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Phytoestrogen (+)-pinoresinol exerts antitumor activity in breast cancer cells with different oestrogen receptor statuses
© The Author(s). 2016
Received: 13 January 2016
Accepted: 19 July 2016
Published: 7 September 2016
Consumption of virgin olive oil (VOO) has been associated with a low breast cancer incidence. Pinoresinol is a phytoestrogen that is typically found in VOO. Considering the role of oestrogen in breast cancer development and progression, we investigated the potential antitumor activity of pinoresinol in breast cancer cells.
To address this question, we treated MDA-MB-231 (oestrogen receptor [ER] negative) and MCF7 (ER+) human breast tumour cells and MCF10A human mammary epithelial cells (ER-) with different concentrations of pinoresinol. The cytotoxic activity, cell proliferation, cell cycle profile, apoptosis induction, reactive oxygen species production and DNA damage were assessed.
Pinoresinol showed cytotoxic, anti-proliferative and pro-oxidant activity in human breast tumour cells, independent of their oestrogen receptor status. In addition, pinoresinol exerted antioxidant activity and prevented DNA damage associated with oxidative stress in human mammary epithelial cells.
Overall, the results suggest that pinoresinol may have antitumor activity in human breast cancer cells independently of oestrogen receptor status. Furthermore, the results show that the pinoresinol has the typical characteristics of a chemopreventive compound.
Growing scientific evidence suggests that the intake of virgin olive oil (VOO), which is the main source of fat in Mediterranean diets, correlates with a low incidence of breast cancer . Among the minor compounds present in VOO that possess different health properties [2–6], we find polyphenols to be a very interesting group because of their biological benefits. It has been reported that polyphenols prevent the development and progression of pathological conditions, such as cancer, neurological and cardio-vascular diseases, diabetes, aging, and so on .
PINO has a chemical structure that is similar to that of oestrogen (i.e., it is a phytoestrogen). Oestrogen is essential for the growth and development of mammary glands and has been linked with the development and progression of breast cancer due to enhanced binding and activation of the oestrogen receptor α (ERα) . For example, the phytoestrogen tamoxifen acts as an oestrogen antagonist in breast tissue and has been shown to slow breast cancer cell proliferation and has been used in clinical practice for breast cancer patients .
Interestingly, ERβ has also been shown to mediate estrogenic action. The specific role of this receptor in human breast cancer remains elusive; however, in contrast to ERα, ERβ has been linked with anti-proliferative and pro-apoptotic activities. In fact, the expression of ERβ is lower in human breast cancer cells compared to normal breast cells, supporting its potential tumour-suppressive role .
Surprisingly, very few studies have noted the role of PINO as a potential agonist or antagonist of oestrogen and the chemopreventive repercussions that PINO treatment may have on hormone-related breast cancer .
The chemical antioxidant activity of PINO also remains unclear. A few studies using DPPH and ABTS assays have shown different antioxidant functions of PINO [21-23]. However, the Oxygen Radical Absorbance Capacity method (ORAC) has not been used in past studies, despite being considered one of the most biologically relevant assays .
Furthermore, the little research that has been done surrounding the effects of this compound on breast cancer cells remains inconclusive. Chin et al.  described a lack of cytotoxic effects and a cytoprotective effect of PINO on MCF7 cells stressed by H2O2 . Other authors have reported anticancer effects of PINO by suppressing the expression of the lipogenic enzyme FASN in HER-2 overexpressing MCF7 cells . Recently, Sepporta et al.  observed that PINO inhibited the growth of MDA-MB-231 cells, but not of MCF7 cells. Importantly, no previous study has examined the effects of PINO on a normal human breast cell line, which would address whether PINO plays a protective role against cancer development.
Therefore, the aim of the present study was to examine whether PINO exerts chemopreventive and/or antitumor activity in breast cancer, specifically because this compound is found in VOO and its consumption has been related with a minor incidence of breast cancer.
Therefore, to determine whether this compound may contribute, at least in part, to the health benefits attributed to VOO on breast cancer incidence and mortality, we studied the effects of PINO on breast cells with different receptor expression patterns. For this purpose, we used the following human mammary cells: highly invasive MDA-MB-231 (oestrogen receptor [ER] and progesterone receptor [PR] negative) breast tumour cells, the minimally invasive MCF7 (ER and PR positive) breast tumour cells and MCF10A human mammary epithelial cells (ER and PR negative).
