Chemopreventive effect of dietary polyphenols in colorectal

cancer cell lines

Joo R. Arajo, Pedro Gonalves, Ftima Martel

Department of Biochemistry (U38-FCT), Faculty of Medicine of Porto, University of Porto, 4200-319 Porto, Portugal
Received 6 September 2010; revised 27 December 2010; accepted 24 January 2011
Abstract
Colorectal cancer (CRC) is the second most fatal and the third most diagnosed type of cancer
worldwide. Despite having multifactorial causes, most CRC cases are mainly determined by dietary
factors. In recent years, a large number of studies have attributed a protective effect to polyphenols and
foods containing these compounds (fruits and vegetables) against CRC. Indeed, polyphenols have been
reported to interfere with cancer initiation, promotion, and progression, acting as chemopreventive
agents. The aim of this review is to summarize the main chemopreventive properties of some
polyphenols (quercetin, rutin, myricetin, chrysin, epigallocatechin-3-gallate, epicatechin, catechin,
resveratrol, and xanthohumol) against CRC, observed in cell culture models. Fromthe data reviewed in
this article, it can be concluded that these compounds inhibit cell growth, by inducing cell cycle arrest
and/or apoptosis; inhibit proliferation, angiogenesis, and/or metastasis; and exhibit anti-inflammatory
and/or antioxidant effects. In turn, these effects involve multiple molecular and biochemical
mechanisms of action, which are still not completely characterized. Thus, caution is mandatory when
attempting to extrapolate the observations obtained in CRC cell line studies to humans.
2011 Elsevier Inc. All rights reserved.
Keywords: Colorectal cancer cell lines; Diet; Polyphenols; Quercetin; Catechins; Chemoprevention
Abbreviations: BT, butyrate; CDK, cyclin-dependent kinase; Cox, cyclooxygenase; CRC, colorectal cancer; CYP, cytochrome
P450; EGCG, epigallocatechin-3-gallate; EGFR, epidermal growth factor receptor; ER, estrogen receptors; ERK,
extracellular signalregulated kinase; FHC, fetal human cell; IGF, insulin-like growth factor; iNOS, inducible
nitric oxide synthase; MAPK, mitogen-activated protein kinases; NF-B, nuclear factor B; PPAR, peroxisome
proliferatoractivated receptors; pRb, phosphorylated retinoblastoma; TNF, tumor necrosis factor; VEGF,
vascular endothelial growth factor.
1. Introduction
The relationship between the more than 8000 polyphe-
nols present in the diet and the prevention of diseases in
humans has been an intense field of research during the last
years [1]. One of the reasons for the growing interest in
studying these compounds resides in their protector role
against colorectal cancer (CRC) [2-6], a high-mortality
pathology in occidental countries [2]. Thus, the aim of this
article is to review the cellular and associated molecular and
biochemical mechanisms by which some polyphenols
(quercetin, rutin, myricetin, chrysin, epigallocatechin-3-
gallate [EGCG], epicatechin, catechin, resveratrol, and
xanthohumol) might protect against CRC, according to the
information provided by cell culture studies.
2. Polyphenols: classification, food sources, and
health-promoting effects
Polyphenols are phytochemicals derived from phenylal-
anine and contain an aromatic ring with a reactive hydroxyl
group [7]. According to their structure, polyphenols can be
divided into different classes [8,9]. The main classes
include flavonoids and phenolic acids (eg, gallic acid and
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E-mail address: fmartel@med.up.pt (F. Martel).
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doi:10.1016/j.nutres.2011.01.006
curcumin), the most abundant in foods, and the less
common stilbenes (eg, resveratrol) and lignans (eg,
secoisolariciresinol) [7,10]. Flavonoids may be further
divided into 7 subclasses: flavones (eg, chrysin), flavonols
(eg, quercetin, myricetin, and rutin), flavanones (eg,
naringenin), anthocyanidins (eg, malvidin), flavan-3-ols
(eg, catechin, epicatechin, and EGCG), isoflavones (eg,
genistein), and chalcones (eg, xanthohumol) [11,12].
