European Journal of Nutrition

https://doi.org/10.1007/s00394-018-1638-9

ORIGINAL CONTRIBUTION

Modulation by hydroxytyrosol of oxidative stress and antitumor

activities of paclitaxel in breast cancer
Nuri El‑azem1 · Mario Pulido‑Moran1,2 · Cesar L. Ramirez‑Tortosa3 · Jose L. Quiles1,4 · Francisca E. Cara5 ·
Pedro Sanchez‑Rovira6 · Sergio Granados‑Principal1,7 · MCarmen Ramirez‑Tortosa1,2 

Received: 25 October 2017 / Accepted: 13 February 2018

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Purpose  The main objective of this study was to test the therapeutic potential of hydroxytyrosol and its combination with
paclitaxel in breast cancer on oxidative stress status.
Methods  Impact on proliferation rates of different chemotherapy administration patterns was assayed in MCF-7 and MDA-
MB-231 breast cancer cell lines. Breast tumor-bearing rats were randomly assigned to Control, Hydroxytyrosol, Paclitaxel
and Paclitaxel plus hydroxytyrosol groups, for 6 weeks. Tumor volume, cell proliferation and several systemic oxidative
stress parameters were measured. Anti-proliferative activity in vitro experiments was correlated with in vivo experiments.
Results  Combination group did significantly reduce tumor volume when compared with paclitaxel alone. Additionally, the
combination improved the antioxidant status without compromising the antitumor activity of standard chemotherapy.
Conclusion  These findings reveal for the first time that hydroxytyrosol is an active partner in combined therapies with pacli-
taxel against breast cancer. Combination with hydroxytyrosol would also ensure a less oxidative impact of chemotherapeutic
drugs that could potentially improve patient wellness.

Keywords  Breast cancer · Hydroxytyrosol · Cell proliferation · Oxidative stress · Multimodal treatment and paclitaxel

Background women worldwide [3]. Fortunately, mortality rates have

been reduced in the last decade due to the extensive study
Breast cancer is the most common type of cancer in women, of this disease and the innovation in new treatments and pre-
with an increasing incidence that is widely larger than other vention diagnostics [4, 5]. Incidence rates vary worldwide
ones [1, 2] and is the leading cause of cancer death among depending on sociocultural and nutritional factors, with a
lower incidence in Mediterranean countries, which are tra-
* MCarmen Ramirez‑Tortosa ditionally characterized by a high consumption of vegetables
mramirez@ugr.es and olive oil [6, 7].
Olive oil (OO) is composed mainly by oleic acid, other
1
“José Mataix” Institute of Nutrition and Food Technology, fatty acids and by different minor elements such as phenolic
Biomedical Research Centre, Health Sciences Technological
Park, University of Granada, 18016 Armilla, Granada, Spain compounds, flavonoids, lignans or secoiridoids. It has been
2 proposed that the beneficial effects of OO can also be attrib-
Department of Biochemistry and Molecular Biology II,
University of Granada, 18016 Armilla, Granada, Spain uted to these minor compounds [8, 9] and not only to oleic
3 acid. Hydroxytyrosol (HT) is the most representative com-
Department of Pathological Anatomy, Hospital Complex
of Jaén, Jaén, Spain ponent of the phenolic group. Many effects have been asso-
4 ciated to HT such as a strong antioxidant ability, antitumor
Department of Physiology, University of Granada, Granada,
Spain properties, antiatherogenic, antimicrobial, antiviral agent,
5 iron chelative, hypolipidemic, antiinflammatory, antithrom-
Department of Nanomedicine, Houston Methodist Research
Institute, Houston, TX, USA botic, hypoglycemic and has also shown protective effects
6 in rat cardiomyocytes in an ischemia–reperfusion model. In
Oncology Service, Jaen City Hospital, Jaén, Spain
addition, it should be noted that HT intake is safe even at
7
Methodist Cancer Center, Houston Methodist Hospital, doses of 500 mg/kg/day [10–14].
Houston, TX, USA