Chemicals and material
The following were purchased from Gibco® Life Technologies Ltd (Paisley, UK): HuMEC Ready Medium (1X), TrypLE™ Express Enzyme (1X) and Minimum Essential Medium (MEM). Foetal bovine serum (FBS) was obtained from PAA Laboratories GmbH (Pasching, Austria). Ethanol 96 % v/v and potassium peroxodisulfate (K2S2O8) (CAS 7727-21-1) were purchased from Panreac Química S.L.U. (Barcelona, Spain). The CellTiter-Blue® Cell Viability Assay was acquired from Promega Corporation (Madison, WI, USA). Round bottom culture plates and cell culture flasks were purchased from Nunc A/S (Roskilde, Denmark). Flat bottom culture plates were from CytoOne (Hamburg, Germany). Fluorescein (FL) (CAS 2321-07-5) was obtained from Life Technologies (Carlsbad, CA, USA). The following were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA): (+)-Pinoresinol (CAS 487-36-5) purity ≥95 %; PBS; (S)-(+)-camptothecin (CPT) (CAS 7689-03-4) purity ≥90 %; 2′,7′-dichlorofluorescin diacetate (DCFH-DA) (CAS 4091-99-0) purity ≥97 %; Sodium pyruvate solution (CAS 113-24-6); MEM Non-essential Amino Acid Solution (NEAA); HEPES buffer solution (CAS 7365-45-9); 2,2-Diphenyl-1-picrylhydrazyl (DPPH) (CAS 1898-66-4); 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (CAS 30931-67-0) purity ≥98 %; (±)-α-Tocopherol (Vitamin E) (CAS 10191-41-0) purity ≥96 %; (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox™) (CAS 53188-07-1) purity 97 % and 2,2′-Azobis (2-methylpropionamidine) dihydrochloride (AAPH) (CAS 2997-92-4) purity 97 %. PBS (1X, Dulbecco’s) and DMSO (CAS 67-68-5) were obtained from AppliChem GmbH (Darmstadt, Germany). The PI/RNase Staining Buffer kit, Annexin V-FITC kit and Comet Assay kit (CAS 50-07-7) were purchased, respectively, from BD Biosciences, Pharmingen (San Diego, CA), Miltenyi Biotec (Bergisch Gladbach, Germany) and Trevigen, Inc. (Gaithersburg, MD, USA). Non-tumorigenic human breast epithelial cells (MCF10A), minimally invasive human breast cancer cells (MCF7) and highly invasive human breast cancer cells (MDA-MB-231) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).
ABTS radical scavenging test
where AC(0) is the absorbance of the blank at t = 0 and AA(t) is the absorbance of the compound/standard at t = 60.
Radical scavenging activity by the DPPH assay
Estimation of the antioxidant capacity against the radical DPPH was carried out according to Brand-William et al.,  with some modifications. An ethanolic solution of DPPH 100 μM (final concentration) was mixed in 96-well plates with ethanolic solutions of PINO or α-tocopherol (antioxidant standard) at 0.03, 0.06, 0.13, 0.25, 0.5, 1 and 2 mole ratios (moles of antioxidant/moles of DPPH). DPPH samples without antioxidants were also measured as blank controls. The absorbance at 520 nm was read every 5 min over 2 h with a TECAN GENios Plus microplate reader. Measurements were performed at least in triplicate in three separate experiments. The radical scavenging activity (% RSA) was calculated as described in Eq. (1) (t = 60).
where f0 is the initial fluorescence at cycle 0 and fn is the fluorescence reading at cycle n.
where Y is the net AUC (AUCsample – AUCcontrol), b is the Y-intercept and m is the slope.
Cell culture and treatments
Human MCF10A (ERα and PR negative) breast epithelial cells were grown in HuMEC Ready Medium. Human MCF7 (ERα and PR positive) and MDA-MB-231 (ERα and PR negative) breast cancer cells were grown in MEM supplemented with 10 % FBS, 1 % Hepes buffer, 1 % NEAA and 1 % Sodium Pyruvate. The cells were cultivated as monolayer cultures in a humidified atmosphere with 5 % CO2 at 37°C and subcultured using TryPLE Express. Cells growing between 90 and 95 % of confluence were used for all experiments. The cells were treated for 24 h with 0.001, 0.01, 0.1, 1, 10 and 100 μM of PINO that was previously dissolved in DMSO (stock concentration 50 mM).
where A corresponds to the relative fluorescence units of each sample. All of the measurements were performed in triplicate and each experiment was repeated at least three independent times.