Quantification of dietary intake of polyphenols is difficult
to estimate, but the studies available point out to an average
ingestion of 0.1 to 1 g/d in occidental Europe and the United
States population [10]. The most important dietary sources of
polyphenols are fruits, vegetables, seeds, and beverages such
as fruit juice, green tea, coffee, cocoa drinks, red wine, and
beer [13].
The growing interest in studying these compounds
resides in the accumulating evidence showing that these
compounds possess a high number of protective biologic
properties: antioxidant, anticarcinogenic, anti-inflammatory
[8,11,14,15], neuroprotector [16], antiallergic, antidiarrheal,
antiulcer, antibiotic [10,17], antilipidemic, vasorelaxing [7],
and antithrombotic properties [8,10,11,14,15]. Because of
these effects, polyphenols may confer protection against
pathologies with very high incidence and mortality in
occidental countries: cardiovascular and neurodegenerative
diseases and cancer [2-6]. In relation to cancer, numerous
case-control [18] and animal and cell culture studies have
corroborated a protector role of polyphenols and of foods and
drinks that contain them (especially fruits and vegetables) in
distinct cancer types (eg, breast, lung, colon, stomach,
esophagus, larynx, and oral cavity) [9,18]. However, in
human prospective studies, a very small [19], or even an
absent, positive association between intake of fruits and
vegetables and reduced risk of cancer [12] has been reported.
3. Colorectal cancer: epidemiology, etiology, risk, and
protective factors
Cancer is the second leading cause of death, after
cardiovascular diseases, in occidental countries [2,20].
Every year, around 10 million people worldwide are
diagnosed with cancer, and approximately 6.2 million die
of this disease [2,21]. Colorectal cancer is the second type of
cancer with the highest mortality (492 000 persons per year)
and the third most diagnosed (945 000 persons per year) in
both American men and women [2,22].
Etiologically, CRC may be hereditary or sporadic or have,
on the background, inflammatory bowel diseases [23]. In
hereditary CRC, inherited genetic mutations occur in critical
genes, such as tumor suppressor genes, genes associated with
DNA mismatch repair, or other genes [23]. Most sporadic
CRCs are due to somatic genetic mutations (normally in the
same genes as those cited for hereditary CRC) that occur as
part of the normal cellular lifespan or because of exposure to
environmental factors such as dietary carcinogens [23].
Dietary factors are responsible for 70% to 90% of CRC
cases, and diet optimization will prevent most of them
[24,25]. Chronic inflammatory bowel disease is also an
etiologic factor in the development of CRC because high
oxidative stress burden present in the inflamed mucosa alters
important cellular functions [20].
Despite the multifactorial etiology of CRC, it is known
that (a) ingestion of a diet rich in calories and lipids
(particularly those of animal origin) [26], red meats, N-
nitroso compounds, and aromatic polycyclic hydrocarbons
present in grilled fish and meat [27]; (b) ingestion of high
amounts of ethanol; and (c) certain diseases (eg, obesity
[28] and diabetes [28,29]) raise the incidence risk of CRC.
On the other hand, it is also known that (a) a diet rich in
fruits, vegetables (and, consequently, polyphenols) [26],
fiber [30], short-chain fatty acids, especially butyrate (BT)
[31]; (b) beverages such as mineral waters [25,32], red
wine [15], and green tea [6,15,33]; (c) micronutrients such
as vitamin A, C, E, and D; folic acid [24,34]; selenium
[35]; and calcium [24]; (d) drugs such as nonsteroidal anti-
inflammatories and selective cyclooxygenase (Cox) inhibi-
tors (eg, aspirin, sulindac, and celecoxib) [24,36], epider-
mal growth factor receptor (EGFR) inhibitors, [21] and
peroxisome proliferatoractivated receptors (PPAR) ago-
nists [37]; (e) estrogen replacement therapy; and (f)
moderate-to-vigorous physical exercise [2,24] reduce the
incidence risk of CRC.