13
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On the other hand, it have been assessed the antitumor
effect of HT both in cancer cell lines [15, 16] and animal
models [17]. These experiments found that HT was able to
inhibit growth and cell proliferation of mammary tumors,
while activating or repressing several genes and pathways
related to cell proliferation and apoptosis.
In addition to direct effects over the modulation of path-
ways related to carcinogenesis, HT is a powerful antioxidant
able to decrease radical oxygen species (ROS) production
and protect against oxidative damage [9]. These ROS play a
pivotal role in the development of cancer as they are able to
strongly induce and promote growth and survival of tumor
cells [18]. Moreover, ROS have been identified as the main
cause of cardiotoxicity induced by certain chemotherapeutic
drugs in breast cancer treatment. As a consequence, pacli-
taxel (PTX) [19] dosage must be limited and tightly con-
trolled to avoid the instauration of adverse cardiac effects in
those patients [19, 20] by a direct interaction between ROS
and cardiomyocytes. A reduction in ROS production and its
levels may prevent or delay the onset of this toxicity, allow-
ing a greater exposure time and/or higher doses of the treat-
ment [19, 20]. Furthermore, have also been demonstrated
that breast cancer patients showing increased systemic anti-
oxidant and DNA repair capacities after chemotherapy, reg-
ister significantly improved survival rates in terms of both
disease relapse and overall survival [21, 22].
For all above described, in this study, the authors have
investigated whether HT could represent a novel compound
to be used in the treatment of breast cancer in combina-
tion with conventional chemotherapeutic drugs (taxanes)
in vivo and in vitro models. Furthermore, HT would be able
to decrease the oxidative damage induced by chemother-
apy that could improve the patient’s wellness and overall
survival.
Materials and methods
Cell culture and proliferation
Estrogen receptor positive (MCF-7) and triple negative
(MDA-MB-231) breast cancer cells lines were purchased
from American Type Culture Collection. Cells were grown
in DMEM (Gibco) supplemented with 10% fetal bovine
serum (ThermoScientific) and 1% antibiotic–antimycotic
(Gibco) at 37 °C with 5% ­CO2. The cell lines were not tested
or authenticated over and above documentation provided by
the ATCC, which includes antigen expression, DNA profile,
short tandem repeat profiling, and cytogenic analysis. Cell
proliferation was assessed with the WST-1 method assay.
Briefly, cells were plated at a density of 2 × 103 cells/well
in a 96-well plate and treated with PTX (0,1, 2.5, 5, 10 nM)
(Teva Pharmaceuticals) alone or combined with HT (0, 10,
13
European Journal of Nutrition
25, 50, 100 µM) (Cayman Chemical) for 72 h. Proliferation
rate was determined by adding premixed WST-1 reagent
(Clontech). After incubation at 37 °C for 4 h, absorbance
was read at 450 nm (reference wavelength 690 nm).
Animal studies
Twenty-four female Sprague–Dawley rats (7 weeks old)
(Harlan Interfauna Iberica), were housed under standard
laboratory conditions (22 ± 2 °C; 12 h/12 h light/dark cycle
and free access to food and water). Mammary tumors were
induced as previously reported by Granados-Principal et al.
[17]. Once the tumors reached 1 cm3, the rats were rand-
omized into 4 different groups (n = 6/group) as follows: (1)