Cell proliferation assay
In all of the cell proliferation experiments performed, the cells were seeded cells onto 96-well plates and allowed to attach before adding PINO or DMSO as the vehicle control. After 24 h of treatments, the medium was replaced by fresh medium and the plates were incubated for another 24 h. Then, CellTiter-Blue® was added, and fluorescence was read after 3 h of incubation with a TECAN GENios Plus microplate reader (Ex. λ485/Em. λ595 nm). The measurements were repeated at 48, 72 and 96 h. The percentage of viable cells was calculated as defined in Eq. (4).
Cell cycle analysis
A total of 1 x 105 cells/mL (for MDA-MB-231 and MCF7 cells) or 5 x 104 cells/mL (for MCF10A cells) were seeded and allowed to attach for 24 h before treating with PINO for another 24 h. The cells were then fixed in cold 70 % ethanol, stored at −20°C for at least 24 h and labelled with a PI/RNase Staining Buffer kit. Cell cycle assessment was conducted by flow cytometry in an EPICS XL-MLC flow cytometer (Beckman Coulter, Spain), and the results were analysed using the FlowJo program (v5.7.2). Each experiment was repeated three independent times.
MDA-MB-231 (1 x 105 cells/mL), MCF7 (1 x 105 cells/mL) or MCF10A (5 x 104 cells/mL) cells were seeded, allowed to attach and treated for 24 h with PINO. The cells and supernatants were collected and labelled with Annexin V-FITC kit according to the manufacturer’s suggestions. As a positive control, the cells were incubated with 1 μM camptothecin (CPT). Apoptosis analysis was carried out using an EPICS XL-MLC flow cytometer, and the results were analysed using the FlowJo program. Each experiment was repeated three independent times.
Detection of reactive oxygen species
Detection of intracellular Reactive Oxygen Species (ROS) was performed using the probe 2’, 7’-dichlorofluorescin diacetate (DCFH-DA) as previously reported by our group . In brief, MCF10A (5.5x103 cells/well), MDA-MB-231 or MCF7 cells (7x103 cells/well) were seeded onto 96-well plates, allowed to attach for 24 h and then treated with PINO for an additional 24 h. After the addition of DCFH-DA (100 μM), the plates were incubated for 30 min at 37 °C and 5 % CO2. Fluorescence was then read for 30 min (Ex. λ485/Em. λ535) with a TECAN GENios Plus microplate reader.
It is well known that the addition of H2O2 increases stress in culture cells . To test whether PINO had a protective role against induced oxidative stress, the assay was also performed after the addition of H2O2 (400 μM) 30 min before quantification.
Both experimental conditions were assayed three independent times, and each measurement was performed in quadruplicate. In all cases, iron free media (MEM or HuMEC) were used.
where Ft0 is the fluorescence at t = 0 min and Ft30 the fluorescence at t = 30 min.
Alkaline single-cell gel electrophoresis (Comet assay)
Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Fisher’s LSD test with the STATGRAPHICS Centurion XVI software (Statpoint Technologies, Inc. Warrenton, VA, USA). The values of p < 0.05 were considered significant. The data are represented as the mean of at least three independent experiments ± SEM and are expressed relative to the untreated controls.
ABTS radical scavenging test
Radical scavenging activity by the DPPH assay
The peroxyl radical scavenging activity of PINO, as measured by ORACFL, showed a protective effect against AAPH-induced peroxyl radical activity. PINO exerted a higher protection than Trolox™. The micromolar TroloxTM equivalents (TE) values were 39.95, 64.93, 114.89 and 214.81 for 12.5, 25, 50 and 100 μM of PINO.
