. Therefore, there is a need for the identification of anti-cancer compounds with negligible effects on non-tumoral cells. Here we show that (–)-oleocanthal (OLCT), a phenolic compound isolated from olive oil, selectively
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impairs MDA-MB-231 cell proliferation and viability without affecting the ability of non-tumoral
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MCF10A cells to proliferate or their viability. Similarly, OLCT selectively impairs the ability of MDA-MB-231 cells to migrate while the ability of MCF10A to migrate was unaffected. The effect of OLCT was not exclusive for triple negative breast cancer cells as we found that OLCT also attenuate cell viability and proliferation of MCF7 cells. Our results indicate that
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OLCT is unable to induce Ca2+ mobilization in non-tumoral cells. By contrast, OLCT induces
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Ca2+ entry in MCF7 and MDA-MB-231 cells, which is impaired by TRPC6 expression silencing. We have found that MDA-MB-231 and MCF7 cells overexpress the channel TRPC6 as compared to non-tumoral MCF10A and treatment with OLCT for 24-72 h
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downregulates TRPC6 expression in MDA-MB-231 cells. These findings indicate that OLCT
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impairs the ability of breast cancer cells to proliferate and migrate via downregulation of
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TRPC6 channel expression while having no effect on the biology of non-tumoral breast cells.
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1. Introduction Triple negative breast cancer is an aggressive type of cancer that represents a therapeutic challenge mostly due to the absence of estrogen and progesterone receptors and lack of HER2 overexpression [1]. Current therapeutic strategies, based on the use of
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chemotherapeutics, are usually accompanied by a number of side effects due to cytotoxic
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actions on normal tissues, which considerably reduce the quality of life. Hence, there is a need for the identification of more selective compounds with anti-cancer properties and negligible effects on non-tumoral cells.
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The olive oil phenolic (–)-oleocanthal (OLCT), a natural product present in extra-virgin olive oil, has recently been reported to exert anti-cancer activity in a variety of human cancer
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types. For instance, OLCT has been reported to attenuate cell growth and to induce apoptosis in colorectal and hepatocellular carcinoma, probably due to the production of
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reactive oxygen species (ROS) [2]. Furthermore, OLCT attenuates tumor growth and metastasis of melanoma in a mouse subcutaneous xenograft model by inhibition of signal
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transducer and activator of transcription 3 (STAT3) phosphorylation, nuclear location and transcriptional activity [3]. OLCT also attenuates cell viability in PC3 prostate and BXPC3
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pancreatic adenocarcinomas [4] and inhibits multiple myeloma cells proliferation by down-
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regulation of the ERK1/2 and AKT signal transduction pathways [5]. OLCT inhibits growth of a number of breast cancer cell lines, including the estrogen receptor-positive (ER+) MCF7 and BT474 or the triple negative MDA-MB-231 cell line through the inhibition of c-Met receptor signaling [6], a receptor tyrosine kinase that is overexpressed in malignant cancer. Moreover, in ER+ breast cancer cells, OLCT has been found to reduce the expression of estrogen receptors and to improve the sensitivity to tamoxifen [7]. Treatment with OLCT has been reported to reduce cell viability in MDA-MB-231 cells [4]. Furthermore, OLCT induces a marked downregulation of phosphorylated mTOR in the
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ACCEPTED MANUSCRIPT MDA-MB-231 cell line, which has been associated with its ability to attenuate cell growth [8]. In the present study we have investigated the role of OLCT in the development of different cancer hallmarks, including proliferation and migration in triple negative MDA-MB-231 cells, as well as in the regulation of intracellular Ca2+ homeostasis. We have also extended our studies to the luminal MCF7 cell line. Our results indicate that OLCT induces a
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concentration-dependent effect on Ca2+ influx via TRPC6 channels. Interestingly, here we
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show that, at micromolar concentrations, OLCT is able to reduce the ability of MCF7 and MDA-MB-231 cells to migrate. Furthermore, OLCT decreases MDA-MB-231 cell proliferation in a concentration-dependent manner, without having any effect in non-tumoral MCF10A cells. The anti-proliferative and anti-migrative effects of OLCT parallel the attenuation of
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TRPC6 expression. These findings shed new light on the mechanisms underlying the anti-
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tumoral effects of OLCT in triple negative breast cancer cells and provide evidence for the
2. Material and methods
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2.1. Materials
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chemotherapeutic potential of this compound in breast cancer.
