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International Journal of
Molecular Sciences
Review
Dietary Phenolics against Breast Cancer. A Critical
Evidence-Based Review and Future Perspectives
MaríaÁngeles Ávila-Gálvez, Juan Antonio Giménez-Bastida , Juan Carlos Espín * and
Antonio González-Sarrías *
Laboratory of Food and Health, Research Group on Quality, Safety and Bioactivity of Plant Foods,
Dept. Food Science and Technology, CEBAS-CSIC, P.O. Box 164, Campus de Espinardo, 30100 Murcia, Spain;
mavila@cebas.csic.es (M.Á.Á.-G.); jgbastida@cebas.csic.es (J.A.G.-B.)
*Correspondence: jcespin@cebas.csic.es (J.C.E.); agsarrias@cebas.csic.es (A.G.-S.)
Received: 21 July 2020; Accepted: 8 August 2020; Published: 10 August 2020
Abstract:
Breast cancer (BC) is the most common malignancy and the leading cause of cancer-related
death in adult women worldwide. Over 85% of BC cases are non-hereditary, caused by modifiable
extrinsic factors related to lifestyle, including dietary habits, which play a crucial role in cancer
prevention. Although many epidemiological and observational studies have inversely correlated
the fruit and vegetable consumption with the BC incidence, the involvement of their phenolic
content in this correlation remains contradictory. During decades, wrong approaches that did not
consider the bioavailability, metabolism, and breast tissue distribution of dietary phenolics persist
behind the large currently existing gap between preclinical and clinical research. In the present
review, we provide comprehensive preclinical and clinical evidence according to physiologically
relevant
in vitro
and
in vivo
studies. Some dietary phenolics such as resveratrol (RSV), quercetin,
isoflavones, epigallocatechin gallate (EGCG), lignans, and curcumin are gaining attention for their
chemopreventive properties in preclinical research. However, the clinical evidence of dietary phenolics
as BC chemopreventive compounds is still inconclusive. Therefore, the only way to validate promising
preclinical results is to conduct clinical trials in BC patients. In this regard, future perspectives on
dietary phenolics and BC research are also critically discussed.
Keywords: breast cancer; polyphenols; in vitro; animal models; clinical trials; dietary phenolics
1. Breast Cancer: General Aspects
Cancer is one of the world’s most substantial health problems and the second leading cause of
death globally, with 9.6 million cancer-related deaths in 2018 [
1
,
2
]. Among women, breast cancer (BC)
is the first cause of cancer-associated death and the most commonly diagnosed malignant tumour
worldwide, accounting for 2.1 million new BC diagnoses in 2018 [
3
]. Although the advances in
diagnosis, treatment and intensive research have significantly increased the survival rates among BC
subjects in the past years, its incidence has increased worldwide, especially in developing countries.
According to U.S. Breast Cancer Statistics, it is estimated that BC impacts nearly 1 in 8 women in their
lifetime [
4
]. Only 10%–15% of all BC cases are hereditary, meaning that the vast majority are caused
by modifiable risk factors, playing a central role in cancer prevention through lifestyle improvement,
including dietary habits [
5
,
6
]. Apart from the dietary and lifestyle habits, other related factors to
BC include age, ethnicity, hormonal status, hormonal therapy, ultraviolet radiation, and circadian
disruption [
7
]. Regarding genetic risk factors, some mutations such as breast cancer susceptibility
gene 1 (BCRA1), BCRA2, and TP53 are generally accepted, although these mutations are rare and only
constitute approximately 5%–10% of all BC rates incidences [5,8,9].
Int. J. Mol. Sci. 2020,21, 5718; doi:10.3390/ijms21165718 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2020,21, 5718 2 of 33
BC is a compilation of distinct malignancies that usually begins in the terminal duct lobular unit
of the mammary gland and progresses in a stepwise manner by multiple molecular alterations. BC is
highly heterogeneous, encompassing from some cases with excellent prognosis to very aggressive
tumours, which usually develop over a long time [
10
,
11
]. Clinically, BC is classified into different
grades and types based on histological characteristics. The histological grade is a well-established
prognostic tool based on the degree of aggressiveness or differentiation of the tumour tissue, including a
combined score for several parameters such as the microscopic evaluation of tubule or gland formation,
nuclear pleomorphism, and the mitotic count (i.e., determination of the proliferation marker Ki-67
by immunohistochemistry). Therefore, BCs with a high histological grade are generally large and
associated with metastasis, rather than those with low histological grade [
12
–
14
]. On the other hand,
the histological type is based on the growth pattern of the tumours including different subtypes,
presenting a higher incidence (around 75%), the so-called invasive or infiltrating ductal carcinomas
of no particular type, compared with other different subtypes such as invasive lobular carcinoma,
mucinous, tubular, medullary, etc. Thus, BCs with smaller tubular carcinomas are generally associated
with an earlier stage of the tumour, compared with invasive ductal carcinomas [
13
,
14
]. Besides, the TNM
is a well-accepted classification to designate the BC stage at the time of diagnosis refers to the size
and invasiveness of the tumour (T), lymph node involvement (N), and presence of distant metastasis
(M) [15].
Regarding molecular basis, BC is broadly categorized into different subtypes based on the
combination (presence or absence) of three receptors for oestrogens (ER), progesterone (PR) and
human epidermal growth factor receptor 2 (HER2). The differential expression of these receptors
strongly determines the prognosis and therapeutic strategies against BC [
11
,
14
]. In this regard,
the incidence of hormone receptor-positive (ER+PR+) is the most common BC subtype, representing
around 70%. It exhibits a better prognosis with high survival and low recurrence rates, compared
with hormone receptor-negative tumours. Moreover, according to the elevated expression of HER2
and related genes, BC is further categorized into different molecular subtypes, such as luminal A
(ER+PR+HER2-) and(or) luminal B (ER+PR+HER2+). HER2+tumours represent a 15%–20% of
BC cases. Interestingly, although this type is more aggressive, associated with an increase in Ki-67
protein, it responds better to the current therapy resulting in high survival rates [
11
,
16
,
17
]. Finally,
the triple-negative BC for these three biomarkers (TNBC) accounts for about 15% of all BCs. It is
characterized by the worst prognosis involving high invasive properties, high relapse rate, and high
resistance to different therapies, what make this subtype highly intractable and invasive (mainly
with the presence of metastasis in other organs) [
11
,
18
]. Six molecular subtypes of TNBC have been
reported according to their gene ontologies and differential gene expression in specific genes involved
in cell cycle, DNA damage response genes, androgen receptors, epidermal growth factor receptor, and
signalling pathways, among others. This highlights the significance of molecular heterogeneity in
BC [19].
Currently, beyond the prevention and early diagnosis, the therapeutic options for BC include
surgical resection, radiation, as well as chemotherapeutic supplementation according to the molecular
subtypes and the BC stage. Thus, for early-stage non-metastatic BC, surgical resection, together with
postoperative adjuvant chemotherapy, radiotherapy, and(or) hormone therapy depending on the
BC subtype, represent the main treatment options. On the other hand, treatments for metastatic BC
(about 20%–45%) generally consist of conventional chemotherapy, including cytotoxic drugs, such as
the anthracycline (doxorubicin), and(or) taxane (paclitaxel) families, combined with cyclophosphamide.
Other treatments include 5-fluorouracil, cisplatin derivatives, gemcitabine, or vinorelbine, as well as
monoclonal antibody therapy, such as bevacizumab that targets the vascular endothelial growth factor
receptor (VEGFR), what in turn inhibits angiogenesis processes [
11
,
20
,
21
]. Fortunately, the knowledge
of predictive biomarkers in the different BC subtypes has led to the use of combined treatments
with adjuvant chemotherapy, allowing for a personalized approach in each patient that increases
the effectiveness of the therapy. In this regard, the main treatments for hormone-dependent BC
Int. J. Mol. Sci. 2020,21, 5718 3 of 33
(ER+/PR+) combined with adjuvant chemotherapy are based on hormonal therapy, which is based
on the use of oestrogen antagonists, such as tamoxifen or fulvestrant and aromatase inhibitors like
anastrozole, letrozole, and exemestane [
22
]. In HER2 overexpressing tumours, the primary treatment
involves monoclonal antibodies such as trastuzumab and pertuzumab and specific HER2 pathway
inhibitors like lapatinib. This treatment could be combined with hormonal therapy, depending on
the hormone receptors’ expression [
20
,
21
,
23
]. As mentioned above, the lack of biomarkers expression
in TNBC is addressed through classical chemotherapy (anthracyclines, taxanes, platinum-containing
chemotherapeutic agents, etc.) as the unique therapeutic option [
11
,
20
,
21
]. However, adverse side
effects of these anticancer drugs alone or in combination are relatively frequent. Furthermore, the further
development of drug resistance processes leads to tumour recurrences after long-time administration.
Therefore, beyond the currently approved chemotherapeutics, natural products (including dietary
compounds) exhibit pleiotropic, multi-target activities and are emerging as possible complementary
chemopreventive molecules against BC with fewer side effects than conventional therapy [6,24].
2. Epidemiological Evidence for Dietary (Poly)Phenols
As stated above, it is well-established that lifestyle improvement, including dietary habits, is the
primary prevention strategy against cancer. Indeed, improper dietary habits (high amounts of fat,
sugar, red meat, and alcohol) may be behind 30%–35% of all the cancer types [
25
,
26
]. In contrast,
epidemiological and observational studies have described the inverse association between high intake of
plant foods and low BC risk [
27
,
28
], or high vegetable intake and low ER-BC risk [
29
,
30
]. Nevertheless,
several studies indicate that this inverse correlation for high consumption of fruit and vegetables
remains still inconsistent or contradictory [
6
,
29
,
31
], and limited evidence has been found according to
the recent European Prospective Investigation into Cancer and Nutrition (EPIC) data [31,32].
Over the past decades, meta-analyses, observational, and epidemiological studies have suggested
the potential role of dietary phenolics in the chemoprevention and chemosensitization in BC [
33
–
35
].
In this regard, significant protective effects against BC have been attributed to some dietary patterns
that involve high intake of phenolics-rich foods, mainly phytoestrogens that can bind to ER, such as
isoflavones in soy-based products in Asian countries [
36
–
38
]. Mediterranean dietary patterns implicate
higher intake of polyphenols than Western diets, especially in postmenopausal women and against
TNBC [39,40].
Many dietary phenolics have been reported to exert anticancer activities by playing pleiotropic
multi-target activities on BC cells and animal models related to cell viability, proliferation, differentiation,
and invasion, as well as by modulating signalling pathways, BC markers, gene, and protein
expression, etc. [41,42]. However, human evidence remains still inconclusive and controversial.