Analysis of cell cycle and apoptosis
Percentage of cell cycle phases was measured by flow cytometry after treatment with PINO
1.72 ± 0.3
60.67 ± 4.8
17.01 ± 2.33
20.59 ± 2.46
0.69 ± 0.11
61.57 ± 0.19
16.72 ± 0.86
21.00 ± 0.74
1.23 ± 0.17
56.45 ± 0.2
20.96 ± 1.29
17.91 ± 1.08
2.57 ± 0.7
60.87 ± 3.37
16.75 ± 1.68
20.23 ± 2.56
0.54 ± 0.03
62.71 ± 0.23
16.70 ± 0.43
20.60 ± 0.73
1.86 ± 0.7
55.32 ± 1.43
22.44 ± 0.34
16.77 ± 1.02
1.80 ± 0.21
59.16 ± 4.53
17.55 ± 2.68
20.71 ± 2.01
0.62 ± 0.14
61.64 ± 1
16.72 ± 0.25
21.58 ± 0.93
1.67 ± 0.42
54.12 ± 2.3
21.79 ± 0.62
17.36 ± 1.47
1.32 ± 0.24
59.33 ± 4.15
18.92 ± 1.68
20.35 ± 2.38
0.49 ± 0.02
60.71 ± 0.51
18.41 ± 0.5
20.61 ± 0.64
1.45 ± 0.22
54.84 ± 1.19
20.47 ± 0.53
19.09 ± 1.1
1.62 ± 0.29
60.63 ± 4.93
18.56 ± 2.45
19.37 ± 2.48
0.49 ± 0.03
62.99 ± 1.28
16.99 ± 0.62
20.12 ± 1.06
1.85 ± 0.25
54.72 ± 0.65
21.23 ± 0.8
17.58 ± 1.16
1.76 ± 0.3
64.85 ± 4.74
17.6 ± 2.38
15.52 ± 2.21
0.57 ± 0.03
61.73 ± 0.65
18.08 ± 0.41
19.62 ± 0.73
1.66 ± 0.12
53.93 ± 1.54
20.23 ± 1.1
19.6 ± 0.84
2.29 ± 0.44
73.39 ± 1.69a
8.34 ± 0.94a
16.06 ± 1.8
0.81 ± 0.19
63.92 ± 1.13
11.96 ± 0.68a
23.38 ± 0.85
1.26 ± 0.23
76.1 ± 2.01a
9.92 ± 1.02a
9.45 ± 2.1a
Percentage of live, apoptotic and dead cells after 24 h after exposure to PINO (0.001, 0.01, 0.1 1 10 and 100 μM)
83.22 ± 5.33
14.82 ± 4.76
1.95 ± 0.6
92,03 ± 2,69
6,97 ± 2,9
1,26 ± 0,61
98.14 ± 0.47
1.33 ± 0.42
0.5 ± 0.08
82.17 ± 4.76
15.56 ± 5.56
2.25 ± 0.94
92,38 ± 1,82
6,2 ± 1,83
1,07 ± 0,62
96.17 ± 0.76
2.73 ± 0.48
1.08 ± 0.29
80.94 ± 8.15
16.24 ± 6.77
2.8 ± 1.57
93,14 ± 2,06
5,98 ± 1,69
0,85 ± 0,42
95.7 ± 1.44
3.45 ± 1.63
0.83 ± 0.37
76.84 ± 9.32
20.15 ± 8.06
2.99 ± 1.34
92,47 ± 1,33
6,49 ± 1,08
1,03 ± 0,39
95.11 ± 2.6
3.99 ± 2.56
0.88 ± 0.16
78.82 ± 7.41
18.05 ± 6.27
3.16 ± 1.29
93,32 ± 1,98
5,86 ± 1,61
0,8 ± 0,43
93.32 ± 1.51
5.85 ± 1.76
0.8 ± 0.25
79.33 ± 7.24
17.75 ± 6.15
2.9 ± 1.19
93,89 ± 1,29
5,4 ± 1,08
0,69 ± 0,28
95.07 ± 1.1
4.17 ± 1.3
0.74 ± 0.2
78.1 ± 6.84
18.46 ± 5.92
3.42 ± 1.09
91,65 ± 1,25
7,48 ± 1,14
0,85 ± 0,18
91.82 ± 2.33a
7.26 ± 2.54a
0.9 ± 0.23
As early as 1980s, it was suggested that lignans might prevent breast cancer and that this effect might be correlated with their phytoestrogenic activity. In addition, consumption of VOO, which contains significant amounts of lignans (e.g., PINO and 1-acetoxypinoresinol) as the major components of its phenolic fraction, has been correlated with a low occurrence of breast cancer . In fact, in the phenolic fraction of VOOs there are several compounds with anti-breast cancer properties as oleouropein , hydroxytyrosol and tyrosol . Certain compounds showed more effectiveness in ER- breast cancer cells than in ER+ breast cancer cells . PINO and 1-acetoxypinoresinol were first detected in VOO by Owen et al.  and differ in their relative amounts according to the different olives varieties used to make the VOO . For example, Brenes et al.  reported that Spanish olive oil contains a range of 20 to 45 mg/kg PINO. Despite the well-established preventative role of phytoestrogens against breast cancer, very little research has been done to elucidate whether PINO plays a chemopreventive role or exhibits antitumor activity in human breast cancer cells. Moreover, the oestrogen receptor status is a key factor to consider in breast cancer therapy. In fact, hormone therapy is only used in oestrogen receptor-positive breast cancer [17, 18]. Accordingly, we attempted to elucidate the effects of PINO on human mammary cells with different oestrogen and progesterone receptor expression, to determine whether this compound may contribute, at least in part, to the reduced incidence of breast cancer associated with VOO consumption. For this purpose, we used the following human breast tumour cells: MDA-MB-231 cells (ER-, PR-) and MCF7 cells (ER+, PR+). Furthermore, non-tumorigenic human mammary epithelial cells were also used in the present study [MCF10A (ER-, PR-)].