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Fura-2 acetoxymethyl ester (fura-2/AM), was from Molecular Probes (Leiden, The Netherlands). Thapsigargin (TG), rabbit polyclonal anti-β-actin antibody (catalog number
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A2066, epitope: amino acids 365-375 of human β-actin), OAG (oleoyl-2-acetyl-sn-glycerol) and bovine serum albumin (BSA) were from Sigma (Madrid, Spain). Rabbit polyclonal antiTRPC6 antibody (catalog number: ACC-120, epitope corresponding to amino acid residues 573–586) was from Alomone (Jerusalem, Israel). Horseradish peroxidase-conjugated antirabbit IgG antibody was from Abcam (Cambridge, U.K.). shRNA control vector was from Origene (Rockville, MD, U.S.A.). Enhanced chemiluminescence detection reagents were from
Pierce
(Cheshire, U. K.). Turbofect transfection reagent
and Live/Dead®
viability/cytotoxicity kit were from Thermo Fisher (Madrid, Spain). BrdU Cell proliferation
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ACCEPTED MANUSCRIPT assay kit was from BioVision (Milpitas, CA, USA). The solvents used for extractions, analytical thin-layer chromatography (TLC) and fast centrifugal partition chromatography (FCPC) separations, such as methanol (MeOH), dichloromethane (DCM), ethanol (EtOH), n-hexane (Hex) and ethyl acetate (EtOAc), were of analytical grade and were purchased from VWR Chemicals (Prolabo, Fontenay-sous-Bois, France). Methanol (MeOH), and
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acetonitrile (ACN) used for high-performance liquid chromatography (HPLC) analyses and
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semi-preparative HPLC purifications were of HPLC grade and were purchased from VWR (Madrid, Spain). Acetic acid (AcOH) used for TLC and HPLC analyses and deuterated chloroform (CDCl3) used to prepare solutions of the purified compound for nuclear magnetic resonance (NMR) analysis were purchased from VWR (Madrid, Spain). Water (H2O) used
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for extractions and chromatographic separations was of ultrapure grade produced by a MilliQ water (1.8 MΩ) equipment (Merck KGaA, Darmstadt, Germany). All other reagents were
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of analytical grade.
The olive oil sample used to isolate OLCT was obtained from ripe olives (Olea europaea
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L. cv. Picual) and was supplied by Drs. Francisco Espínola and Manuel Moya (Department
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of Chemical Engineering, University of Jaén, Spain).
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2.2. Extraction of phenolic compounds from olive oil The phenolic extract was obtained following a procedure based on that described by the
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International Olive Council [9]: an olive oil sample (300 g) was extracted in a separatory funnel with a mixture of MeOH/H2O 8:2 (v/v) (3 250 mL); the upper phases and the oily phase were combined again and sonicated at room temperature for 15 min. The upper phases were decanted from the rest and were centrifuged at 3000 g for 25 min with a centrifuge model Mixtasel-BL Selecta (JP Selecta, Barcelona, Spain). The resulting supernatant phases were combined and evaporated under vacuum at temperatures not higher than 40 ºC. The dry extract (1.50 g) was stored under argon at –20 ºC until use.
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ACCEPTED MANUSCRIPT 2.3. Isolation and identification of (–)-oleocanthal (OLCT) OLCT was isolated by a combination of FCPC and semi-preparative HPLC separation techniques. FCPC separations were performed on a FCPC-200® instrument (Kromaton Technologies, Angers, France) with a total column volume capacity of 200 mL and equipped with an Alltech 627 isocratic pump (Alltech Associates, Deerfield, IL, USA) and an UV-Vis
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Linear UVIS 200 detector (Linear Instrument Co., Reno, NV, USA) set at 280 nm. HPLC
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separations were performed with a semi-preparative C18 reversed-phase Spherisorb ODS2 column, 250 mm 10 mm i.d., 5 µm (Waters), on a Waters 600E instrument (Waters) equipped with a diode array detector (DAD) (Waters CapLC 2996 photodiode array
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detector).
The dry extract previously obtained (1.50 g) was first fractionated with the FCPC-200®
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instrument using a quaternary biphasic solvent system composed of Hex/EtOAc/EtOH/H2O (2:3:2:3, v/v/v/v) in ascending mode, at a flow rate of 9 mL/min and a rotation speed of 1200
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rpm. Seventeen fractions of 12 mL each were collected and monitored by TLC and analytical HPLC. For TLC, a mixture of DCM/EtOH/AcOH (95:5:0.2, v/v/v) was used as
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developing solvent, and a UV lamp (254 nm) to visualize spots. For analytical HPLC, the separation was performed with a C18 reversed-phase Spherisorb ODS-2 column, 250 mm
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× 3 mm i.d., 5 μm (Waters Chromatography Division, Mildford, MA, USA) and carried out by a step gradient with mixtures of MeOH/ACN (1:1, v/v, solvent A) and H2O/AcOH (99.8:0.2,
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v/v, solvent B) at a flow rate of 0.7 mL/min. The gradient program consisted in (a) a linear gradient from 4 to 50% A in 40 min, (b) a linear gradient from 50 to 60% A in 5 min, (c) a linear gradient from 60 to 100% A in 15 min, (d) 100% A for 10 min, (e) and other 12 min to return to the initial conditions. As result of the FCPC separation, a sample of 180 mg of OLCT, with a purity of 73% (according to HPLC; see below), was obtained. Then, this sample was re-purified by semi-preparative HPLC with solvents ACN/AcOH (99.8:0.2, v/v, solvent A) and H2O/AcOH (99.8:0.2, v/v, solvent B) and flow rate of 5 mL/min. A linear gradient from 20 to 25% A in 50 min afforded pure (–)-oleocanthal (25 mg). This pure OLCT
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ACCEPTED MANUSCRIPT was used for structural identification (see next paragraph) and also to quantify the OLCT content of the 180-mg sample obtained from the FCPC separation (see above). To determine the OLCT content of that sample, an external-standard method was followed using the HPLC peak area of OLCT at 280 nm. For that, a standard calibration curve was previously constructed with 6 concentrations of pure OLCT (10–500 mg/L in MeOH) using
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the same conditions described above for the analytical HPLC analyses. The equation
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formulated and the coefficient of determination (R 2) were: y = 3124.6 x – 5755.1; and R2 = 0.998.