control (saline + sterile water by gavage), (2) HT (0.5 mg/
kg/day, 5 days/week for 6 weeks by gavage) (3) PTX (2 mg/
kg/week for 6 weeks, i.v.) and (4) PTX-HT: combo group
treated with the same doses and timing than HT and PTX
groups. Tumor volume was recorded twice a week. 1 week
after the last dosage, animals were weighed, anaesthetized
with intraperitoneally administered ketamine (Sigma), and
killed by aortic bleeding and thoracotomy. A 21 G needle
was employed for an expert technician to collect whole
blood samples directly from the bifurcation of the aorta
with a downward direction of the bevel, pulling the plunger
enough slowly to collect as much sample as possible with-
out causing hemolysis and blood clotting. Whole blood was
collected into EDTA containing tube (Vacutainer® EDTA
Tubes BD, Franklin Lakes, NJ) and centrifuged at 1000×g
for 15 min to obtain plasma which was aliquoted and frozen
at − 80 °C until its use. The animals were handled according
to the principles of the Helsinki Declaration, Spanish animal
welfare legislation and after the approval of procedures by
the Ethical Committee in Animal Experimentation of the
University of Granada (CEEA 264–2008) (Spain).
Immunohistochemistry
For immunohistochemical techniques, tumor sections were
deparaffinized in xylene and rehydrated through graded
alcohols to water. Antigen retrieval was performed with the
PTLink module (Dako, Glostrup, Denmark) using Dako
low-pH Antigen Retrieval fluid (Dako) followed by several
washes in water before being placed onto an Autostainer
Plus Link (Dako) where the remainder of the immunohis-
tochemical staining was performed using Envision FLEX
(Dako). Briefly, sections were first placed in washing buffer
followed by blockade of endogenous peroxidase with 3%
hydrogen peroxide for 5 min. Then, primary antibody Ki-67
(Clone SP6, Máster Diagnóstica, Granada, Spain) was
applied for 20 min, followed by another buffer wash, and
visualized with 3,3′-diaminobenzidine for 10 min. Following
a water wash, sections were counterstained in hematoxylin
European Journal of Nutrition
for 7 min, washed in water, dehydrated, and cover-slipped.
The results were compared to negative controls to eliminate
false positives. Tissues were blindly evaluated by a pathol-
ogist. Ki-67 proliferation was assessed from at least 1000
tumor cells. The most peripheral tumor areas were selected
as they constitute front infiltration of the neoplastic.
Alkaline comet assay
Alkaline comet assay was used to measure the DNA dam-
age in isolated plasma lymphocytes as previously described
[23]. Briefly, cells were incubated in lysis buffer for 1 h, and
subjected to alkaline DNA unwinding for 40 min. After elec-
trophoresis, the nucleoids were stained with 4′,6-Diamidino-
2-phenylindol dihydrochloride (Sigma) and scored using a
Leica DMLS fluorescence microscope (Leica Microsys-
tems). One hundred comets from each gel were scored using
computerized image analysis (Komet 3.0; Kinetic Imaging
Ltd.) and the percentages of fluorescence in the comet tail
(representing the fraction of DNA in the tail), head (rep-
resenting the fraction of DNA in the head) and Olive Tail
Moment (OTM) (indicating the smallest detectable size of
migrating DNA and the number of relaxed/broken pieces)
were measured.
Total antioxidant capacity (TAC) and protein
oxidative damage
TAC was assessed using the method described by Vera et al.