3. Bioavailability of Dietary Phenolics and Their Occurrence in Human Breast Tissues
The bioavailability of dietary phenolics is an essential factor to consider, either increasing or limiting
the overall anticancer effects against systemic cancers such as BC. In line with this, understanding the
metabolic/molecular forms and concentrations of those phenolics that can reach the human breast
tissue, is crucial in order to suggest their possible role in the
in vivo
beneficial effects, as well as to
(re)design physiologically relevant
in vitro
studies, thus increasing the knowledge of their possible
chemopreventive mechanisms of action.
Without considering the food matrix and(or) food processing related factors, the rate and extent
of intestinal absorption of each phenolic compound depend on the chemical structure that determines
its solubility, membrane permeability, and its microbial and(or) phase-II metabolism [43–45].
Dietary phenolics are usually characterized by their poor bioavailability, limiting their distribution
in systemic tissues in their native form, mainly as glycosides and complex oligomeric structures.
Once ingested, they are transformed into a variety of diverse bioavailable metabolites that can
remain in the systemic circulation for a few days [
46
]. Most phenolics are hydrolyzed and further
metabolized by either the intestinal enzymes or by the gut microbiota. After absorption, the resulting
Int. J. Mol. Sci. 2020,21, 5718 4 of 33
polyphenol-derived metabolites undergo extensive phase-II metabolism inside the enterocytes to form
sulphates, glucuronides, methyl-derivatives, or other conjugates. Therefore, in contrast to their parental
molecules that rarely appear in the systemic circulation, significant concentrations of phenolic-derived
conjugated metabolites can be detected in plasma and target systemic tissues where might trigger
biological effects, including anticancer activity [46–48].
However, it was found that conjugated forms of phenolics or microbial metabolites (phase-II
derivatives) display much lower or even no anticancer effects than their corresponding precursor
unconjugated forms [
49
–
51
]. Therefore, to date, whether dietary phenolics may protect against the
progression of BC (despite the low bioavailability and high metabolism of these compounds) is unclear,
and several fundamental questions about their mechanism of action remain unanswered.
Regarding the occurrence of phenolic-derived metabolites in human breast tissue, the evidence
is limited to a low number of human intervention studies, in which the sample size is also small
(from 3 to 31 volunteers) and the characteristic of the volunteers are different between the studies
(Table 1). In contrast, the number of animal studies is higher, but the results reported are contradictory.
The methodologies used for quantifying the metabolites in most of these studies showed low accuracy
and should be considered with caution (Table S1).
Similar to animal studies, the most robust human evidence is reflected in investigations with
isoflavones. Four clinical studies, conducted in women undergoing aesthetic breast, have allowed us to
establish the identity and quantity of the isoflavone metabolites that can occur in human breast tissues
after the intake of soy-based foods. Overall, these studies showed that unconjugated daidzein, genistein,
and the daidzein microbial-derived metabolite, equol, reached concentrations in the low nanomolar
range, reporting higher levels for equol (in those equol-producer individuals) and daidzein than
genistein [
52
,
53
]. However, no significant differences between daidzein and genistein were observed in
another study in women undergoing breast biopsy or cancer surgery [
54
]. Notwithstanding, it should
be noted that the breast tissues were hydrolyzed by enzymatic treatment in these studies. Thus,
the levels of these phenolics were overestimated, i.e., the unconjugated phenolics did not reach the
tissue as such, but the enzymatic treatment in the tissue generated them.
In this regard, Bolca et al. identified and quantified the distribution of isoflavones in non-hydrolyzed
and enzymatically hydrolyzed adipose and glandular breast tissues after the intake soy milk or
supplement by healthy women undergoing an aesthetic breast reduction [
55
]. As expected, the results
showed overall total glucuronidation of 98% (900–1150 pmol/g total isoflavone glucuronides), mainly
genistein-7-O-glucuronide and daidzein-7-O-glucuronide, whereas only traces of free genistein and
daidzein were observed (20–25 pmol/g), but not for the microbial daidzein-derived metabolites (equol or
O-DMA). However, after enzymatic hydrolysis, equol and dihydrodaidzein were also detected in a
few subjects, in addition to high levels of unconjugated daidzein and genistein.
According to this fact, in those animal studies where non-hydrolyzed samples were also analyzed
compared with hydrolyzed ones, isoflavones-derived metabolites were mostly detected (>60%) in
their physiologically conjugated forms, mostly glucuronides, and the unconjugated forms were even
undetectable in some cases (Table S1) [
56
–
58
]. Besides, it should be considered that only an animal
study reported the distribution in breast tumour tissues of isoflavones metabolites, and described
higher levels in tumour than in normal (liver and colon) tissues, for both total and free genistein,
and daidzein to a lesser extent [
59
]. Bolca et al., following the same approach and methodology,
also observed extensive glucuronidation (>90%) for prenylflavonoids in breast adipose/glandular
tissues [
60
]. However, low concentrations (picomolar, ranging from 0.8 to 83.7) of total isoxanthohumol
(IX), xanthohumol (XN) and 8-prenylnaringenin (8-PN, only in those 8-PN producers) were quantified
in hydrolyzed tissues from healthy women undergoing an aesthetic breast reduction after hop intake
for 5 days [
60
]. This result was in agreement with those reported in rodent models where 8-PN and
6-PN glucuronides were predominantly found in the mammary gland tissue after intraperitoneal
administration of prenylflavonoids or hop extract (Table S1) [61,62].
Int. J. Mol. Sci. 2020,21, 5718 5 of 33
In recent years, new human studies have further advanced in the identification of metabolites
in human breast tissue, through the combination of the analysis in hydrolyzed and non-hydrolyzed
samples and the evaluation of the distribution in both normal and malignant tissues. This fact is
especially relevant since breast tumour and normal tissues have been reported to show differences in
phase-II enzymes expression such as lower and higher activities of UDP-glucuronosyltransferases and
glutathione S-transferases, respectively [
63
], as well as increased the
β
-glucuronidase activity when
exposed to inflammatory or hypoxic conditions within the tumour microenvironment [63,64].
Thus, in two pre-surgery non-controlled dietary interventions with silybin and tea extracts
conducted in patients with newly diagnosed BC, higher concentrations of total silybin and EGCG
were detected in tumour tissues than in the adjacent normal tissues. In both studies, similar to other
phenolics, the total concentration of conjugated forms was higher compared with free silybin and
EGCG (4- and 2-fold, respectively), suggesting a high rate of phase-II metabolism, although free EGCG
was not detected in tumour rather than normal tissues. However, the quantification of conjugates
was not performed due to the lack of conjugated standards [
65
,
66
]. Similar results (considering the
limitations of the conjugated forms quantification) related to the identification of methyl and sulphate
of catechins have been observed in two animal studies conducted in mice fed green tea extracts.
However, it is noteworthy that besides the higher quantification of EGCG, other catechins such as
epicatechin and epigallocatechin were also detected in hydrolyzed samples (Table S1) [67,68].
Finally, a recent pre-surgery-controlled intervention study with a higher number of BC patients
that consumed a cocktail of plant extracts (cocoa, pomegranate, lemon, orange, grapeseed, olive,
and RSV) identified a total of 39 and 33 metabolites in normal and tumour tissues, respectively.
However, although the qualitative and quantitative metabolic profiling of phenolics was quite similar
in normal and tumour tissues, higher levels were found for most phenolics in tumour tissues.In both
tissues, phenolic-derived metabolites, including RSV, urolithins, and hesperetin, showed levels in
the range of the low nM, and were mainly glucuronidated and(or) sulphated (>85%). Among these
conjugates, the percentage of sulphated was slightly lower in normal tissues (31%) than in tumour
(42%) [69].
More recently, the same authors tried to overcome one of the main limitations of these human
studies, i.e., the long pre-surgery fasting that could hamper: (i) the detection of other phenolic-derived
metabolites (including different metabolic forms), (ii) the occurrence of higher concentrations that
could be reached in the breast tissues without fasting period. In this study, conducted in rats fed
equivalent doses to those used in the human study, the analyses at shorter times described the presence
of conjugated, but not free metabolites, derived from RSV, hesperetin, urolithins. These results are
in agreement with the data described in the previous human study, although some differences were
observed: higher concentrations (range of low
µ
M), a higher proportion of RSV sulphates than
glucuronides (opposite to what is observed in humans), as well as the occurrence of other metabolites
at shorter times such as hydroxytyrosol glucuronide and sulphate [
70
]. These results corroborate
some limitations of human studies (associated with hospital protocols) and confirm that human and
animal studies carried out under similar conditions contribute to increasing our knowledge on the
bioavailability of dietary phenolics.
Regarding other phenolics with high potential anticancer activity like curcumin (diferuloylmethane)
or lignans, the evidence is even more limited, with insufficient studies. Only studies with rodent
models have detected their presence in breast tissues (Table S1). Like other phenolics, the accumulation
of free and conjugated gut microbiota-derived enterolignan metabolites, mainly enterolactone and
enterodiol glucuronides, has been identified in the mammary tissues of rats and pigs (Table S1) [
71
,
72
].
Int. J. Mol. Sci. 2020,21, 5718 6 of 33
Table 1. Dietary phenolics and derived metabolites identified in human breast tissues.
Group and Sample Size Diet/Compound Administration Extraction and Analytical Conditions Identified and(or) Quantified Phenolic Metabolites References
8 women undergoing breast
biopsy or cancer surgery
Intake of four soy-supplemented bread
rolls per day, providing approximately
45 mg of isoflavonoids, for 14 days prior to
surgery (n =4) Placebo group (n =4).
Breast cancer tissues were extracted and
further hydrolyzed by enzymatic
treatment 1. Quantitative analyses were
performed by GC/MS using specific
standards.
The mean daidzein concentration in
non-soy-supplemented patients was 0.021
(range 0.017–0.028) nmol/g compared with 0.145
(range 0.083–0.218) nmol/g in soy-supplemented patients.
[54]
3 healthy women
undergoing breast
reductions
Soy-based food supplement containing
100 mg of genistein/genistin, 37 mg of
daidzein/daidzin, and 15 mg of
glycitein/glycitin (more than 90% as
glycosides (single and triple dose, n =1
each) or a placebo tablet (n =1) for 5 days
before aesthetic breast surgery.
Breast tissues were extracted and further
hydrolyzed by enzymatic treatment 1.
Analyses were performed by HPLC-DAD
and the compounds were identified by
comparison of the retention time with the
respective standards (UV spectra).