The structure of the purified OLCT was elucidated by proton nuclear magnetic resonance (1H NMR) and carbon nuclear magnetic resonance ( 13C NMR) spectra recorded on a Bruker
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Avance DPX 400 spectrometer (Bruker Daltonik GmbH, Rheinstetten, Germany), operating
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at 400 MHz and 100 MHz, respectively, and using CDCl 3 as solvent (containing tetramethylsilane (TMS) as internal reference). The NMR data (see supplementary information) of this compound agreed with those reported in the literature for oleocanthal
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[10].
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2.4. Cell culture and transfection
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MCF10A, MCF7 and MDA-MB-231 cell lines were obtained from ATCC (Manassas, VA, USA), and cultured at 37 ºC with a 5% CO 2 in DMEM-F12 (MCF10A) or DMEM (MCF7 and
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MDA-MB-231), supplemented with 20% (v/v) horse serum and 10% (v/v) fetal bovine serum, respectively, and 100 U/mL penicillin and streptomycin. Cells were transfected with expression plasmids for the shTRPC6 or scramble plasmids as described previously [11-12] using Turbofect transfection reagent and were used 48 h after transfection. Plasmids were used for silencing experiments at 1 µg/mL. 2.5. Measurement of cytosolic free-calcium concentration ([Ca2+]c) Cells were loaded with fura-2 by incubation with either 2 μM fura 2/AM for 30 min at 37 ºC
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ACCEPTED MANUSCRIPT for MCF7 and MDA-MB-231 cells or 5 μM fura 2/AM for 45 min at 37 ºC for MCF10A cells. Coverslips with cultured cells were mounted on a perfusion chamber and placed on the stage of an epifluorescence inverted microscope (Nikon Eclipse Ti-2, Amsterdam, The Netherlands) with image acquisition and analysis system for videomicroscopy (Nikon NISElements AR). Cells were continuously superfused with HEPES-buffered saline (HBS)
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containing (in mM): 125 NaCl, 5 KCl, 1 MgCl2, 5 glucose, 25 HEPES, and pH 7.4,
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supplemented with 0.1% (w/v) BSA. Cells were alternatively excited with light from a xenon lamp passed through a high-speed monochromator (Optoscan ELE 450, Cairn Research, Faversham, U.K.) at 340/380 nm. Fluorescence emission at 505 nm was detected using a cooled digital sCMOS camera (Zyla 4.2, Andor, Belfast, U.K.) and recorded using NIS-
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Elements AR software (Nikon, Amsterdam, The Netherlands). Fluorescence ratio (F340/F380) was calculated pixel by pixel, and the data are presented as F/F0, where F is
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the experimental fura-2 340/380 nm fluorescence ratio and F0 is the mean basal fura-2 340/380 fluorescence ratio [13]. TG- or OLCT-evoked Ca2+ release and influx was
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measured as the integral of the rise in ΔF/F 0 for 2½ min after the addition of the agent in the
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absence or presence of extracellular Ca2+, respectively.
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2.6. Determination of cell proliferation
Cells were seeded at a concentration of 5 103/well (MCF7 and MDA-MB-231) or 1
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103 /well (MCF10A) into 96-well plates. Twenty four hours later cells were treated with OLCT and after 0, 24, 48 and 72 h, cell proliferation was assessed by a specific cell proliferation assay kit based on the measurement of BrdU incorporation during DNA synthesis according to the manufacturer’s instructions (BioVision, Milpitas, CA, USA) as described previously [14]. Data are presented as the percentage of proliferation relative to control. 2.7. Wound healing assay
For wound healing assay, MCF10A, MCF7 and MDA-MB-231 cells were seeded in 35-
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ACCEPTED MANUSCRIPT mm 6 well multidish to obtain confluence after 24 h. Next, cells were cultured in medium supplemented with 1% serum and a wound was created using a sterile 200-µL plastic pipette tip. Photographs were taken immediately or at the times indicated using an inverted microscope (Nikon Eclipse TS100, Japan). Migration of cells was quantitated using Fiji
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ImageJ (NIH, USA).