[22]. To determine it, the aliquoted plasma samples were
gradually thawed and only once defrosted. The results were
expressed as µM Trolox.
Plasma levels of protein carbonyl groups were assessed
using Protein Carbonyl Kit (Cayman Chemical), according
to the manufacturer’s instructions. Total protein concentra-
tion in the plasma samples was measured using Biuret Col-
orimetric Assay Kit (Spinreact). The results were expressed
as nmol of carbonyl proteins per mg of total proteins in
plasma.
Statistical analysis
Data were analyzed using the IBM SPSS Statistics for
Windows Version 22.0 (IBM Corp., Armonk, NY, USA).
Data are presented as mean ± SEM. Statistical significance
between two groups was analyzed by two-tailed Student’s
t test. Experiments with more than three groups were ana-
lyzed with one-way ANOVA (analysis of variance) followed
by Bonferroni’s post-hoc test. Statistical analysis of tumor
volume and differences between combination groups in vitro
was assessed by two-way ANOVA and Bonferroni’s post-
hoc test. A p value less than 0.05 was considered as sig-
nificant. Tumor cells lines experiments were performed in
triplicate for each assay condition. In addition, all experi-
ment were carried out at least five times.
Results
Effect of PTX in combination with HT on tumor cell
proliferation
It was assessed the effects of HT, both alone and in combina-
tion with different concentrations of PTX on the proliferation
rates of MCF-7 (Fig. 1a) and MDA-MB-231 (Fig. 1b) cell
lines for 72 h. HT alone was able to significantly decrease
cell proliferation in MCF-7 at 50 µM and dramatically at
100 µM doses (Fig. 1a). In MDA-MB-231 cells, cell pro-
liferation decreased at all doses (10, 25, 50, and 100 µM)
(p < 0.05) (Fig. 1b). MDA-MB-231 cells were also more
sensitive to PTX treatment alone (5 and 10 nM), and the
combination of HT (100 µM) and PTX led to a significant
reduction in cell proliferation (p < 0.05) (Fig. 1b).
Combination of HT with PTX on tumor growth
in a rat model of breast cancer
Based on in vitro data, it was next investigated whether
combination of HT with PTX was able to reduce tumor vol-
ume in a breast cancer rodent model to ascertain the results
in cells. Daily injections of HT in combination with PTX
(2 mg/kg/week) were given to breast tumor-bearing rats for
6 weeks. Tumor growth was decreased in HT alone, PTX,
and very substantially in the combination group (Fig. 2a,
p < 0.001).
Consistent with in vitro results, it was observed reduced
tumor growth in PTX-HT group compared to PTX group
throughout the experiment (Fig.  2a, p < 0.001). Conse-
quently, it was correlated these results with tumor cell
proliferation by immunohistochemistry (Ki-67). Lower
proliferation levels were found in HT alone and PTX-HT
groups compared to the control and to PTX groups (Fig. 2b,
p < 0.05). PTX group exhibited higher percentage of pro-
liferating cells that was efficiently decreased by the com-
bined treatment with HT (Fig. 2b, p < 0.05). HT per se could
decrease cell proliferation at the same level as combined
group. Overall, these results demonstrate that HT did not
alter the effectiveness of standard chemotherapeutic drugs.
Moreover, it has an evident effect when it is combined with
PTX in vivo.
Impact on oxidative status
To assess whether co-treatment with HT is able to
decrease the oxidative stress induced by chemotherapy,
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 European Journal of Nutrition