Genistein and equol concentrations were 4.16 µg/g and
52.98
µ
g/g, respectively, after soy-based food supplement
(single dose), whereas daidzein was below the limit of
detection.
Genistein, equol and daidzein concentrations were
35.1 µg/g, 681.7 µg/g and 16.0 µg/g, respectively, after
soy-based food supplement (triple dose).
[52]
28 volunteers before
aesthetic breast surgery
Soy-based food supplement containing
100 mg of genistein/genistin, 37 mg of
daidzein/daidzin, and 15 mg of
glycitein/glycitin (more than 90% as
glycosides, for 5 evenings before aesthetic
breast surgery (n =9). Placebo group
(n =19).
Breast tissues were extracted and further
hydrolyzed by enzymatic treatment 1.
Analyses were performed by HPLC-MS
and the compounds were quantified using
specific standards.
The median daidzein and equol concentrations were
7.03 nmol/L and 2.44 nmol/L, respectively,
in soy-supplemented subjects. Genistein was not
detectable.
The median daidzein concentration was 5.44 nmol/L in
the placebo group. Equol and genistein were only
detectable in some subjects of the placebo group,
with equol ten-fold values higher than genistein.
No significant differences were found between the
2 groups.
[53]
31 healthy women
undergoing an aesthetic
breast reduction
Soy milk (n =11; 250 mL containing
16.98 mg genistein and 5.40 mg daidzein
(per dose)), soy supplement (n =10;
5.27 mg genistein and 17.56 mg daidzein
aglycone (per dose), or control (n =10,
no soy product). 3 doses of soy milk or soy
supplements were taken daily for 5 days
before an aesthetic breast reduction.
Breast tissues were dissected into fractions
(adipose and glandular tissue) and were
both non-hydrolyzed and hydrolyzed by
enzymatic treatment 1. Quantitative
analyses were performed by
HPLC-MS/MS and the compounds were
quantified using specific standards of
aglycones, but not conjugated forms.
Total isoflavones showed a breast adipose/glandular
tissue distribution of 40:60. In hydrolyzed breast adipose
and glandular tissues, total genistein and daidzein
concentrations ranged between 92.33–493.8 pmol/g and
22.15–770.8 pmol/g. Total equol and dihydrodaidzein
were only detected in few subjects (up to 559.4 pmol/g
and up to 368.8 ±171.1 pmol/g.
In non-hydrolyzed breast adipose and glandular tissues
only traces of genistein and daidzein aglycones were
observed (20–25 pmol/g total isoflavone aglycones),
whereas an overall total glucuronidation of 98% was
estimated (900–1150 pmol/g total isoflavone
glucuronides), mainly genistein-7-O-glucuronide and
daidzein-7-O-glucuronide. Neither glucuronides nor
aglycones of microbial daidzein metabolites (equol and
O-DMA) were detected after isoflavone supplementation.
[55]
21 healthy women
undergoing an aesthetic
breast reduction
Hop-supplemented group (containing
2.04 mg xanthohumol (XN), 1.20 mg
isoxanthohumol (IX), and 0.1 mg
8-prenylnaringenin (8-PN) per
supplement) (n =11) or control group
(n =10). Three supplements were taken
daily for 5 days before surgery.
Breast tissues were dissected into fractions
(adipose and glandular tissue) and were
both non-hydrolyzed and hydrolyzed by
enzymatic treatment 1. Qualitative
analyses were performed by
HPLC-MS/MS and the compounds were
quantified using specific standards for
aglycones, but not conjugated forms.
Total prenylflavonoids showed a breast
adipose/glandular tissue distribution of 38:62.
Total XN and IX concentrations ranged between 0.26 and
5.14 pmol/g and 1.16 and 83.67 pmol/g in hydrolyzed
breast tissue, respectively. 8-PN was only detected in
samples of moderate and strong 8-PN producers
(0.78–4.83 pmol/g). An extensive glucuronidation was
observed (90%).
[60]
Int. J. Mol. Sci. 2020,21, 5718 7 of 33
Table 1. Cont.
Group and Sample Size Diet/Compound Administration Extraction and Analytical Conditions Identified and(or) Quantified Phenolic Metabolites References
12 early breast cancer
patients
Orally bioavailable complex of
silybin–phosphatidylcholine (2.8 g/day)
for 4 weeks prior to surgery.
Breast tissues (tumour and normal) were
extracted and further hydrolyzed both
with and without enzymatic treatment 1.
Qualitative analyses were performed by
HPLC-MS/MS and the free and total
silybin were quantified using specific
standard.
The median total and free silybin concentration in breast
cancer tissues were 131 ng/mg (IQR, 35–869) and 33 ng/g
(IQR, 4–158), respectively.
Concentrations were higher in the tumour as compared
with the adjacent normal tissue (total and free silybin
concentration were 11 ng/mL (IQR, 0–34) and 0 ng/g
(IQR, 0–19), respectively).
[65]
12 early breast cancer
patients
Patients received 300 mg of a caffeine-free
green tea catechin extract, equivalent to
44.9 mg of epigallocatechin-3-O-gallate
(EGCG) daily, for 4 weeks prior to surgery.
Breast tissues (tumour and normal) were
extracted and further both
non-hydrolyzed and hydrolyzed by
enzymatic treatment 1. Quantitative
analyses were performed by
HPLC-MS/MS and the compounds were
quantified as free (unconjugated) EGCG,
thereafter through enzymatic hydrolysis
as total EGCG (free and conjugated) using
EGCG standard.
Total EGCG was detectable in all tumour tissue samples
(median total EGCG 3.18 ng/g (IQR, 2.76–4.58)) and
higher amount than in adjacent normal tissue (under the
limit of detectability, minimum, 0 ng/g; maximum,
2.85 ng/g).
Free EGCG concentrations were under the limit of
detectability in tumour tissue but present in adjacent
normal breast tissue (median =1.07 ng/g; IQR, 0–1.25).
[66]
27 breast cancer patients
Breast cancer patients consumed a cocktail
of plant extracts (cocoa, pomegranate,
lemon, orange, grapeseed, and olive) plus
resveratrol, providing 37 different
phenolics (473.7 mg), theobromine and
caffeine (19.7 mg) (n =19) from diagnosis
to surgical resection (6 ±2 days). Control
group did not consume extracts (n =8).
Normal and tumour glandular breast
tissues were extracted and further both
non-hydrolyzed and hydrolyzed by
enzymatic treatment 1. Quantitative
analyses were performed by
UPLC-ESI-QTOF-MS and carried out by
peak area integration of their EIC using
calibration curves of specific (free and
conjugated metabolites) standards.
A total of 39 and 33 metabolites were identified in normal
and tumour tissues, respectively. Some representative
metabolites detected in tumour tissues (median and
range, pmol/g) were urolithin-A-3-O-glucuronide (26.2;
3.2–66.5), 2,5-dihydroxybenzoic acid (40.2; 27.7–52.2),
RSV-3-O-sulphate (86.4; 7.8–224.4),
dihydroRSV-3-O-glucuronide (109.9; 10.3–229.4), and HP
30-O-glucuronide 12.9 (2.7–14.1).
No significantly different conjugation profiling was
found in tumour vs. normal tissues. Overall,
all compounds were mostly glucuronidated and(or)
sulphated in tumour and normal tissues, 85.5% and
86.6%, respectively. Among these conjugates, the
percentage of sulphated was slightly higher in tumour
(42%) than in normal tissues (31%). Quantitative analysis
after hydrolysis was only possible for 2,5- and
2,6-dihydroxybenzoic acids, HP, urolithin A, isourolithin
A, and urolithin B.
[69]
1
enzymatic treatment was performed using
β
-glucuronidase/sulfatase enzymes. Abbreviations: DAD, diode array detection; EGCG, epigallocatechin-3-gallate; EIC, extracted
ion chromatogram; GC, gas chromatography; HP, Hesperetin; HPLC, high performance liquid chromatography; IQR, interquartile range; MS, mass spectrometry; O-DMA,
O-desmethylangolensin; RSV, resveratrol; UPLC-ESI-Q-TOF, ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight; UV, ultraviolet.
Int. J. Mol. Sci. 2020,21, 5718 8 of 33
Overall, based on the current evidence, the distribution of phenolic-derived metabolites in breast
tissue is similar to that observed in plasma, yet at lower concentrations. However, in contrast to most
phenolics, curcumin, beyond its poor bioavailability, remains a challenge. Thus, despite curcumin is
rapidly metabolized to conjugated forms, mainly glucuronides, recent animal studies have identified
free curcumin at significantly higher concentrations (up to 15-fold) than curcumin glucuronide in breast
tumour tissues from tumour-bearing mice models, after oral and intravenous administration [
73
].
Besides, similar to that observed for other phenolics, curcumin levels in healthy mammary tissues
were significantly lower than tumours tissues [
73
]. This is in agreement with another recent animal
study where the concentration of free curcumin in serum was lower than its glucuronide. However,
the absolute amount and the percentage of total curcumin was significantly increased in bones (up to
3
µ
M), whereas curcumin glucuronide was barely detectable due to the high glucuronidase activity [
74
].
At present, this fact has not been confirmed in human studies hitherto.
On the other hand, it is also important to note that recent preclinical studies have explored
(non-nutritional) strategies to increase polyphenols’ bioavailability and stability to overcome the fast
clearance of most phenolic-derived metabolites [
46
]. Different techniques are included within these
alternative approaches, such as encapsulation (micelles, liposomes, nanoparticles, etc.) and phenolic
loaded self-microemulsifying drug delivery system (SMEDDS) or self-nanoemulsifying drug delivery
system (SNEDDS), which are used for different phenolics (curcumin, quercetin, RSV, etc.) in both
bioavailability and chemopreventive animal studies (Tables S1 and S2). Similar approaches to increase
cytotoxicity and bioavailability have also been assayed in BC cell models [75–77].
4. Evidence of the Chemopreventive Potential of Phenolic Compounds in Physiologically
Relevant Preclinical Studies
Following the statement “First
in vivo
and then
in vitro
” [
78
], we must be cautious with all the
accumulated evidence on
in vitro
polyphenols’ effects against BC. In this regard, innumerable
in vitro
studies have been carried out with doubtful physiological relevance, i.e., regardless of bioavailability,
metabolism, and tissue distribution of dietary phenolics or their derived metabolites. Therefore, in the
present review, we focus only on those physiologically relevant
in vitro
and
in vivo
studies conducted
with dietary phenolics that could elucidate whether they are responsible for the effects attributed to
plant-based foods.