4. Colorectal cancer: the molecular perspective
Intestinal epithelium is a dynamic tissue with an elevated
regeneration capacity: in the inferior two thirds of the
colonic crypt, colonocytes proliferate, and in the upper two
thirds of the crypt, they undergo differentiation and finally
apoptosis [22,38]. The balance between proliferation,
differentiation, and apoptosis maintains the tissue homeo-
stasis, and deregulation of these processes has a determinant
role in CRC development [22,39]. In general, carcinogen-
esis is a complex process that involves multiple phases
where molecular and cellular alterations, particularly of
genetic origin, may occur [21,23]. In a simple manner, 3
phases have been described: initiation, with exposure of the
normal cell, particularly the nucleus, to the carcinogenic
agent causing a genetic alteration; promotion, a phase
longer than the former, in which survival and replication of
damaged cells occur; and progression, which is character-
ized by deregulation of cellular proliferation and differen-
tiation, reduction of apoptosis of damaged cells (tumor
growth), and increase of metastatic and angiogenic potential
[18,40,41]. Human CRC is particularly associated with a
progressive inhibition of apoptosis [42], this being an
important mechanism by which colonocytes with damaged
DNA escape normal clearance mechanisms and grow to
become invasive tumors [43,44].
During the last 3 decades, chemoprevention has been a
field of intense research. Chemoprevention is the use of
synthetic or natural compounds, in pharmacologic doses, to
78 J.R. Arajo et al. / Nutrition Research 31 (2011) 7787
reduce the risk of development or recurrence of cancer [21].
As the carcinogenic process may be delayed or interrupted in
each of its phases, several anticarcinogenic mechanisms may
be displayed. Chemopreventive blocker agents act immedi-
ately before or after initiation of carcinogenesis, and
chemopreventive suppressor agents act after initiation,
during the prolonged phases of promotion and progression
[18]. Dietary polyphenols modulate different cellular
processes (pleiotropic effects) on cancer cells, acting as
chemopreventive blocker agents, chemopreventive suppres-
sor agents, or both [45] (Table 1).
Although not completely proved to occur in vivo, Ramos
[12] pointed out the 6 main common chemopreventive
effects that polyphenols can exert on cancer cells: (1)
antioxidant effect, (2) antiproliferation and antisurvival
effect, (3) induction of cell cycle arrest, (4) induction of
apoptosis, (5) anti-inflammatory effect, and (6) inhibition of
angiogenesis and metastasis. Each of these effects will be
analyzed next.
4.1. Antioxidant effect
The most well-known property of polyphenols is their
antioxidant capacity [11-13]. This property depends on the
hydroxylation status of their aromatic rings [45]. Antioxi-
dant effects of polyphenols include the following: (a)
scavenging of free radicals [11,85], (b) chelation and
stabilization of divalent cations, and (c) modulation of
endogenous antioxidant enzymes (induction of phase II and
inhibition of phase I [cytochrome P450 {CYP}] enzymes)
[11,16,85]. Because polyphenols are redox-sensitive com-
pounds [86], depending on cell type, dose, and/or time of
treatment, they can also act as pro-oxidants, enhancing
reactive oxygen species production, especially H
2
O
2
, and,
therefore, reducing cell growth [12].
4.2. Antiproliferative and antisurvival effects
One of the most representative studies concerning
polyphenols ability to inhibit cellular proliferation was
performed by Kuntz et al [50]. Of the 36 polyphenols tested,
30 demonstrated antiproliferative activity in the absence of
cell cytotoxicity in human CRC cell lines. The most
important signaling pathways regulating cell proliferation
and survival involve phosphatidyl-inositol-3-kinase/protein
kinase B, growth factor receptors/Ras/mitogen-activated
protein kinases (MAPK), and, especially, nuclear factor
B (NF-B) [12]. Constitutive activation of NF-B is
common in cancer, inhibition of its activation being a key
chemoprevention target [87].