Fig. 1  Effects of different con-

centrations of paclitaxel (0, 1,
2.5, 5 and 10 nM) in combina-
tion with different concentra-
tions of hydroxytyrosol (HT)
(0, 10, 25, 50 and 100 µM) on
cell proliferation in MCF-7
(a) and MDA-MB-231 (b)
cell lines. Data are expressed
as the mean ± SEM. Bars not
sharing superscripts letters are
statistically different (p < 0.05)
for each concentrations of
paclitaxel

DNA damage was measured in isolated lymphocytes, as
well as antioxidant markers in plasma.
Alkaline comet assay was employed to investigate DNA
damage in lymphocytes that is depicted herein as OTM
(Fig. 3). The results show that HT alone caused the low-
est DNA damage level. PTX did not cause an additional
increase in DNA damage compared to control. PTX-HT
group exhibited a lower level of DNA damage compared to
control group and a slight downward trend facing to PTX.
Plasma TAC (Fig. 4) was measured using the ABTS
assay. It was found that both HT combined with PTX and
HT alone did significantly increase the plasma antioxi-
dant capacity compared to PTX group (Fig. 4, p < 0.05),
although they did not show significant differences with
control group. However, PTX combined with HT showed
levels of TAC significantly higher than PTX group.
In relation to protein carbonyls (Fig. 5), the combined
treatment of HT with PTX decreased protein carbonyls
levels significantly (p < 0.05) compared to PTX group.
However, HT group showed significantly a high capacity
to decrease the oxidation of protein compared to PTX
group (p < 0.05).
Discussion
Current pharmacological treatments for breast cancer consist
highly effective drugs that are able to reduce tumor volume
but with several important side effects that could compro-
mise patient wellness. Indeed, it have previously been shown
that breast cancer patients experience high levels of DNA,
protein and lipid oxidative damage, which are sustained
along different clinical interventions, including the adminis-
tration of anthracyclines and taxanes as part of their chemo-
therapeutic treatment [21, 22]. In terms of toxicity derived
from overproduction of ROS, PTX induces the production
of ROS [24] through a yet unknown mechanism, but it is
believed that it could increase mitochondrial permeability
and ROS formation in a similar way as docetaxel [25].

13
European Journal of Nutrition
Fig. 2  Progress of the trucut mammary tumor volume during the
experimental times (6 weeks) and cell proliferation (Ki67) in a rat
model of breast cancer. Differences between control, HT, PTX and
PTX + HT group (n = 6 per group) (a). Percentage of Ki-67 positive
cells in control, HT, PTX and PTX + HT groups of rats (n = 6) with
experimental breast cancers at the end of the treatments (b). Results
HT has been recently emerging as a promising agent
against breast cancer due to the high antitumor activity
both in vitro [26, 27] and in vivo models [17, 28]. Addi-
tionally, HT has been extensively studied as a powerful
antioxidant [9] that could help to ameliorate the high oxi-
dative damage induced by chemotherapeutics [29]. To the
best of our knowledge, this is the first report studying the
effect of HT in combination with standard chemotherapeu-
tic (PTX) using in vitro and in vivo models of breast can-
cer. Antitumor effects of HT in breast, thyroid and colon
cell lines [27, 30] and in vivo models of breast cancer [17,
are expressed as mean ± SEM. Statistical significance is indicated as
symbols (†differences between control group and treatment groups,
p < 0.001) and (*differences among the treatment groups, p < 0.05).
HT hydroxytyrosol (n = 6), PTX paclitaxel (n = 6), PTX-HT paclitaxel
plus hydroxytyrosol. Bars for cell proliferation not sharing super-
scripts letters are statistically different, p < 0.05
28] are widely known. Nonetheless, there is lack of studies
showing potential effect of HT in breast cancer models.
Trying to unscramble the antiproliferative character, it
was observed how HT per se was able to decrease the per-
centage of growth both MCF-7 cells and MDA-MB-231 at
50 µM and 100 µM. It is essential to note that when HT was
combined at 100 µM, the largest decrease of proliferation
was obtained (Fig. 1a).
Similarly, in MDA-MB-231 cells, when HT is combined
at 100 µM with PTX 5 nM, is again achieved better anti-
proliferative effect than if only PTX were used (Fig. 1b).A
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 European Journal of Nutrition

Fig. 3  DNA damage of isolated 1.2

lymphocytes of experimental c
breast cancer rats (n = 6 per
group) treated with PTX, HT 1

Olive Tail Moment

or their combination specified
bc
as OTM. Data are expressed
as mean ± SEM. Statistical 0.8
significances: bars not sharing
superscripts letters are statisti- b
cally different p < 0.05. HT 0.6
hydroxytyrosol, OTM Olive tail
moment, PTX paclitaxel, PTX-
HT paclitaxel plus hydroxyty- 0.4
a
rosol
0.2

0
CONTROL HT PTX PTX-HT

Fig. 4  Total antioxidant capac-

ity (TAC) of plasma from
experimental breast cancer rats
(n = 6 per group) treated with
PTX, HT or their combination
specified as µM of Trolox. Data
are expressed as mean ± SEM.
Bars not sharing superscripts
letters are statistically different,
p < 0.05. HT hydroxytyrosol,
PTX paclitaxel, PTX-HT pacli-
taxel plus hydroxytyrosol