The cytotoxic activity of PINO on human breast tumour cells is a debated issue. Previously, Chin et al.  described that PINO has a cytotoxic effect against MCF7 breast cancer cells (ED50 = 4.74 μM); however, in a later article , the same author found no cytotoxic effects. Surprisingly, the range of concentrations used in both studies was not specified. In addition, the cytotoxic effects of PINO in MDA-MB-231 cells have not been previously reported. In contrast, we tested a wide range of PINO concentrations and showed that there was cytotoxic activity at different concentrations in both human breast tumour cells tested. While PINO showed cytotoxic activity in both types of human breast tumour cells tested, the effect was more pronounced in negative oestrogen receptor tumour cells compared to oestrogen receptor-positive tumour cells (Figs. 4 and 5). In addition, for the first time, we describe the effects of PINO on human mammary epithelial cells. Our results suggest that PINO ranging between 0.001 and 0.1 μM, which could be considered as physiological doses, has a much greater cytotoxic effect on breast tumour cells compared to mammary epithelial cells, suggesting an anti-tumour effect of this compound with a minor damage to non-tumorigenic tissue.
Little research has been performed to understand the effects of PINO on human breast cancer cell proliferation. Sepporta et al.  found that PINO inhibited the growth of MDA-MB-231, but not of MCF7 cells; however, their study was limited to 100 μM, which is not considered to be a physiological concentration. In contrast, we tested a wide range of PINO concentrations, ranging from 0.001 to 100 μM, and showed that low concentrations of PINO elicited a significant antiproliferative effect on both human breast tumour cell lines tested. Future work is needed to clarify the mechanisms of inhibition of breast cancer cells growth only at low doses.
Oestrogen has been associated with the promotion and growth of breast cancer. In line with this result, most human breast cancers that are oestrogen-dependent undergo regression when deprived of the supporting hormone . Our results, therefore, are very interesting because although PINO is a phytoestrogen with an oestrogen-like chemical structure, it produced a decrease in the proliferation of human breast tumour cells. Thus, PINO could have oestrogen antagonist activity, like tamoxifen, which inhibits breast cancer cells proliferation. However, in the experimental cell model we designed, we used cell culture media without oestrogen supplementation, suggesting that PINO is not likely to act as an oestrogen antagonist. Interestingly, a previous prospective study showed that high dietary intakes of plant lignans, such as PINO, were associated with reduced risks of ER+/PR+ postmenopausal breast cancer . We do not believe that the anti-proliferative effects of PINO are mediated by interactions with ERα because this receptor is not expressed in MDA-MB-231 breast tumour cells. Furthermore, the cell proliferation reduction was higher in MDA-MB-231 cells than in ERα + MCF7 breast cancer cells. On the other hand, it is unlikely that the anti-proliferative effects of PINO could be due to the activation of ERβ because both breast cancer cells tested MDA-MB-231 and MCF7, express low levels of this receptor . Additionally, it has been suggested that ERβ exerts anti-proliferative effects in breast cancer cells in the presence of ERα, but exerts proliferative effects in the absence of ERα . If this were true, treatment with PINO would result in an increase of MDA-MB-231 breast cancer cell (ERβ low/ ERα negative) proliferation. Instead, we found an anti-proliferative effect, which was even greater than that observed in MCF7 breast tumour cells (ERβ low/ ERα positive). Based on these results, we hypothesize that the anti-proliferative effects of PINO in the breast cancer cells assayed are independent of both ERα and ERβ status.