4.3. Induction of cell cycle arrest
Deregulated cell cycle is a hallmark of cancer. Cell
cycle control is a highly regulated process that involves
the modulation of cell cycle regulatory proteins, including
cyclins (cyclin A, B, Ds, or E); cyclin-dependent kinases
(CDKs) (CDK 1, 2, 4, or 6); and CDK inhibitors, such as
p21
WAF1
, p27
KIP1
, p53, and phosphorylated retinoblasto-
ma (pRb) [16,43,88]. Any alteration of cell cyclespecific
proteins by polyphenols can affect growth and prolifera-
tion of cancer cells. In addition, cell cycle checkpoints,
such as G
1
/S and G
2
/M, are also important targets for
polyphenols [40].
4.4. Apoptosis
Programmed cell death (apoptosis) is a protective
mechanism against cancer, by removing genetically dam-
aged cells from the epithelium before they undergo clonal
expansion. Thus, resistance to apoptosis is another hallmark
of cancer [48]. The 2 major pathways that initiate apoptosis
are (a) extrinsic, mediated by death receptors CD95/Fas/
Apo1, tumor necrosis factor (TNF) receptor 1, TNF receptor
2, and death receptors 3 to 6 [23,89] and (b) intrinsic
(mitochondria-mediated) [90]. In the mitochondria, propa-
gation of the apoptotic signal is regulated by proteins such as
Bcl-2 family members (Bcl-2, Bcl-xL, and Bcl-w, which
exert antiapoptotic effects, and Bid, Bad, Bak, Bax, and Bim,
which exert proapoptotic effects) [23,89,91]. A third
apoptotic pathway, the endoplasmic reticulum stress
pathway, has recently been described [89].
4.5. Anti-inflammatory effect
The association between inflammation and CRC
involves key inflammatory mediators such as NF-B,
TNF, inducible nitric oxide synthase (iNOS), lipoxygenase
[11,92], PPAR- [93], and, particularly, Cox. Constitutive
Cox-1 and inducible Cox-2 are key isoenzymes involved in
prostaglandin biosynthesis. Inhibition of Cox, particularly
of Cox-2, may inhibit tumor cell growth, proliferation,
angiogenesis, metastasis [11,92], and inflammation and
induce apoptosis [7,92].
4.6. Antiangiogenic and antimetastatic effects
Angiogenesis, the formation and growth of new blood
vessels from preexisting microvasculature [78], is a key
stage in tumor growth, invasion, and metastasis. In an
intimate way, metastasis involves the interplay of extracel-
lular matrix degradation, proteolysis, cell adhesion, cell
migration, angiogenesis, and invasion [12].
Because the chemopreventive effect of polyphenols is
dependent on the particular compound selected, on its
concentration, time of treatment, and cell type studied, each
polyphenol must be analyzed individually. Thus, clarification
of the molecular mechanisms by which polyphenols might
exert a potential anticarcinogenic effect turns out to be an
important challenge [12]. The intestine is considered to be a
promising site for chemoprevention because it is exposed to
higher doses of dietary polyphenols compared to most other
tissues, which are exposed to similar or inferior levels to those
found in plasma. In fact, most polyphenols are probably too
hydrophilic to penetrate the gut wall by passive diffusion,
having, thus, low systemic bioavailability [10,13]. After the
79 J.R. Arajo et al. / Nutrition Research 31 (2011) 7787
ingestion of 250 to 500 mg of polyphenol supplements, the
lumen of the colon can be exposed to concentrations around
0.1 to 3 mmol/L [13,22,57,94], whereas the plasma
concentrations are around 1 mol/L [56].
The aim of this review is to summarize the cellular and
molecular chemopreventive effects of some dietary poly-
phenols in CRC cell lines. The selected polyphenols
(quercetin, rutin, chrysin, myricetin, catechin, epicatechin,
EGCG, resveratrol, and xanthohumol) are some of the most
representative in the human diet and have been previously
extensively studied by our group [47,59,95-97].