Fig. 5  Proteins oxidation deter- 1 b

mined as plasma protein carbon-
yls levels in rats with experi- 0.9
mental breast cancer (n = 6 per
Protein Carbonyls (nmol/mg)

group) treated with PTX, HT 0.8 ab

and their combination. Data are 0.7 ab
expressed as the mean ± SEM.
Bars not sharing superscripts 0.6 a
letters are statistically different
(p < 0.05). HT hydroxytyrosol, 0.5
PTX paclitaxel, PTX-HT pacli- 0.4
taxel plus hydroxytyrosol
0.3
0.2
0.1
0
CONTROL HT PTX PTX-HT

13
European Journal of Nutrition
strong point in favor of HT was the large decrease produced
on tumor volume (most of all) when was associated to PTX,
showing a good agreement with the previously detailed in
in vitro studies and now supported by a reduced proliferation
rate, well below the control and PTX groups and at the same
level as the HT alone. This enhanced On the other hand, the
breast cancer cell line MCF-7 (estrogen receptor positive)
has more effected with HT (100 µM) alone or combined with
PTX than the breast cancer cell line MDA-MB-231 (triple
negative). These evidences are in according with Sirianni
et al. (2010) [31]. These authors demonstrated that HT can
have a chemopreventive role in breast cancer cell prolifera-
tion through the inhibition of rapid signals dependent on
estrogen involved in uncontrolled tumor cell growth.
Based on these results, it was attempted to demonstrate
whether this effect was equal or similar when animal vari-
ables were introduced, such that all normal biological pro-
cesses may act together over HT and vice versa. All the
rats developed with DMBA breast cancer with expression
of estrogen receptor in according to Alvarado et al. [32]
study and after 6 weeks of treatment in Sprague–Dawley
rats, both tumor volume (Fig. 2a) and cell proliferation (by
Ki-67) (Fig. 2b) were evaluated. It was found how HT and
PTX produced a smaller tumor volume than control which
was increasing over time, as obviously expected the initial
idea of antitumor character of the HT and its great effect
when is associated to conventional highly toxic chemother-
apy, even in animals where all biological aspects and pos-
sible interactions come into play. In this sense, our finding
are in accordance with [33] where HT inhibited the cancer
cell growth via Epidermal Growth Factor Receptor (EGFR)
at the same level than other conventional drugs, showing a
possible cooperation mechanism as well as the beneficial
character of the association of HT and other anticancer
drugs without compromising the antitumor activity. These
results are perfectly correlated with lower tumor cell prolif-
eration, expressed as percentage of Ki-67, in PTX-HT and
HT groups compared to PTX or control groups as showed
the Fig. 2b.
The association between proliferative index and tumor
growth is well established. Tumors with high Ki-67 expres-
sion increase more than tumors with low Ki-67 expression.
However, the relation proliferative index/tumor growth is
different when the tumors have received chemotherapy.
Burcombe et al. [34] have described that in median day-21,
Ki-67 was higher in pathological responders (30.3 versus
14.1%, p = 0.046) and there were no association between
pathological response and changes in Ki-67 throughout the
study period, in patients with breast cancer in neoadjuvant
chemotherapy. Moreover, the reduction in Ki-67 index after
one cycle of treatment was not sustained and was often fol-
lowed by a rebound increase in cell proliferation by the time
of surgery although the clinic-pathological responses were
good. More recent studies with neoadjuvant chemotherapy
and treatment with taxanes showed that a lot of tumours with
medium pathological response decreased the tumor volume
but the proliferative index measured as Ki-67 did not change
or even increased after the treatment compared with its basal
value [35]. All described above is in according with our
results where the proliferative index in rat with experimental
breast cancer was the same than in control group. As has
been demonstrated by Enomoto et al. [36] and Diaz-Botero
et al. [37] these results show a bad prognostic although in
our study this effect is eliminated by the HT mechanism of
action.
Overall, these results demonstrate for the first time that
HT is able to enhance the antitumor capacity of chemothera-
peutic drugs in breast cancer provoked in rats with DMBA
which produced estrogen receptor positive tumor. Due to
the ethical difficulties to try HT as a unique antitumor drug
in future clinical trials, must be highlighted its great effect
during the treatment against breast cancer, promoting a
decrease in adverse effects associated to conventional anti-
tumor drugs.
Seeking answers to whether the antioxidant character
of HT, when combined with PTX exerted the same effect,
it was found that this association, although DNA damage
expressed as OTM levels (Fig. 3) did not decrease to the
same levels as HT (the lowest), it was done regarding the
control group, and shows a clear downward trend compared
to PTX alone, which made it easier to think that if PTX
produced greater damage to DNA, by associating with HT
there was an improvement in the results. Maybe they were
together mitigating the adverse effects related to high levels
of oxidative stress which can promote several deleterious
effects, thus avoiding genetic instability and mutations that
affect the control of cell [38], neurodegenerative disorders
[39], diabetes, cardiovascular diseases, or metabolic syn-
drome [40], associated at least in part to PTX.
These data were supported further by observing a similar
pattern in plasma TAC (Fig. 4). HT had the highest levels,
even compared to the control group that, as other studies
showed, tumors themselves promote a heightened state own
body’s defense against the ROS generated by pathology
[21, 22]. PTX had the lowest antioxidant capacity values,
similar as occur in [41] and once again, associating HT,
improved results as a greater possibility of plasma antioxi-
dant response. If higher levels of plasma TAC imply a lower
oxidative damage to different biological structures and mol-
ecules [42], this gave points in favor of the possibility that
HT palliate the side effects associated to PTX.
All together allow to elucidate that are not isolated or
random results, HT combined has the ability to improve the
response to the ROS produced by chemotherapeutic further
enhancing its antitumor and antiproliferative effects, improv-
ing the antioxidant capacity and most importantly, without
13