In the last decades,
in vivo
animal studies have been an essential tool in investigating the
biological effects of natural compounds against BC. More than 200
in vivo
studies, included in this
review (Table S2), describe the effects of different phenolic compounds on BC. Isoflavones (genistein
and daidzein), as well as lignans (flaxseed and secoisolariciresinol diglucoside), encompass the highest
number of studies (almost 50%), whereas the effects of other classes, including anthocyanins or flavones
have been less investigated so far. In general, diets supplemented with phenolics (anthocyanins,
flavanones, flavones, flavonols, flavan-3-ols, RSV, curcumin, lignans, and hydrolysable tannins) were
associated with reduction of tumour growth, incidence (percentage of animals that develop breast
tumours), multiplicity (tumours per mouse), metastasis (mainly to lungs), and higher tumour latency
period (time between carcinogen exposure and tumour detection), which in turn are related to a higher
survival rate. However, these effects are less clear regarding the consumption of isoflavones.
A significant number of investigations challenges the reported evidence concerning the benefits
of soy products and isoflavones consumption in the prevention or treatment of BC. At least 15 studies
(out of 52 included in Table S2) described the absence of protection or even the increase of tumour
growth, incidence, metastasis, or reduction of tumour latency. These contradictory results have led the
food scientists to search for new approaches (i.e., a combination of different phenolic compounds) to
reduce the stimulatory effects of isoflavones on human breast tissue. In this regard, the combination
of flaxseed, enterolactone, and(or) enterodiol with soy protein or genistein has demonstrated to be
effective attenuating tumour growth promotion in animal models [79,80].
Int. J. Mol. Sci. 2020,21, 5718 9 of 33
On the other hand, the combination of phenolics is not limited to lignans-rich foods and isoflavones.
Different mixtures such as quercetin-RSV-genistein, curcumin-EGCG, and curcumin-RSV have been
shown to be effective in reducing tumour growth and modulating apoptosis and angiogenesis signalling
pathways [
81
–
83
]. In line with this, emerging strategies using self-nanoemulsifying drug delivery
systems (SNEDDS) showed higher plasma concentrations of mixtures of phenolics related to more
efficient anti-breast cancer effects [84].
Regarding the mechanism of action, the reduction on tumour growth associated with the
consumption of the different dietary phenolics has been reported to be closely related to higher
levels of apoptosis and lower cell proliferation and angiogenesis (together with the modulation of
molecular mechanisms associated). Additional mechanisms described in the numerous preclinical
studies included in Table S2 support the potential protective role of the phenolics against BC.
One of these mechanisms is the modulation of morphological changes in mammary tissue
structures. Isoflavones [
85
,
86
] and resveratrol [
87
] have shown the capacity to reduce the number of
terminal end buds (TEBs). The reduction of the TEBs density is considered a protective mechanism
since these structures are targets of carcinogens like DMBA. Thus, the reduction of tumour growth
in DMBA-treated rats fed grape juice concentrate was related to a decrease in the formation of
DMBA-DNA adducts [
88
]. RSV also exhibited protection through the reduction of lipid peroxidation
and DNA damage related to its antioxidant activity [
89
]. Further mechanisms by which phenolics exert
their effects on breast tumours involve the regulation of oestrogen/progesterone levels and receptors,
including ER, PR, and HER2. The reduction of 17
β
-oestradiol and progesterone levels was described
in breast cancer animal models fed diets containing hesperetin and soy protein isolated [
90
,
91
]
as well as luteolin and genistein in the presence of letrozole and tamoxifen, respectively [
92
,
93
].
Likewise, pomegranate emulsion, flaxseed, and secoisolariciresinol modulated the expression of ER,
PR, and HER2 [94–96].
Moreover, the benefits of the phenolic compounds against BC are also linked to their
anti-inflammatory effects. Dual inhibition of 5-LOX and COX-2 [
97
] and the modulation of cytokines
regarding BC [
98
] are interesting strategies in the treatment or prevention of this disease. Inhibition
of 5-LOX (and its product LTB4) and COX-2 via NF-
κ
B inhibition by resveratrol consumption
has been reported to be involved in BC prevention in DMBA-treated rats [
89
,
99
]. Naringenin
and soy protein modulated the expression of genes involved in regulating the inflammatory
response and cytokine signalling, including MCP-1 and IL-6 [90,100], while wogonoside and the mix
resveratrol-quercentin-genistein reduced the level of IL-6, TNF-
α
, and the TNF receptor-associated
factors TRAF2 and TRAF4 [
84
,
101
]. Several studies have highlighted the role of the immune system
regarding the protective effects of phenolic compounds against BC. Thus, quercetin and silybin
failed to protect against tumour development in immunodeficient mice. However, they were able
to regulate the accumulation of immune cells (i.e., T-cells) as well as the biosynthesis of cytokines
(i.e., TNF-
α
, IL-1
β
, IL-2, IL-4, IL-10, and INF-
γ
) in immunocompetent BALB/c mice [
102
,
103
]. Further
evidence is supported by experimental animal models fed EGCG and naringenin-enriched diet showing
accumulation and activation of T-cells in response to tumour-promoting stimuli [104,105].
Regarding
in vitro
studies, Table 2highlights the limited current evidence on the anticancer
effects for relevant phenolic-derived metabolites against different BC cell models. All in vitro studies
corroborated that phase-II conjugation reduced the cytotoxic and(or) antiproliferative effect against
different BC cell models and oestrogenic/anti-oestrogenic effects in ER+MCF-7 cells. The most robust
evidence has been reported in few studies for phase-II metabolites of RSV, quercetin, and isoflavones
indicating potential effects but lower than their counterpart unconjugated forms. Besides, a few
in vitro
studies made a comparison between BC and non-tumourigenic cells reporting the lack of cytotoxic
or antiproliferative effects in normal cells, unlike BC cell lines, at the tested concentrations and times
(Table 2) [
106
–
109
]. On the other hand, to date, no effect has been reported for phase-II metabolites to
other phenolic groups such as flavanones, urolithins and catechins, although the number of
in vitro
Int. J. Mol. Sci. 2020,21, 5718 10 of 33
studies conducted is still scarce. However, these results should be interpreted with caution since the
concentrations of conjugates in most in vitro studies are supra-physiological (>10 µM) (Table 2).
Although the conjugated metabolites are less bioactive than their free forms, and it remains
difficult to attribute their role in the chemopreventive
in vivo
effects, new approaches are being
investigated to confirm their possible
in vitro
anticancer activity. For instance, recent results have been
obtained assaying concentrations close to those detected
in vivo
(up to 10
µ
M) of RSV-derived phase-II
conjugates [
109
]. This study attributed the anticancer activity of RSV conjugates to cellular senescence
induction in BC cells and, therefore, deserves further investigation.
To the best of our knowledge, there are no
in vitro
studies conducted with phase-II curcumin
and enterolignans metabolites despite the large number of studies conducted with their unconjugated
compounds or enriched extracts. The lack of availability of these relevant metabolites may be behind
this gap.
On the other hand, it should not be discarded the potential role of phase-II metabolites (mainly
glucuronides and sulfates) as a source of bioactive aglycones in breast tumour tissues. This process
might occur via deconjugation under specific stimuli such as inflammation within the tumour
microenvironment, where the ratio glucuronyltransferase/glucuronidase is lower than in normal
tissues [
64
]. However, results that support this hypothesis are still limited to a few
in vitro
and animal
studies [74,110].
Int. J. Mol. Sci. 2020,21, 5718 11 of 33
Table 2. In vitro studies conducted with relevant phenolic-derived metabolites on breast cancer (BC) models.
Breast Cellular Model Compound Assayed Dose/Duration Main Outcomes References
Resveratrol
MCF-7, ZR-75-1, MDA-MB-231
(breast cancer cell lines) and
MCF-10A (normal cell line)
RSV, RSV 3-sulph, RSV 4
0
-sulph,
RSV 3,40-disulph. 1–350 µM; 48 h
RSV: IC50 =67.6–82.2 µM against all breast cancer cells;
IC50 =20 µM against MCF-10A.
RSV 3-sulph: IC50 =189–258 µM against MCF-7 and
MDA-MB-231; IC50 =228.3 µM against MCF-10A.
RSV 40-sulph and RSV 3,40-disulph: no cytotoxic effect
against breast cancer cells; IC50 =202.4–202.9 µM
against MCF-10A.
[111]
MCF-7
RSV, RSV 3-sulph, RSV 4
0
-sulph,
RSV 3,5 disulph, RSV
3,40-disulph, RSV
3,40,5-trisulph.
340 µM; 72 h Cytotoxic effect (only RSV and RSV 3-sulph). [112]
Saccharomyces cerevisiae strain
Y190 co-transformed with ERα
LBD and hTif2 coactivator and
MCF-7
RSV, RSV 3-gluc, RSV 3-glur,
RSV 3-sulph, RSV 40-sulph. 0.05–100 µM; 18–24 h
RSV 3-sulph showed anti-oestrogenic activity at 10 and
50
µ
M (with a marked preference for ER
α
at 10–100
µ
M),
and weak oestrogenic activity.
RSV showed oestrogenic activity in MCF-7 (5–10 µM).
[113]
MCF-7 and MDA-MB-231
RSV, RSV 3-glur, RSV 3-sulph,
RSV 40-sulph, DH-RSV,
DH-RSV 3-glur.
10 and 50 µM; 3 and 7 days
↓Proliferation in MDA-MB-231 cells (only RSV and
DH-RSV).
Oestrogenic/anti-oestrogenic effect in MCF-7 (only RSV
and DH-RSV).
[51]
MCF-7, MDA-MB-231 and
MCF-10A (normal cell line)
RSV, RSV 3-glur, RSV 3-sulph,
RSV 40-sulph, DH-RSV,
DH-RSV 3-glur.
0.5, 1, and 10 µM;
1–14 days
The effects were only observed in MCF-7 cells for all
compounds: ↓Clonogenicity; cell cycle arrest;
senescence induction; modulation of p53/p21 and
p16/Rb pathways.
In MDA-MB-231 cells (only RSV at 10 µM):
↓clonogenicity.
[109]
Flavanones
MCF-7 and normal mammary
epithelial cells H184B5F5/M10
Baicalein, baicalein 7-O-sulph,
and baicalein-8-sodium
sulphonate.
50, 100, and 200 µM; 24 h
Effects in MCF-7 cells: ↓cell viability; ↑LDH release;
induction of cell cycle arrest; induction of morphological
changes; ↑apoptosis; ↑ROS; ↑caspase-3, -9 activity.