Previously, it has been shown that persistent ROS induction in non-tumorigenic cells may lead to cancer initiation, progression and spreading via activation and maintenance of signalling pathways that regulate cellular proliferation, survival, angiogenesis and metastasis . However, we have not found previously published results regarding the antioxidant capacity of PINO in mammary cells. Our results suggest that PINO may prevent cancer development, as it diminished ROS levels in MCF10A mammary epithelial cells.
On the other hand, it is known that cancer cells possess higher intracellular ROS levels than non-tumorigenic cells and that enhanced ROS levels may be exploited to promote cancer cell death . In fact, many of the commonly used chemotherapies are based on increasing oxidative stress above a toxic threshold level to selectively kill cancer cells . In line with this concept, PINO may be used as a potential effective adjuvant to cancer therapies, as it was found to promote ROS generation in breast cancer cells, while it tended to diminish ROS induction in mammary epithelial cells. MCF7 cells were shown to be particularly sensitive to increased ROS levels after H2O2-induced oxidative stress, which could be related with the levels of DNA damage observed under basal conditions and after oxidative shock. Under basal conditions, PINO also caused DNA damage in MDA-MB-231 cells; however, in contrast, PINO treatment prevented DNA damage in non-tumorigenic mammary epithelial cells, suggesting that PINO treatment may protect DNA in a pro-tumorigenic environment, thereby inhibiting breast cancer initiation and progression. Surprisingly, ER negative cells showed reduced DNA damage in response to H2O2, whereas ER positive cells showed an increase in DNA damage.
Very few reports have studied the chemical antioxidant capacity of PINO, and the results have varied considerably. For example, Kuo et al.  obtained a significant DPPH free radical scavenging activity for PINO, but these results differ from the work done by Chin et al.  and Vuorela et al. , which demonstrated a much higher IC50. Our results suggest that PINO harbours a radical scavenging activity at concentrations of 10 μM or above for ABTS. This capacity was also shown using the DPPH method and is line with work published by Chin et al. . In the ORAC assay, which is considered to be the most biologically relevant assay , PINO also showed antioxidant activity in a dose dependent manner.
Here, we showed that PINO possesses a chemical antioxidant capacity and may have a therapeutic potential to prevent breast cancer development via the reduction of intracellular oxidative stress and DNA damage in human mammary epithelial cells. Furthermore, we showed that PINO promotes an increase in the ROS levels of breast cancer cells after H2O2 treatment. In sum, this work suggests that PINO may act as adjuvant to pro-oxidative chemotherapies.
Finally, we showed that PINO has anti-tumour effects at low concentrations by promoting cytotoxic, anti-proliferative and pro-oxidant activities in breast cancer cells, independent of their oestrogen receptor status.
ABTS, 2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); DCFH-DA, dichlorofluorescin diacetate; DPPH, 2,2-Diphenyl-1-picrylhydrazyl; ER, oestrogen receptor; FBS, foetal bovine serum; HER2, human epidermal growth factor receptor 2; NEAA, non-essential amino acids; ORAC, oxygen radical absorbance capacity; PINO, (+)-pinoresinol; PR, progesterone receptor; ROS, reactive oxygen species; RSA, radical scavenging activity; VOO, virgin olive oil.
This study was supported by the “Junta de Andalucía” (Proyecto de Excelencia PI10-AGR-6724) and by a grant of “Consejería de Economía, Innovación y Ciencia” (Ayuda de personal investigador en formación. Convocatoria 2010).
This study was financially supported by the “Junta de Andalucía” (Proyecto de Excelencia PI10-AGR-6724) and by a grant of “Consejería de Economía, Innovación y Ciencia” (Ayuda de personal investigador en formación. Convocatoria 2010).
Availability of data and materials
The relevant datasets supporting the conclusions of this article are included within the article. The whole data including all images obtained, histograms, analyses or raw data are available from the corresponding author on reasonable request.
Conception and design: JJG, AL-B, CS-Q; Development of methodology: AL-B, CS-Q; Sample processing: AL-B, CS-Q; Analysis of data: AL-B, JJG, MD-R; Writing of the manuscript: AL-B, JJG; Revision of the manuscript: JJG, GB, MD-R. All authors read and approved the final manuscript.
The authors declare that they have no competing interest.
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