countering any effect, as occurring with other antioxidants
tested as resveratrol [43] or quercetin [44].
These unbalanced ROS levels also affect to proteins. As
has been shown in Fig. 5, protein carbonyls levels were a
similar pattern than OTM parameter. These results are in
according with Panis et al. (2012) [41] and Pulido-Moran
et al. (2018) [45]. Moreover, when HT and PTX were com-
bined, there were significant differences with PTX group,
thanks to HT effect.
In conclusion, considering all above, it can be generally
concluded that HT shows the best results due to its decreased
cell proliferation and increased capacity for an antioxidant
response thanks to enhance all defense systems against ROS,
something that favor the reduction of damage to lipids, pro-
teins and nucleic acids in breast cancer with expression of
estrogen receptors. Since it would be unethical use as a sin-
gle compound for a future therapy in humans, it is combined
with other chemotherapeutic agents as paclitaxel which by
themselves generate a very high redox imbalance, promoting
damage to all macromolecules of both tumor and nontumor
cells. Therefore, this association compensates the adverse
effects linked to chemotherapeutics as Paclitaxel. HT even
allows lower doses of chemotherapy, reducing the associated
toxic effects, without losing effectiveness.
Acknowledgements  This research was supported by Grants from
Excelentísima Diputación de Jaén, CEAS Foundation 30.C0.244500
and Junta de Andalucía PI-0210/2007.
Compliance with ethical standards  12. Medina-Martinez MS, Truchado P, Castro-Ibanez I, Allende A
Ethical standards  Manuscript submitted for publication has been
approved by the appropriate ethics committee and have, therefore, been
performed in accordance with the ethical standards laid down in the
1964 Declaration of Helsinki and its later amendments.
Conflict of interest  The authors have declared no conflict of interest.
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