Effects on H184B5F5/M10 cells: No cytotoxic.
[114]
Int. J. Mol. Sci. 2020,21, 5718 12 of 33
Table 2. Cont.
Breast Cellular Model Compound Assayed Dose/Duration Main Outcomes References
MCF-7 and MDA-MB-231 HP, HP 30-glur, HP 7-glur, HP
30-sulph, HP 7-sulph. 10 and 50 µM; 3 and 7 days
↓
Proliferation in MDA-MB-231 cells (only HP at 50
µ
M).
Oestrogenic/anti-oestrogenic effect in MCF-7 (only HP at
10 and 50 µM).
[51]
Quercetin
MCF-7
Quer, Quer-3-O-gluc, Quer
3-O-glur, Quer 40-O-sulph,
Tamarixetin, Isorhamnetin.
50 µM; 48 h ↓
Cell proliferation (no effect of Quer-3-O-gluc and Quer
3-O-glur). [115]
Saccharomyces cerevisiae
expressing ERα-Tif2 or ERβ-Tif2
and MCF-7
Quer, rutin, isoquercitrin, Quer
3-O-glur, Quer 3-O-sulph.
0.1–100 µM;
18–24 h
Quer 3-O-glur (IC50 =2.1 ±0.38 µM) and Quer (IC50 =
2.4 ±0.93 µM) showed oestrogenic activity in MCF-7
cells.
Quer 3-O-glur showed ERα-Tif2 (IC50 =103 ±2.24 µM)
and ERβ-Tif2 (IC50 =96 ±1.2 µM) agonistic activity.
Isoquercitrin showed similar ER
β
-Tif2 agonistic activity
than Quer 3-O-glur.
Quer showed weak ERβ-Tif2 (IC50 =5.3 ±0.9 µM)
agonistic activity.
[116]
MDA-MB-231 Quer 3-O-glur (alone or
together with A and NA).
10−10–10−4M (binding
assay) and 0.01–1 µM (cell
assay); 1–24 h
Quer 3-O-glur showed competitive binding of [3H]-NA
to β2-AR (10-4–102µM). ↓ROS formation; ↓HMOX1,
MMP-2, and MMP-9 gene expression; ↓intracellular
cAMP level; ↓p-ERK 1/2 and p-P38; ↓RAS activation;
↓invasive capacity of MDA-MB-231; ↓MMP-9 activity.
[117]
MCF-10A (normal cell line)
Quer and Quer 3-O-glur (alone
or together with 4-OHE2and
NA).
10−10–10−4M (binding
assay) and 0.05–10 µM (cell
assay); 2 h
Quer and Quer 3-O-glur showed competitive binding of
[3H]-NA to α2-AR (10−4–102µM).
↓γ-H2AX and AP sites activation.
[118]
MCF-7 and normal mammary
epithelial cells H184B5F5/M10
Quer, Isorhamnetin, and
Isorhamnetin 3-O-glucuronide. 25, 50, and 100 µM; 24–48 h
Effects in MCF-7 cells:
↓Cell viability; ↑LDH release: induction of cell cycle
arrest; induction of morphological changes; ↑apoptosis;
↑ROS.
Effects on H184B5F5/M10 cells: No cytotoxic effects.
[107]
MCF-7 and normal mammary
epithelial cells H184B5F5/M10
Quer, Quer 3-O-glur, and Quer
3-O-sulph. 25, 50 and 100 µM; 24–48 h
Effects in MCF-7 cells: ↓cell growth; ↑LDH release;
↑ROS; ↑apoptosis; induction of cell cycle arrest;
induction of morphological changes.
Effects on H184B5F5/M10 cells: No cytotoxic effects.
[108]
Int. J. Mol. Sci. 2020,21, 5718 13 of 33
Table 2. Cont.
Breast Cellular Model Compound Assayed Dose/Duration Main Outcomes References
Urolithins
MCF-7 and MDA-MB-231
Uro-A, Uro-A 3-glur, Uro-A
8-glur, Uro-A 3-sulph, IsoUro-A,
IsoUro-A 3-glur, IsoUro-A
9-glur, Uro-B, Uro-B 3-glur,
Uro-B 3-sulph.
10 and 50 µM; 3 and 7 days
↓Proliferation in MCF-7 (Uro-A and IsoUro-A) and
MDA-MB-231 (free forms and conjugates at 50 µM).
Oestrogenic activity in MCF-7 (only free forms at 10 and
50 µM). Anti-oestrogenic activity in MCF-7 (Uro-A and
its conjugates, IsoUro-A and its conjugates, and Uro-B at
10 and 50 µM).
[51]
Catechin and epicatechin
MCF-7
Epi, Epi-30-O-sulph,
30-O-methyl-Epi,
40-O-methyl-Epi, catechin,
30-O-methyl-catechin.
100 µM; 48 h ↓Cell proliferation (only 40-O-methyl-Epi). [115]
Isoflavones
MCF-7
GEN, GEN 40-O-glur, GEN
7-O-glur, GEN
40-O-sulph-7-O-glur, GEN
40-O-sulph, GEN 7-O-sulph,
GEN 40,7-di-O-sulph, DAZ,
DAZ 40-O-glur, DAZ 7-O-glur,
DAZ 40-O-sulph-7-O-glur, DAZ
40-O-sulph, DAZ 7-O-sulph,
DAZ 40,7-di-O-sulph, Glycitein,
Glycitein 7-O-glur, DH-DAZ,
DH-DAZ 7-O-glur, O-DMA,
O-DMA 7-O-glur.
10–1000 µM; 24 h
Low stimulatory MCF-7 growth, β-gal induction, and
binding to ERs: sulphates of GEN and glucuronides of
glycitein and DH-DAZ.
Moderate binding, but low (or lack) MCF-7 growth and
β-gal induction: glucuronides of GEN, DAZ, and
O-DMA.
Moderate MCF-7 growth and β-gal induction, but low
(or lack) binding: DAZ 40-O-sulph-7-O-glur.
O-DMA was the most active compound stimulating
MCF-7 growth, binding to hERβ, and inducing β-gal.
[119]
MCF-7
GEN, GEN 7-O-glur, DAZ, DAZ
7-O-glur. 16 µM; 72 h ↑Cytotoxicity (only DAZ). [120]
Int. J. Mol. Sci. 2020,21, 5718 14 of 33
Table 2. Cont.
Breast Cellular Model Compound Assayed Dose/Duration Main Outcomes References
Isoflavones
MCF-7
GEN, GEN 7-O-sulph, GEN
40-O-sulph, DAZ, DAZ
7-O-sulph, DAZ 40-O-sulph,
equol, equol-7-sulph (in the
presence of 4 ×10−10 M
[2,4,6,7-3H] oestradiol).
10−7–10−4M; 18–24 h
(binding and gene
expression assay) and 7 or
14 days (proliferation assay)
The sulphation in position 7 reduced the oestrogen
capacity of GEN and equol in all assays.
GEN 40-O-sulph showed lower proliferative activity (at
1 and 10 µM), and similar or even higher binding
affinity to ER (compared to GEN).
DAZ 40-O-sulph showed lower binding affinity to ER
and increased proliferative activity (at 10 µM). DAZ
7-O-sulph showed similar or even higher affinity to ER
and similar proliferative activity (at 1 and 10 µM)
(compared to DAZ).
[121]
HS578T, MDA-MB-231, and
MCF-7
Puerarin, GEN, DAZ, and mix
of DAZ glucuronides/sulphates.
12.5–100 µM
(free compounds) and 2.35
µM the mix of DAZ
conjugates; 24–72 h
Effect of only with free compounds:
(IC50 =29–71 µM) ↓cell viability; induction of cell cycle
arrest; ↑apoptosis; ↑caspase-3 activity.
Effect of mix of DAZ conjugates: (IC50 =2.35 µM) ↓cell
proliferation; induction of cell cycle arrest; ↑apoptosis;
↑caspase-9, p53, p21, and Bax.
[122]
MCF-7, T47D, and MCF-10A
(normal cell line)
GEN, GEN 7-O-sulph, GEN
40-O-sulph, GEN 7-O-glur.
5.12 ×10−3–80 µM for GEN
and 2.2–4.5 µM for
conjugates; 3 days
Dissimilar effects of GEN cell proliferation: ↑at low
concentrations and ↓at higher concentrations. GEN
7-O-glur stimulated cells growth. This effect was related
to deconjugation. GEN 7-O-sulph, GEN 40-O-sulph
exerted no effects.
[106]
T47D and T47D-ERβ
(tetracycline dependent ERβ
expression)
GEN, GEN 7-O-glur, DAZ, DAZ
7-O-glur. 10−5–1000 µM; 48 h
Dissimilar effects on proliferation: ↓at low
concentrations and ↑at higher concentrations.
Lower proliferative potency than 17β-oestradiol:
PC
50
in T47D-wt: 17
β
-oestradiol =4.2
×
10
−6µ
M; GEN =
0.19 µM; GEN 7-O-glur =21.4 µM; DAZ =0.186 µM;
DAZ 7-O-glur =107 µM. PC50 in T47ERβ:
17β-oestradiol =2.45×10−7µM; GEN =0.2 µM; GEN
7-O-glur =7.8 µM; DAZ =0.035 µM; DAZ 7-O-glur =
400 µM.
[123]
Int. J. Mol. Sci. 2020,21, 5718 15 of 33
Table 2. Cont.
Breast Cellular Model Compound Assayed Dose/Duration Main Outcomes References
Isoflavones
MCF-7 and T47D
DAZ, DAZ 40-O-sulph, DAZ
7-O-sulph, DAZ 4
0
,7-di-O-sulph,
equol, O-DMA.
0.1, 1, and 10 µM; 1–24 h
Different effects on NGB: ↑equol, O-DMA, DAZ
7-O-sulph, DAZ 40,7-di-O-sulph and ↓DAZ and DAZ
40-O-sulph. Effects of DAZ, DAZ 40-O-sulph and equol
on protein phosphorylation: ↑p-ERα/ERα
(all compounds);
↑
p-Akt/Akt (only equol; 1 h treatment);
↑p-p38/p38 (all molecules; 1 and 24 h treatment). DAZ,
DAZ 40-O-sulph and equol preserve PAX activity in
MCF-7 cells (↓NGB, ↑PARP-1 cleavage, and ↓cell
number) in the presence of 17β-oestradiol.