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2.8. Western blotting
The Western blotting was performed as described previously [15]. Briefly, cell lysates were resolved by 10% SDS-PAGE and separated proteins were electrophoretically
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transferred onto nitrocellulose membranes for subsequent probing. Blots were incubated overnight with 10% (w/v) BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) to block
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residual protein binding sites. Immunodetection of TRPC6 or β-actin was achieved by incubation overnight with the anti-TRPC6 antibody diluted 1:500 in TBST or by incubation for
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1 h with the anti-β-actin antibody diluted 1:2000 in TBST. The primary antibody was removed and blots were washed six times for 5 min each with TBST. To detect the primary antibody,
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blots were incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody diluted 1:10000 in TBST and then exposed to enhanced chemiluminescence
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reagents for 5 min. The density of bands was measured using C-DiGit Chemiluminescent
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Western Blot Scanner.
2.9. Cell viability
Cell viability was assessed using Live/Dead® viability/cytotoxicity kit. Cells were incubated for 45 min with 2 µM calcein-AM and 4 µM propidium iodide following the manufacturer´s instructions. Cells were washed and resuspended in fresh HBS. Coverslips with cultured cells were mounted on a perfusion chamber and placed on the stage of an epifluorescence inverted microscope (Nikon Eclipse Ti-2) with image acquisition and analysis system for
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ACCEPTED MANUSCRIPT videomicroscopy (Nikon NIS-Elements AR). Samples were excited at 430 nm and 555 nm for calcein and propidium iodide, respectively, and the resulting fluorescence was recorded at 542 nm (for viable cells) and 624 nm (for dead cells).
2.10. Statistical analysis
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Analysis of statistical significance was performed using one-way analysis of variance
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(ANOVA) combined with the Tukey test. P<0.05 was considered to be significant for a difference.
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3. Results
3.1. Isolation of (–)-oleocanthal (OLCT) from olive oil
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The isolation of OLCT from olive oil has been carried out in two steps: (a) a liquid-liquid extraction of an olive oil sample based on the method proposed by the International Olive
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Council (IOC) to make determinations of phenols in olive oil [9]; (b) a liquid-liquid chromatography of the resulting dry phenolic extract followed by a semi-preparative HPLC
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re-purification of the fractions enriched in OLCT (Fig. 1).
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OLCT has usually been isolated from olive oil by silica gel column chromatography and preparative thin-layer chromatography [16], by preparative HPLC [17] and by a combination
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of size-exclusion chromatography and HPLC [18]. Due to the known OLCT sensitivity to decomposition in contact with solid stationary phases [19] and upon exposure to oxygen and light [20] we have pre-purified OLCT using the fast centrifugal partition chromatography (FCPC) technique. Thus, an olive oil sample obtained from olives of cultivar Picual was extracted with a mixture of methanol/water 8:2 to finally give a dry extract enriched in phenolics (extraction yield: 0.5%), which was fractionated by FCPC. After several attempts with different solvent systems [21] we found that a biphasic solvent system composed of nhexane/ethyl acetate/ethanol/water (2:3:2:3, v/v/v/v) allowed us to get semi-pure OLCT
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ACCEPTED MANUSCRIPT fractions (purity: 73% according to HPLC) that could be further purified by semi-preparative HPLC on a C18 reversed-phase column to finally yield pure OLCT (>98%). The nuclear magnetic resonance (NMR) data for this pure compound agreed with those reported in the literature [10].
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3.2. Effect of OLCT in MCF7, MDA-MB-231 and MCF10A cell viability and proliferation
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OLCT has been reported to impair growth of different breast cancer cell lines, including MDA-MB-231 cells [6]. Hence, we have explored the effect of treatment with OLCT on cell viability by using the cell-permeant dye calcein and propidium iodide. Breast cancer MCF7 and MDA-MB-231 cells and non-tumoral MCF10A cells were treated with increasing
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concentrations of OLCT (1-20 µM) or the vehicle (Control) and 24 h later calcein and propidium iodide fluorescences were assessed. As shown in Fig. 2A, our results indicate
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that almost 100% of the untreated MCF10A cells show calcein fluorescence and calcein staining was found to be unaffected by treatment of MCF10A cells with OLCT, at least for 24
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h (p < 0.05; n = 4). However, treatment of MCF7 and MDA-MB-231 cells with OLCT significantly attenuated calcein staining in a concentration-dependent manner. Calcein
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staining was 99 ± 1, 83 ± 3 and 63 ± 8% of control in MCF7 cells and 97 ± 1, 81 ± 1 and 82 ±
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2% of control in MDA-MB-231 cells after treatment for 24 h with 1, 10 and 20 µM OLCT (Fig. 2A; p < 0.05; n = 4). Conversely, propidium iodide staining in MCF7 and MDA-MB-231 cells
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was 1 ± 1, 17 ± 3 and 37 ± 8% of control and 3 ± 1, 19 ± 1 and 18 ± 2% of control, respectively, after treatment for 24 h with 1, 10 and 20 µM OLCT (Fig. 2A; p < 0.05; n = 3). These findings indicate that OLCT reduces MCF7 and MDA-MB-231 cell viability without affecting the viability of MCF10A cells. We have further explored the effect of OLCT in MCF10A and MDA-MB-231 cells proliferation using the BrdU cell proliferation assay kit. As shown in Fig. 2B, cell treatment with 10 and 20 µM OLCT did not significantly modify MCF10A cell proliferation at least during 72 h (p < 0.05; n = 6). Interestingly, treatment of MDA-MB-231 cells with 10 µM OLCT
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ACCEPTED MANUSCRIPT abolished cell proliferation during the first 72 h as compared to MDA-MB-231 cells treated with the vehicle (Fig. 2B; p < 0.05; n = 6). Similar results were observed after treatment with 20 µM OLCT (Fig. 2B). Therefore, our observations reveal that OLCT selectively impairs MDA-MB-231 breast cancer cell proliferation compared to the effect on non-tumoral cells.
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3.3. Effect of OLCT in MCF7, MDA-MB-231 and MCF10A cell migration
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Next, we assessed the effect of OLCT in the ability of non-tumoral MCF10A cells and both MCF7 and MDA-MB-231 breast cancer cells to migrate using the well-established wound healing assay. Cells were seeded, scratched, and cultured in medium supplemented with 1% serum to prevent further cell growth. Cell migration was estimated as described in
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Materials and Methods. MCF10A, MCF7 and MDA-MB-231 cells were treated with increasing concentrations of OLCT (1-20 µM) or the vehicle as control. As shown in Fig. 3, in the
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absence of OLCT all the cells significantly reduced the wound size during the first 48 h (p < 0.05; n = 5). Addition of OLCT (1-20 µM) to MCF10A cells did not significantly attenuate cell
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migration (Fig. 3A; p < 0.05; n = 5). Treatment with OLCT significantly attenuated MCF7 and MDA-MB-231 cell migration in a concentration-dependent manner, reaching the maximal
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effect at the concentration 20 µM but with significant effects also at 10 µM (Figs. 3B and C; p
cell migration.
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< 0.05; n = 5), which indicates that treatment with OLCT specifically impairs breast cancer
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3.4. OLCT induces Ca2+ entry in MCF7 and MDA-MB-231 breast cancer cells
Calcium signaling has long been reported to play a relevant role in cell proliferation [22]. Hence, we have assessed the ability of OLCT to mobilize Ca2+ in non-tumoral MCF10A cells and both MCF7 and MDA-MB-231 breast cancer cells. First of all, we have recorded Ca2+ release from the intracellular stores induced by the SERCA inhibitor TG and Ca 2+ entry via the activation of store-operated Ca2+ entry. As shown in Fig. 4A-C, treatment of fura-2loaded MCF10A, MCF7 and MDA-MB-231 cells with TG (1 µM) in a Ca2+-free medium
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ACCEPTED MANUSCRIPT results in a transient increase in the fura-2 340/380 nm fluorescence ratio due to passive Ca2+ efflux from the intracellular Ca2+ stores. Subsequent perfusion with HBS containing 1 mM Ca2+ resulted in a further rise in the fura-2 340/380 nm fluorescence ratio indicative of Ca2+ entry. In cells treated with DMSO instead of TG no significant Ca 2+ release or entry was detected (Fig. 4A-C). Treatment of MCF10A, MCF7 and MDA-MB-231 cells, perfused
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with a Ca2+-free medium, with increasing concentrations of OLCT (1-20 µM) induced a
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negligible effect, if any, in the fura-2 fluorescence ratio, thus suggesting that OLCT does not evoke Ca2+ release from the intracellular stores (Fig. 4D-L). In the presence of extracellular Ca2+, OLCT was unable to induce a detectable Ca2+ influx in non-tumoral MCF10A cells (Figs. 4D-F). By contrast, OLCT was found to stimulate Ca2+ entry in MCF7 and MDA-MB-
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231 cells in a concentration-dependent manner (Figs. 4G-L). Similar results were obtained when the cells were treated with OLCT in the presence of 1 mM extracellular Ca2+ (Fig. 5).
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These findings indicate that OLCT selectively stimulates Ca 2+ influx in MCF7 and MDA-MB231 breast cancer cells but not in MCF10A cells.
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Next, we explored the mechanism involved in the activation of Ca 2+ influx by OLCT.