Effects of the mix of metabolites:
1
Mix of sulphates metabolites:
↓
NGB by in the presence
or absence of 1 µM DAZ; the mix of sulphates together
with 1 µM DAZ preserve PAX effects (↑PARP-1
cleavage) in the presence of 17β-oestradiol.
2Mix of gut metabolites: ↑NGB by equol +O-DMA
(1 µM each) in the presence or absence of 1 µM DAZ;
↓PAX effects (↑PARP-1 cleavage) in the presence of
17β-oestradiol.
[124]
1
Mix of sulphate metabolites composition: DAZ 4
0
-O-sulph +DAZ 7-O-sulph +DAZ 4
0
,7-di-O-sulph (1
µ
M each);
2
Mix of gut metabolites composition: equol +O-DMA (1
µ
M each).
Abbreviations: A, adrenaline;
α2
-AR,
α2
-adrenergic receptor;
β2
-AR,
β2
-adrenergic receptor; cAMP, cyclic adenosine monophosphate; DAZ, daidzein; DH-DAZ, dihydrodaidzein; DH-RSV,
dihydroresveratrol; Epi, epicatechin; ER+, oestrogen receptor+; ERE-CAT, oestrogen response element chloramphenicol acetyl transferase;
γ
-H2AX, H2A histone family member X
phosphorylated on Ser139; GEN, genistein; gluc, glucoside; glur, glucuronide; hER
α
, human oestrogen receptor alpha; HMOX1, heme oxygenase 1; HP, hesperetin; hTif2, coactivator
receptor-interacting domain; LBD, ligand binding domain; LDH, lactate deshydrogenase; NA, noradrenaline; NGB, neuroglobin; O-DMA, O-desmethylangolensin; PAX, paclitaxel; PC,
potency concentration; PARP-1, poly [ADP-ribose] polymerase 1; Quer, quercetin; ROS, reactive oxygen species; RSV, resveratrol; 4-OHE2, 4-hydroxy-oestradiol; sulph, sulphate.
Int. J. Mol. Sci. 2020,21, 5718 16 of 33
5. Dietary (Poly)Phenols and Clinical Studies in Breast Cancer Patients
Table 3summarizes the current evidence regarding the effects of several dietary (poly)phenols or
phenolic-containing products in breast cancer patients. The main search criteria used were as follows:
(i) clinical trials dealing with BC patients (either with active disease or cured patients to prevent
recurrence), (ii) evaluation of the effects of orally administered (poly)phenols or phenolic-containing
products (pure standards, plant extracts or foodstuffs) on tumour biomarkers or other clinically-relevant
outcomes related to BC, including the decrease of side-effects associated with chemo- or radiotherapy
(i.e., intravenous administration or articles dealing with only ‘hot flashes’, ‘wellness’ or other general
determinations were excluded). Articles dealing with either healthy or at-risk volunteers (including
postmenopausal women) were omitted, as were those studies conducted with non-dietary sources.
Trials, where metabolites disposition was evaluated in malignant breast tissue from patients, were also
included only when some effects on tumour-related biomarkers were evaluated.
As expected, the evidence on the anticancer effects of dietary phenolics in BC patients is limited,
fragmented, and inconclusive. In this context, the most studied polyphenols are EGCG, as a component
of green tea extracts, and curcuminoids present in curcumin extracts.
The pioneer pilot study of Thompson et al. revealed the increase of apoptosis, and the reduction of
both Ki-67 (a cell proliferation marker) and c-erB2 (also known as HER-2) expression in 19 BC patients
that consumed 25 g/day flaxseed vs. 13 patients that consumed placebo for approximately 37 days
from the diagnosis until the resection of the tumour [
125
]. Despite the promising results, it took almost
10 years for a new study on flaxseed to be published by
McCann et al
. [
126
]. This new study was also a
pre-surgery pilot study in which postmenopausal women diagnosed with ER+BC consumed flaxseed
(25 g/day) alone or combined with the aromatase inhibitor anastrozole or placebo, between tumour
biopsy and resection (mean of 19 days). The authors found a reduction of endogenous oestrogen
production (androstenedione in the flaxseed +anastrozole group, and dehydroepiandrosterone in
the flaxseed group) (Table 3) [
126
]. Aromatase inhibitors block the enzyme aromatase that catalyzes
the conversion of androgens into oestrogens, the primary source of endogenous oestrogens in
postmenopausal women. This is relevant since anti-estrogen therapy aims to prevent access of the
tumour to oestrogen and has been considered the standard of care in ER+tumours [127].
Green tea extracts are the most widely tested polyphenol-containing products to date in BC patients
(Table 3). The main active component is supposed to be EGCG, but the specific association between
EGCG and the reported outcomes has not been unequivocally proven so far. Green tea extracts have
been assayed to explore the effects on cancer biomarkers after short treatments (pre-surgery) [
66
,
128
]
as well as after longer treatments (6 months) to assess the maximum tolerability dose (MTD) [
129
] and
effects on some cancer-related biomarkers [
129
,
130
]. The MTD was established at 1200 mg/day for
6 months in patients with ER- and PR- breast tumours, but no significant changes were observed on ER,
Ki-67, IGF-1, IGFBP-3, and mammographic density vs. placebo after 6 months [
129
]. The same authors
explored the effects on more biomarkers in the same group of patients, but no effects were observed
on VEGF, oxidative damage, and inflammatory markers, among others, but only a transient decrease
of serum HGF in green tea consumers vs. placebo [
130
]. The other pre-surgery studies reported a
significant decrease of Ki-67 (only in healthy breast tissue, but not in the malignant one), while no effects
were observed on apoptosis (caspase-3) and angiogenesis (CD34) in both healthy and malignant breast
tissues after consuming green tea extract (2.2 g/day for 35 days) [
128
]. More recently,
Lazzeroni et al
.
evaluated the disposition of EGCG in normal and malignant tissues from 12 patients with ER+
tumours, but the authors did not find any effect on cell proliferation, angiogenesis, oxidative stress,
inflammation or other markers [
66
]. However, they found a significant increase (36%) in testosterone
levels. Although testosterone can be aromatized to oestradiol, which increases proliferation and hence,
BC risk, testosterone has also been associated with BC prevention [131,132].
Curcumin extracts, also containing the related curcuminoids, demethoxycurcumin and
bisdemethoxycurcumin, have also been assayed in BC patients. The first trial was conducted by
Bayet-Robert et al. in 14 patients with metastatic breast cancer to evaluate the MTD or oral curcumin
Int. J. Mol. Sci. 2020,21, 5718 17 of 33
in combination with intravenous administration of docetaxel (Table 3) [
133
], a chemotherapeutic drug
used in metastatic BC. The authors found a high MTD of oral curcumin (8 g/day) in combination with
the drug (100 mg/m
2
), administered every 3 weeks for six cycles, with a total of 63 cycles, and observed
a significant decrease of plasmatic CEA and VEGF from the 3rd cycle. However, no clear association
was found between markers decrease and the occurrence of curcumin in plasma [
133
]. Later,
Ryan et al
.
observed the protection of oral curcumin (6 g/day) administration vs. placebo after 6 months against
radiation dermatitis in BC patients (n =30) with prescribed radiation therapy (RT) [
134
]. The same
authors tried to confirm their previous exploratory results in an ambitious trial that enrolled 686 patients
with prescribed RT [
135
]. Nevertheless, curcumin did not reduce radiation dermatitis severity at
the end of RT compared to placebo after 6 months (30 RT sessions). Overall, this is a warning to
consider the minimum number of patients necessary to conclude the possible effects associated with
the consumption of phenolics.
Other trials with BC patients and phenolics or phenolic-containing products, included the assay
of grapeseed [
136
], milk thistle [
65
], and red clover [
137
] extracts, walnuts [
138
], and also a product
containing a mixture of EPA, DHA hydroxytyrosol and curcumin [
139
]. In one of the pioneer studies
dealing with polyphenols and BC, Brooker et al. did not find any effect on tissue hardness due to RT in
69 BC patients after consuming a grapeseed proanthocyanidins extract (300 mg/day) for 12 months vs.
placebo [
136
]. Lazzeroni et al. described the disposition of some metabolites in breast tissue. However,
they did not observe any effect on NO, IGF-1, and Ki-67 in 12 patients with ER+breast cancer after
consuming 2.8 g/day of a silybin-containing milk thistle extract in combination with phosphatidylcholine
for 4 weeks between biopsy diagnosis and resection [
65
]. The only study dealing with food and BC has
been conducted so far by Hardman et al. [
138
]. These authors conducted a pre-surgery trial in 10 BC
patients that consumed around 30 g walnuts/day for 15 days (between tumour biopsy and resection).
Surprisingly, with only 10 patients, and taking into account the huge inter-individual variability
in gene expression and the previously reported artefacts [
140
,
141
], they observed the activation of
genes participating in pathways that promote apoptosis and cell adhesion and inhibition of pathways
that promote cell proliferation and migration (involving 456 genes in the tumour) due to walnut
consumption [
138
]. Recently, Mart
í
nez et al. described the anti-inflammatory effect (decrease of
plasma CRP vs. placebo) in BC patients with no active disease, and receiving adjuvant hormonal
therapy, after consuming a mixture containing 460 mg fish oil (EPA, DHA), olive extract (125 mg
hydroxytyrosol) and 50 mg curcumin extract for 30 days [
139
]. Although the authors described a
relevant decreased of plasma CRP from 8.2 mg/L to 5.3 mg/L, unfortunately, the final CRP levels
were in the boundary of those associated with patient’s survival [
142
] and still associated with high
cardiovascular risk [
143
]. Besides, the responsible compound for the anti-inflammatory effect observed
was not identified from such a complex mix. Finally, Ferraris et al. recently evaluated the effect of a
red clover extract containing isoflavones (80 mg/day) to reduce side-effects of tamoxifen treatment in
patients with ER+BC under tamoxifen treatment for 2 years. Although the consumption of red clover
extract was safe, no effects were observed in the targets related to tamoxifen treatment (menopausal
rating score, sex hormone levels, endometrial thickness, and breast density, among others). Only BMI
and waist circumference were significantly reduced in the red clover group vs. placebo [137].
Int. J. Mol. Sci. 2020,21, 5718 18 of 33
Table 3. Clinical trials with dietary (poly)phenols or phenolic-containing foodstuffs in patients with breast cancer.
Cohort and Sample Size Trial Design Objective Outcomes References
Newly biopsy-diagnosed
breast cancer patients (n =32),
with no hormone therapy
Design: Pre-surgery, randomized double-blind,
placebo-controlled clinical trial.
Product and dose: Patients (n =19) consumed
flaxseed (25 g/d) or placebo (n =13).