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Several Ca2+-permeable channels have been reported in breast cancer cells, including Orai1 and 3, TRPC1, 3 and 6, TRPV4 and 6, TRPM7 and 8 [23]. Functional TRP channel
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expression has been reported in breast and prostate cancer cells, which play an important role in the development and progression of cancer [24-25] and TRPC6 expression has been
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found to be enhanced in breast carcinoma samples in comparison with normal tissue [26]. Furthermore, we have recently reported that TRPC6 plays a relevant role in Ca2+ influx as well as in proliferation and migration of MCF7 and MDA-MB-231 cells [27]. Hence, we have tested the expression of TRPC6 at the protein level in MDA-MB-231 cells by Western blotting and analyzed its involvement in Ca2+ influx stimulated by OLCT. As shown in Fig. 6A, our results indicate that TRPC6 is overexpressed in MCF7 and MDA-MB-231 cells at the protein level as compared to non-tumoral MCF10A cells (p < 0.05; n = 3). The functional expression of TRPC6 in MDA-MB-231 cells was confirmed by stimulation with the diacylglycerol analog,
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ACCEPTED MANUSCRIPT and TRPC6 activator, OAG. In the presence of 1 mM extracellular Ca2+, treatment of MCF10A, MCF7 and MDA-MB-231 cells with 100 µM OAG induces a transient increase in the fura-2 fluorescence ratio solely in breast cancer cells, indicative of Ca2+ influx via TRPC6 channels (Fig. 6C-E). Furthermore, treatment of MCF10A, MCF7 and MDA-MB-231 cells with 100 µM OAG for 24 h did not alter cell viability (supplemental Fig. 1). In order to confirm
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whether TRPC6 is involved in OLCT-evoked Ca2+ entry we have transfected MCF7 and
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MDA-MB-231 cells with shTRPC6 or control vector. As depicted in Fig. 6B, transfection with shTRPC6 significantly attenuated TRPC6 expression in these cells (p < 0.05; n = 3). Furthermore, TRPC6 expression silencing significantly inhibited Ca2+ entry evoked by 10 and 20 µM OLCT (Fig. 6F-M; p < 0.05), which indicates that OLCT-induced Ca2+ entry
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strongly depends on the activation of TRPC6 channels in MCF7 and MDA-MB-231 breast
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cancer cells.
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3.5. OLCT induces attenuation of TRPC6 expression in MDA-MB-231 cells
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ACCEPTED MANUSCRIPT TRPC6 has been previously reported to promote HGF-induced cell proliferation of human prostate cancer DU145 and PC3 cells [28]. In agreement with this study, we have found that HGF-mediated Ca2+ entry in MDA-MB-231 cells is entirely dependent on the expression of TRPC6 channels (supplemental Fig. 2). Hence, in order to ascertain the involvement of TRPC6 on OLCT-mediated anti-proliferative and anti-migrative effects we have assessed
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the effect of treatment with OLCT on TRPC6 channel expression. MDA-MB-231 cells were
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treated with 20 µM OLCT for 10 min, 24, 48 and 72 h or the vehicle (DMSO) and then subjected to Western blotting with specific anti-TRPC6 antibody. Interestingly, cell treatment with OLCT significantly attenuated TRPC6 channel expression in a time-dependent manner as compared to vehicle-treated cells where the expression of TRPC6 was unaffected during
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the time investigated (Fig. 7; p < 0.05; n = 3). These findings indicate that OLCT induces downregulation of TRPC6 expression, which might be responsible for the inhibition of cell
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proliferation and migration.
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4. Discussion
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Our results and previous reports [6] show that OLCT induces selective anti-proliferative and anti-migrative effects on the triple negative MDA-MB-231 and the luminal MCF7 cell
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lines, without having any effect on the non-tumoral MCF10A cells. In MDA-MB-231 cells this mechanism involves the downregulation of TRPC6 channel expression, a channel that plays
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a relevant functional role in MCF7 and MDA-MB-231 cells [27]. The lack of effect of OLCT in non-tumoral MCF10A cells might be attributed to the low TRPC6 expression in these cells; subsequently, OLCT was unable to evoke Ca2+ mobilization in MCF10A cells or to alter the ability of these cells to proliferate or migrate. In agreement with previous studies [4], we have found that OLCT also reduces the viability of MCF7 and MDA-MB-231 cells without having any significant effect on MCF10A cell viability. These findings make OLCT a promising agent for anti-cancer strategies in cells where the different cancer features require TRPC6 channel function, especially in triple negative breast cancer, which is not sensitive to
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ACCEPTED MANUSCRIPT hormonal therapies and the current chemotherapeutics are not free from serious side effects [29]. We have isolated OLCT from olive oil by a combination of liquid-liquid extraction and liquid-liquid chromatographies. Liquid-liquid extraction and solid-phase extraction are the most used techniques to extract phenolic compounds from olive oil [30], whereas silica gel
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column chromatography (CC) is the most applied one to isolate pure phenolics [31]. We
including olive tree extracts
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have also routinely used silica gel CC to isolate phenolic compounds from many plants [32], and also in combination with size-exclusion
chromatography (SEC) and semi-preparative high-performance liquid chromatography (HPLC) [33]. However, more recently we have turned our attention towards countercurrent
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chromatography (CCC), since this technique is becoming a good alternative to traditional
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solid support chromatographic techniques for natural products purifications [34]. The absence of a solid stationary phase in CCC gives to this liquid-liquid chromatography several advantages, such as better sample recovery, lesser solvent consumption, minimum
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peak tailing, shorter separation times, etc. [35]. During the course of our work an integrated
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process for the recovery of OLCT and other high added-value phenolics from olive oil was published [36]. This optimized procedure combined a centrifugal partition extraction of olive
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oil and a gradient elution FCPC purification of the resulting total phenolic fraction, using the same quaternary solvent system than us.
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Our results indicate for the first time that OLCT induces a transient Ca2+ entry in MCF7 and MDA-MB-231 cells by activation of TRPC6. This statement is based on the observation that Ca2+ influx evoked by OLCT is impaired by attenuation of TRPC6 expression using siRNA. Later on, exposure to OLCT results in downregulation of TRPC6 expression in MDAMB-231 cells, which might be responsible for the anti-proliferative and anti-migrative roles of OLCT in these cells, as previously reported for prostate cancer cells [28] Downregulation of TRPC6 expression by different agents has been reported to exert diverse effects in a number of cell types. For instance, a recent study has reported that TRPC6 expression is
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ACCEPTED MANUSCRIPT downregulated by astragaloside IV, a saponin isolated from Astragalus membranaceus, to prevent high glucose-induced podocyte apoptosis and diabetic nephropathy [37]. Losartan, a type 1 angiotensin II receptor antagonist, reduces proteinuria and podocyte injury induced by angiotensin II via downregulation of TRPC6 [38]. Furthermore, ATRA (all-trans-retinoic acid) attenuates proteinuria in a rat model of glomerulosclerosis by downregulation of TRPC6
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expression [39]. We have recently reported that silencing TRPC6 expression results in
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attenuation of the ability of MCF7 and MDA-MB-231 cells to proliferate and migrate due to the impairment of Ca2+ entry via Orai channels [27]. These findings, together with the role of TRPC6 in cell proliferation in different cell types [28, 40-41], might provide an explanation to
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the anti-proliferative effects of OLCT in MDA-MB-231 cells.
TRPC6 channels have been reported to be involved in receptor-operated Ca2+ entry, both
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as a STIM1-regulated store-operated channel [42] and as a second messenger-operated channel [43]. The different expression of TRPC6 in non-tumoral and tumoral cells might
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provide an explanation to the different effect of OLCT in both cell types, as non-tumoral cells show a low TRPC6 expression as compared to MCF7 and MDA-MB-231 cells.
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Summarizing, our results demonstrate for the first time selective activation of TRPC6-
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dependent Ca2+ influx and TRPC6 downregulation by olive oil-derived OLCT at low micromolar concentrations in breast cancer cell lines, which might be responsible for the
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inhibitory effects of OLCT on cell proliferation and migration. Interestingly, OLCT was without effect on cell proliferation and migration of the non-tumoral breast cell line MCF10A, most likely due to the low expression, and, thus, low dependence of TRPC6. The selective effect of OLCT on breast cancer cells over non-tumoral cells indicate that OLCT might be taken into consideration as a potential anti-cancer agent with low side effects for the treatment of triple negative breast cancer.
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ACCEPTED MANUSCRIPT
ACKNOWLEDGEMENTS This work is supported by MINECO (Grant BFU2016-74932-C2-1-P) and Junta de
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Extremadura-FEDER (Fondo Europeo de Desarrollo Regional Grants IB16046 and
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GR18061). I.J. is supported by contract Juan de la Cierva (Ministerio Economia y Competitividad, Spain; IJCI-2015-25665). R.D.-B. is supported by MINECO. Part of the work was supported by the Centro de Instrumentación Científico-Técnica of the University of
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Jaén.
Author contributions: J.A.R. conceived the project and wrote the manuscript. R.D.-B., M.E.,
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J.J.L. and I.J. performed the experiments and analyzed the data. J.O-V, S.S. and J.A. isolated OLCT. G.M.S., S.S. and J.A. helped write the manuscript and were involved in data
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discussion. All authors reviewed and approved the manuscript.
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DISCLOSURES
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The authors declare no conflict of interests.
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ACCEPTED MANUSCRIPT FIGURE LEGENDS
Figure 1. Schematic procedure for the extraction of olive oil, fractionation of the phenolic extract and purification of (–)-oleocanthal (OLCT).
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Figure 2. OLCT impairs cell viability and proliferation in MDA-MB-231 cells.
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(A) MCF10A, MCF7 and MDA-MB-231 cells were treated with OLCT (1-20 µM) or the vehicle. Twenty four hours later cells were loaded with calcein and propidium iodide and cell staining was visualized using an inverted microscope as described in Material and Methods. (B) MCF10A and MDA-MB-231 cells were treated with 10 and 20 µM OLCT or the vehicle
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(Control) and cell proliferation was assessed for a further 24, 48 and 72 h using the BrdU cell proliferation assay kit, as described in Material and Methods. Bar graphs represent
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MCF10A and MDA-MB-231 cell proliferation after 24, 48 and 72 h presented as BrdU uptake rate in a.u. and expressed as mean ± SEM. *p < 0.05 compared to the corresponding
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control.