Follow-up: 37 and 39 days in the flaxseed and
placebo groups, respectively.
Effect of flaxseed on tumour
biomarkers.
Significant reductions in Ki-67 labelling
index (34.2%) and in c-erbB2
expression, (71%), and an increase in
apoptosis (30.7%) were observed in the
flaxseed, but not in the placebo group.
[125]
Patients (n =66) with tissue
hardness due to radiotherapy
for early breast cancer (at least
24 months prior to trial entry),
with no active disease
Design: Phase II, placebo-controlled,
randomized trial.
Product and dose: Capsules containing a grape
seed proanthocyanidin extract 100 mg three
times a day orally, or placebo.
Follow-up: 6 months.
Effect on the surface area of
palpable breast induration after
12 months. Secondary
endpoints: change in
photographic breast appearance
and patient self-assessment of
breast hardness, pain and
tenderness.
No significant difference between
treatment and control groups in terms
of external assessments of tissue
hardness, breast appearance or patient
self-assessments of breast hardness,
pain or tenderness.
[136]
Patients (n =14) with
metastatic breast cancer
Design: Open label, phase I, non-controlled
trial.
Product and dose: Intravenous docetaxel plus
oral curcumin (escalated dose until toxicity
limit is reached).
Follow-up: Docetaxel 100 mg/m2was
administered every 3 weeks (w) for six cycles.
Curcumin was given orally for 7 consecutive
days (d) (from d-4 to d+2) for six cycles (a total
of 63 cycles).
Establishment of the maximal
tolerated dose (MTD) of oral
curcumin plus intravenous
docetaxel.
MTD of curcumin was 8 g/day, in
combination with docetaxel 100 mg/m
2
administered every 3 w for six cycles.
Recommended curcumin dose: 6 g/d
for 7 consecutive d every 3 w in
combination with a standard dose of
docetaxel.
Decrease of plasmatic CEA and VEGF.
[133]
Patients (n =40) with resected
stage I-III ER- and PR- breast
cancer with no active disease
Design: Randomized, phase IB,
double-blinded, placebo-controlled,
and dose-escalation study.
Product and dose: Capsules (green tea extract)
containing EGCG (treated group, n =30).
Daily oral dose was 800 mg (n =16), 1200 mg
(n =11), and 1600 mg (n =3) EGCG. Control
group (n =10): placebo.
Follow-up: 6 months.
Establishment of the MTD for
EGCG.
MTD was 1200 mg/d EGCG for
6 months.
No significant change in SHBG, IGF-1,
IGFBP-3, ER, Ki-67 proliferation index
or mammographic density
[129]
Int. J. Mol. Sci. 2020,21, 5718 19 of 33
Table 3. Cont.
Cohort and Sample Size Trial Design Objective Outcomes References
Patients (n =28) with ductal
carcinoma in situ or primary
invasive stage I or II breast
cancer
Design: Pre-surgery, controlled study.
Product and dose: 3 capsules/d (green tea
extract) containing EGCG (treated group,
n=13). Daily oral dose was 940 mg EGCG.
Control group (n =15): no capsules.
Follow-up: Average duration of green tea
intake was 35 days.
Short-term effects of green tea
supplementation on cancer
biomarkers.
Significant decrease of Ki-67 in the tea
group, but only in normal tissue.
No effects on apoptosis (caspase-3),
and angiogenesis (CD34) markers in
benign or malignant tissue.
[128]
Patients (n =30) with
non-inflammatory breast
cancer or carcinoma in situ
and prescribed RT without
concurrent chemotherapy
Design: Randomized, double-blind,
placebo-controlled clinical trial.
Product and dose: Patients (n =14) consumed
6 g/d curcumin extract (a daily dose of 4.7 g
curcumin, 0.9 g demethoxycurcumin, and
0.15 g bisdemethoxycurcumin) or placebo
(n =16).
Follow-up: 6 months (30 RT sessions)
Effect of curcumin to reduce
RDS.
Significant reduction of RDS and moist
desquamation at the end of treatment
vs. placebo (mean RDS =2.6 vs. 3.4;
and 28.6% vs. 87.5%; respectively).
[134]
Postmenopausal women (n =
24) with newly-diagnosed,
and resectable, ER+breast
cancer
Design: Pre-surgery, 2 ×2 factorial,
randomized, placebo-controlled trial.
Product and dose: 25 g/d ground flaxseed +
1/d placebo pill (n =6); 1 mg/d anastrozole
(aromatase inhibitor) (n =7); 25 g/d ground
flaxseed +1 mg/d anastrozole (n =6); or 1/d
placebo pill control (n =5).
Follow-up: Mean of 18.8 days.
Effects of flaxseed and the
aromatase inhibitor, anastrozole,
on steroid hormones and
tumour-related biomarkers.
Mean ERβexpression was
approximately 40% lower from pre- to
post-intervention in the flaxseed (FS) +
anastrozole (AI) group only. Significant
negative association for
androstenedione in the FS +AI group
vs. placebo, and for
dehydroepiandrosterone with AI
treatment.
[126]
Patients (n =34) with resected
stage I-III ER- and PR- breast
cancer with no active disease.
(Ancillary study to that of
Crew et al. [129]
Design: Randomized, phase IB,
double-blinded, placebo-controlled, and
dose-escalation study.
Product and dose: Capsules (green tea extract)
containing EGCG (treated group, n =26).
Daily oral dose was 800 mg (n =14), 1200 mg
(n =11), and 1600 mg (n =1) EGCG. Control
group (n =8): placebo.
Follow-up: 6 months
Effect of EGCG on cancer
biomarkers risk.
Significant transient decrease of serum
HGF (only after 2 months of treatment)
in EGCG consumers. No significant
effects on VEGF, serum cholesterol and
triglycerides, oxidative damage,
and inflammatory biomarkers.
[130]
Int. J. Mol. Sci. 2020,21, 5718 20 of 33
Table 3. Cont.
Cohort and Sample Size Trial Design Objective Outcomes References
Patients (n =12) with newly
diagnosed breast cancer, ER+,
not eligible for neoadjuvant
treatment
Design: Pre-surgery (non-controlled) dietary
intervention.
Product and dose: Silybin-phosphatidylcholine
complex (2.8 g/d) given orally.
Follow-up: 4 w before surgery.
Effects on NO, IGF-1 and Ki-67.
No effects on NO, IGF-1 and Ki-67 were
observed. [65]
Patients (n =12) with newly
diagnosed breast cancer, not
eligible for neoadjuvant
treatment.
Design: Pre-surgery (non-controlled) dietary
intervention.
Product and dose: Catechin (65.1 mg/d) and
EGCG (44.9 mg/d) given orally (300 mg tea
extract).
Follow-up: 4 w before surgery.
Effect on cell proliferation,
angiogenesis, oxidative stress,
chronic inflammation, and
adiposity-related endocrine
mechanism.
Significant increase of testosterone.
No effect in the rest of markers. [66]
Patients (n =578) with
non-inflammatory breast
cancer or carcinoma in situ,
and prescribed fractionated
radiation therapy (RT)
without concurrent
chemotherapy
Design: Phase II, randomized, double-blind,
placebo-controlled clinical trial.
Product and dose: Patients (n =283) consumed
either 6 g/d curcumin extract (a daily dose of
5.4 g curcumin, 0.48 g dimethoxy curcumin,
and 0.12 g bisdemethoxy curcumin), or placebo
(n =295).
Follow-up: 6 months (30 RT sessions).
Confirmatory study on the
effect of curcumin to reduce
radiation dermatitis severity.
Curcumin did not reduce radiation
dermatitis severity at the end of RT
compared to placebo.
[135]
ER+and(or) PR+
postmenopausal women
(n =45) with resected breast
cancer at early stage (with no
active disease), and receiving
adjuvant hormonal therapy
Design: Open-label, single-arm
(no placebo-controlled).
Product and dose: Three daily capsules,
containing 460 mg of fish oil (EPA and DHA),
125 mg of olive extract (12.5 mg
hydroxytyrosol), and 50 mg extract of
curcumin (47.5 mg curcuminoids)
Follow-up: 30 days.
Effect on inflammation and
pain.
Significant decrease of plasma CRP
(from 8.2 ±6.4 mg/L at baseline to
5.3 ±3.2 mg/L), and pain (21.5%) after
30 days.
[139]
Int. J. Mol. Sci. 2020,21, 5718 21 of 33
Table 3. Cont.
Cohort and Sample Size Trial Design Objective Outcomes References
Breast cancer patients (n =10)
Design: Pre-surgery, two arms, controlled
study.
Product and dose: Patients (n =5) consumed
walnuts (around 60 g/d) or not (i.e., controls,
n=5).
Follow-up: About 15 days.
Effect of walnut consumption
on gene expression in breast
cancer tissue.
Significant change of 456 genes in the
tumour due to walnut consumption.
Activation of pathways that promote
apoptosis and cell adhesion, and
inhibition of pathways that promote
cell proliferation and migration.
[138]
Patients (n =81) with
histologically confirmed
operable ER+breast cancer,
with no distant metastasis,
and receiving tamoxifen.
Design: Randomized, double-blind, and
placebo-controlled trial.
Product and dose: Patients (n =42) consumed
a red clover extract (80 mg isoflavones/d) or
placebo (n =39).
Follow-up: 2 years
Effect of isoflavones from red
clover extract and lifestyle
change to reduce side-effects of
tamoxifen treatment.
The reductions in BMI and waist
circumference were significantly
greater in the treatment than placebo
group. No differences between groups
in the rest of determinations: MRS,
HDLc, insulin, total cholesterol, LDLc,
triglycerides, insulin resistance, sex
hormone levels, endometrial thickness
and breast density.
[137]
Abbreviations: CEA, carcinoembryonic antigen; c-erbB2 (or HER2), Humanized epidermal growth factor receptor 2; DHA, docosahexanoic acid; EGCG, epigallocatechin gallate; EPA,
eicosapentaenoic acid; ER: oestrogen receptor; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor 1; IGFBP-3, Insulin-like growth factor binding protein-3; MRS, menopausal
rating score; MT, malignant tissue; NT, normal tissue; PR: progesterone receptor; RDS, Radiation Dermatitis Severity score; RT, radiation therapy; SHBG, Sex hormone binding globulin;
VEGF, vascular endothelial growth factor.
Int. J. Mol. Sci. 2020,21, 5718 22 of 33
6. Identifying the Existing Gaps to Address Future Preclinical and Clinical Research
As thoroughly detailed in the present review, despite substantial progress made during the
last decades in advancing our knowledge of how dietary phenolics exert their biologic effects and,
consequently, how they may be involved in the prevention and treatment of BC, to date, minimal
success has been achieved.