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Figure 3. OLCT attenuates MCF7 and MDA-MB-231 cell migration. MCF10A (A), MCF7 (B) and MDA-MB-231 (C) cells were treated with OLCT (1-20 µM) or the
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vehicle (Control) and were subjected to wound healing assay as described in Methods. Images were acquired at 0, 24 and 48 h from the beginning of the assay. The dotted lines
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define the areas lacking cells. The bar graphs represent the wound size, in micrometers, at the different conditions, expressed as the mean ± SEM of three independent experiments. * p < 0.05 compared to the time = 0 h.
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p < 0.05 compared to the corresponding time in
vehicle-treated cells. Figure 4. OLCT evokes Ca2+ entry but not Ca2+ release in MCF7 and MDA-MB-231 breast cancer cells.
(A-C) Fura-2-loaded MCF10A (A), MCF7 (B) and MDA-MB-231 (C) cells were perfused with
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ACCEPTED MANUSCRIPT a Ca2+-free medium (100 µM EGTA added) and then stimulated with TG (1 µM) or the vehicle (DMSO) followed by reintroduction of external Ca2+ (final concentration 1 mM) to initiate Ca2+ entry. Data are mean ± SEM of 50 cells/day/5-7 days. MCF10A (D-F), MCF7 (GI) and MDA-MB-231 cells (J-L) were perfused with a Ca2+-free medium (100 µM EGTA added) and then treated with increasing concentrations (1-20 µM) of OLCT followed by
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reintroduction of external Ca2+ (final concentration 1 mM) to visualize Ca2+ entry. Data are
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mean ± SEM of 50 cells/day/5-7 days. Dashed lines represent Ca2+ mobilization in vehicletreated cells.
Figure 5. Ca2+ mobilization by OLCT in non-tumoral MCF10A cells and MCF7 and
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MDA-MB-231 breast cancer cells.
Fura-2-loaded MCF10A (A-C), MCF7 (D-F) and MDA-MB-231 cells (G-I) were perfused with
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a medium containing 1 mM Ca2+ and then stimulated with increasing concentrations of OLCT (1-20 µM) or the vehicle (DMSO; dashed lines). Data are mean ± SEM of 50
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cells/day/5 days.
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Figure 6. TRPC6 mediates Ca2+ entry induced by OLCT.
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(A) MCF10A, MCF7 and MDA-MB-231 cells were lysed and whole cell lysates were subjected to 10% SDS-PAGE and Western blotting with the anti-TRPC6 and anti-β-actin
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antibodies, as described in Material and Methods. Blots are representative of 3 separate experiments. Bar graphs represent TRPC6 expression presented as the percentage of MCF10A cells. (B) MCF7 (left panel) and MDA-MB-231 cells (right panel) were transfected with shTRPC6 or scramble (shRNAcv) plasmids. Forty eight hours later cells were lysed and subjected to Western blotting with the anti-TRPC6 antibody, followed by reprobing with anti-β-actin antibody for protein loading control. Blots are representative of 3 separate experiments. Bar graphs represent TRPC6 expression presented as the percentage of Control (cells treated with shRNAcv). (C-E) Fura-2-loaded MCF10A (C), MCF7 (D) and
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ACCEPTED MANUSCRIPT MDA-MB-231 cells (E) were perfused with a medium containing 1 mM Ca2+ and then stimulated with OAG (100 µM) or the vehicle (DMSO). Data are mean ± SEM of 50 cells/day/5 days. (F-K) MCF7 (F-H) and MDA-MB-231 (I-K) cells were transfected with shTRPC6 or scramble (shRNAcv) plasmids, as indicated. After 48 h cells were loaded with fura-2 and perfused with Ca2+-free HBS. Cells were treated with increasing concentrations
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of OLCT (1-20 µM) followed by perfusion with HBS containing 1 mM Ca 2+ to estimate Ca2+
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entry. Data are mean ± SEM of 50 cells/day/5-7 days. (L and M) Data represent Ca2+ entry evoked by OLCT (1-20 µM) in MCF7 (L) and MDA-MB-231 (M) cells transfected with shTRPC6 or the corresponding control plasmid.
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Figure 7. OLCT downregulates TRPC6 expression.
MDA-MB-231 cells were treated for 10 min, 24, 48 or 72 h with OLCT (20 µM; B) or the
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vehicle (DMSO; A) and lysed. Whole cell lysates were subjected to 10% SDS-PAGE and Western blotting with the anti-TRPC6 and anti-β-actin antibodies, as described in Material
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and Methods. Blots are representative of 3 separate experiments. Bar graphs represent
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compared to control.
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TRPC6 expression presented as the percentage of control (untreated cells). * p < 0.05
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ACCEPTED MANUSCRIPT Highlights 2+ • (–)-Oleocanthal (OLCT) induces Ca entry in MCF7 and MDA-MB-231 cells via TRPC6 channel. • OLCT attenuates viability and impairs proliferation and migration in MDA-MB-231 cells. • OLCT downregulates TRPC6 expression in MDA-MB-231 cells.
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• The effects of OLCT are selective for breast cancer cells which show high TRPC6 expression.
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