Overall, several reasons have led to the vast gap between
in vitro
and
in vivo
evidence. Among the
main reasons, it should be highlighted the low number of well-conducted human studies reported so
far. Furthermore, it is essential to point on several central issues found in clinical trials, and pending
aspects that should be addressed in future clinical studies:
(i) There is a need for multidisciplinary teams beyond researchers (oncologists, surgeons, pathologists,
etc.) to conduct dietary interventions in BC patients. Indeed, this issue is inherent in every clinical
trial dealing with cancer patients. In the case of ‘dietary interventions’, oncologists usually oppose
the assay of high concentrations of dietary phenolics to patients undergoing chemotherapy due to
the limited knowledge regarding the possible interactions between dietary compounds, including
phenolics, and chemotherapy drugs. Overall, this challenging logistic yields a low number of
recruited BC patients. Consequently, a restricted number of samples to explore or validate results,
which is even more challenging when assaying molecules with relatively low anticancer activity
compared to standard chemotherapy drugs.
(ii)
The high heterogeneity of the results obtained. Inter-individual variability is perhaps one
of the most critical aspects to establish definite conclusions in clinical studies. The low
number of volunteers, as well as the specific selection criteria of the volunteers selected
in the different studies, are key factors to understand the high variability reported. Genetic
polymorphisms or differences in the composition or functionality of the gut microbiota will
also contribute to the different response of individuals to interventions with polyphenol-rich
foods [48,144–146]
. In this regard, a highly variable metabolism between individuals has been
described for some phenolic compounds. This allows for grouping the population according
to the excretion of the metabolized compound into “high or low producers”, as it occurs in
the metabolism of the phenolic precursor-derived metabolite pairs hesperidin-hesperetin [
147
],
lignans-enterolignans [
148
], ellagic acid-urolithins [
149
] and procyanidins-valerolactones [
150
,
151
].
Besides, specific metabotypes, attributed to the different composition and functionality of the gut
microbiota, have been described in the case of urolithins [
146
,
152
] or isoflavones [
153
], allowing
the stratification of individuals based on the production of specific metabolites associated with a
particular gut microbiota ecology. Thus, the inter-individual variability related to bioavailability
and metabolism of dietary phenolics is essential to comprehend the results in clinical studies and
to identify whether the possible beneficial effects can be extrapolated to the whole population
or only to certain specific individuals [
146
]. Future research should be conducted to evaluate
associations between specific phenolic-related gut microbiota metabotypes and protective effects
against BC.
(iii)
The difficulty of attributing chemopreventive effects to phenolics. Most human studies have
been conducted with phenolic-containing products or extracts, which makes it challenging to
identify whether single phenolics are (or not) actually responsible for the possible anti-tumour
effect. Along this line, it is important to consider the differences in phenolic composition between
plant foods, the difficulty in the estimation of the dietary intake of phenolics modulated by
several factors such as food matrix, food processing-related factors, as well as the interaction
between phenolics and the gut microbiota, and also other food components, such as proteins
and carbohydrates that will undoubtedly influence the bioavailability and subsequent potential
chemopreventive effects [45,154].
Another element that can help establish causality between effect and polyphenols is the
determination of the derived metabolites in the target breast tissue. However, the characterization of the
Int. J. Mol. Sci. 2020,21, 5718 23 of 33
metabolic profiling of phenolic-derived metabolites in breast tissues remains poorly explored. Several
reasons contribute to this gap: the lack of pure standards (mostly phase-II conjugates) that prevent their
accurate quantification, and the accessibility to enough mammary biopsies to perform the analyses.
Besides, the lack of standardized analytical protocols contributes to reporting incomplete or even
misleading identifications/quantifications (heterogeneity of samples, hydrolysis vs. non-hydrolysis
approaches, etc.).
Finally, it should be taken into account that phenolics generally exert relatively low biological
activity, which makes difficult to discern their possible anticancer effects. In this regard, clinical studies
should be focused on looking for effects upon long-term consumption of phenolic-rich foods. Therefore,
in the coming years, there is a need to improve and re-design clinical trials to demonstrate the efficacy
of dietary phenolics against BC unequivocally.
(iv)
There is not enough evidence regarding the possible interaction between dietary phenolics and
conventional chemotherapy drugs. Despite the preclinical evidence that supports the potential
benefits of dietary phenolics as possible adjuvants in BC management [
155
,
156
], to date, only three
clinical studies have been conducted using a combination of phenolics and chemotherapeutic
drugs [
126
,
137
,
139
]. Therefore, there is a need to evaluate the potential interactions between
phenolics and chemotherapeutic drugs to support phenolics’ usefulness as adjuvants in BC
treatment and further follow-up of patients to prevent relapses.
As far as chemoprevention is concerned, the assayed conditions in animal models that cannot
be extrapolated to humans, and the non-physiological
in vitro
approaches were predominant in the
past. Nowadays, the trend is to perform physiologically relevant studies, as highlighted in recent
guidelines [
78
,
157
]. Regarding BC, attention should be paid to the following gaps and pitfalls performed
in the past strategies for phenolics’ chemoprevention determination in animal and cellular BC models:
(i)
The difficulty of extrapolating the results obtained from animal research to humans. As in the
case of human studies, several problems can be found in the BC animal models to evaluate the
chemopreventive effect of phenolics: the methodology issues (low number of animals, and lack
of controls, etc.), the heterogeneity of data, the difficulty of attributing chemopreventive effects to
phenolics (use of whole-foods or extracts instead of single phenolics, determination of phenolics
in breast tissues, etc.).
Besides, other intrinsic aspects should be considered and improved in animal research: any breast
tumour-induction process (chemically-induced BC, murine mutant models, etc.) can reflect the
heterogeneity of human BCs. It is habitual to use inadequate doses and exposure times for single
phenolics, food, or extracts. Instead, doses should be equivalent to those achievable in humans
after the regular intake of the phenolic-containing foods. The systemic administration of phenolics
or food extracts (intravenous, intraperitoneal, etc.) is not representative of a dietary context and
omits the significant handicaps of bioavailability and metabolism. Finally, the suitability of the
specific animal model, according to the capability of each species to metabolize phenolic compounds,
is of particular interest, especially in polyphenol-related gut microbiota metabotypes (urolithin and
isoflavone metabotypes).
Besides, we must add to all the above logical inter-species differences (rats, mice, pigs, etc.),
and even within strains from the same animal model, which contribute to the great challenge of
extrapolating
in vivo
results to BC patients [
158
]. Nevertheless, animal research remains an essential
step before clinical trials, mainly in evaluating the interaction with chemotherapeutics.
(ii)
In vitro
studies must have physiological relevance to establishing potential effects and conclusions.
Different aspects of
in vitro
research should be avoided: the use of both unrealistic concentration
and metabolic forms of phenolics; the unsuitability of BC cell models using a single cell line instead
of considering the heterogeneity of cell lines using multiple cell lines with different mutations and
other genetic characteristics (ER/PR positive or negative, etc.), or even more realistic advanced
Int. J. Mol. Sci. 2020,21, 5718 24 of 33
models such as primary cell cultures, organoids, etc.; and testing single phenolic or derived
metabolite without considering the real mixture of compounds present
in vivo
, as well as the
food matrix effect, avoiding the possible synergistic, antagonistic, or additive effects among
them. Therefore, following these principles, transferring the information on the pharmacokinetic
properties and bioavailability of phytochemicals in breast tissues plays one of the critical roles for
a better evaluation of their mechanism of action involved in their BC chemoprevention.
7. Concluding Remarks
According to the detailed literature survey, including only physiologically relevant preclinical
studies and clinical studies, we can conclude that some dietary phenolics, especially RSV, quercetin,
isoflavones, EGCG, lignans, and curcumin, seem to be the most promising candidates. However,
the clinical evidence is still minimal when compared with that obtained in preclinical studies.
As stated above, the increase and development of highly robust, well-designed clinical studies
in BC patients and the quantitative and qualitative determination of phenolic-derived metabolites
that reach breast tissues will make possible the continuous progress of the preclinical research.
This approach will allow a more in-depth understanding of the molecular mechanisms involved in
their chemopreventive and(or) chemotherapeutic properties and, perhaps, allowing the establishment
of preventive nutritional strategies.
In the “omics” era, complementary approaches, including in silico and molecular docking studies,
as well as high-throughput “omics” technologies, such as (epi)genomics, proteomics, and metabolomics
are rapidly gaining interest to identify new ligands for specific cancer molecular targets in order to
improve the therapeutic potential of both phenolics and their metabolic derivatives [
159
,
160
]. Besides,
the analysis of the composition and functionality of the intra-tumour microbiota in breast cancer
has been reported to be useful to predict different responses between tumour types and subtypes
against immunotherapy [
161
]. Whether the microbiota of breast cancer, particularly rich and diverse,
could modulate dietary phenolics’ effects deserves further research.
Supplementary Materials:
Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/16/
5718/s1. Table S1: Dietary phenolic-derived metabolites identified in breast tissues in animal model studies. Table
S2: Animal studies conducted with dietary phenolics and/or phenolic-rich extracts on BC models.
Author Contributions:
J.C.E. and A.G.-S. conceived and designed the study; M.
Á
.
Á
.-G., J.A.G.-B., J.C.E. and
A.G.-S. contributed to the discussions and preparation of the manuscript; J.A.G.-B., J.C.E. and A.G.-S. wrote the
article. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by the projects PID2019-103914RB-I00 (MICINN, Spain), 19900/GERM/15
(Fundaci
ó
n S
é
neca de la Regi
ó
n de Murcia, Spain), and 201870E014 and 201770E081 (CSIC, Spain). J.A.G.B.
was supported by a Juan de la Cierva contract (IJCI-2016-27633) from the Ministry of Science, Innovation and
Universities (Spain) and a Standard European Marie Curie Fellowship from the European Commission. This project
has received funding from the European Union’s Horizon 2020 research and innovation programme under the
Marie Sklodowska-Curie grant agreement No 838991.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
BC Breast Cancer
DMBA 7,12-Dimethylbenz[a]anthracene
EGCG Epigallocatechin Gallate
ER Oestrogen Receptor
HER2 Human Epidermal growth factor Receptor 2
MTD Maximum Tolerability Dose
PR Progesterone Receptor
RSV Resveratrol
TNBC Triple Negative Breast Cancer
Int. J. Mol. Sci. 2020,21, 5718 25 of 33
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