5. Quercetin and rutin
The flavonol quercetin (3,3,4,5,7-pentahydroxyflavone)
is the most frequently occurring polyphenol in foods [85] and
one of the most largely studied [11,13]. Quercetin is found in
abundance in onions, tea [13], apples, broccoli, berries [11],
and red wine [12]. Despite being converted to its glycoside
rutin by the -glycosidase enzymes of colonic microorgan-
isms [51], this reviewwill focus on quercetin rather than rutin
because the former has been studied in more detail.
Rutin and quercetin have shown a range of different effects
in in vivo animal models of CRC, inhibiting [44,46,48],
promoting [98], or not influencing [58] CRC development.
Because of these contrasting effects, discussion whether
quercetin was carcinogenic or not was stimulated [22]. In
vitro, quercetin was shown to inhibit growth and proliferation
of CRCcells such as the human adenocarcinoma HT-29 [44],
COLO 201, LS-174T [16,50], HCT-116 [56], SW480 [55],
and Caco-2 cells [50] and, in a much lower extent, of
nontransformed cells such as rat intestinal epithelial (IEC-6)
cells [51], and human fetal colon cells (FHC) [44,54]. In
addition, it was found to be cytotoxic for actively proliferating
cells and to cause undifferentiated cancer cell lines to
differentiate [57,62] (Table 1). Compared to quercetin, rutin
demonstrated a less significant effect, either inhibiting [50] or
not altering [44] CRC cell proliferation (Table 1).
Several mechanisms explaining the in vitro antiprolifera-
tive effect of quercetin have been proposed. These include:
(a) cell cycle arrest in the G
0
/G
1
, G
2
/M, and S phase [44,52];
(b) inhibition of proliferation signal transduction pathway
associated enzymes [58] (eg, MAPK [46], phosphatidyl-
inositol-3-kinase [56], l-phosphatidylinosotol-4-kinase [99],
Table 1
Mechanisms involved in the chemopreventive effect of polyphenols in colorectal cell lines
Polyphenol Cell type Cellular mechanism References
Quercetin HT-29, Caco-2, SW480, HCT-116,
IEC-6, FHC, VACO-235, COLO 201,
LS-174T, T84, DLD-1, LT97, rat
and mouse colonocytes
Cell growth inhibition and cytotoxic activity;
reduction or stimulation of cell proliferation;
decrease of cell migration; induction of cell cycle
arrest, differentiation, apoptosis, and autophagy
[12,16,18,22,44,46-58]
Rutin HT-29 and Caco-2 Inhibition or no alteration of cell proliferation,
differentiation, and apoptosis
[44,47,50,59]
Myricetin HT-29, Caco-2, SW480, T84,
VACO-235, COLO 205
Cell growth inhibition; reduction of cell
proliferation; induction of apoptosis;
antimetastatic properties
[47,50,59-61]
Chrysin HT-29, Caco-2, SW480 Cell growth inhibition; reduction of cell
proliferation; induction of cell cycle
arrest and apoptosis
[47,50,59,62]
EGCG HT-29, Caco-2, SW480, SW837,
SW426, HCT-116, FHC, T84, murine
colon 26-L5, mouse colon 26
Inhibition of cell growth, proliferation, neoplastic
transformation, invasion, and angiogenesis;
induction of cell cycle arrest and apoptosis
In a few cases, proliferation is increased.
[12,45,47,59,63-72]
Epicatechin HT-29 Weak or absent growth-inhibitory and
apoptotic activity
[12,70,72]
Catechin HT-29 Weak or absent growth-inhibitory and
apoptotic activity
[72,73]
Resveratrol HT-29, SW480, Caco-2, SW620,
HCT-116, CCL 220.1, WiDr
Inhibition of cell growth; induction of
apoptosis; arrest of proliferation, cell cycle,
and neoplastic transformation
[17,47,52,57,59,74-81]
Xanthohumol HT-29, CCL 220.1, HCT-116derived
40-16
Inhibition of cell growth, proliferation,
invasiveness, and angiogenesis; induction
of apoptosis, terminal differentiation, and
cell cycle arrest
[78,82-84]
Combination of polyphenols HT-29, Caco-2, SW480 Inhibition of cell growth and proliferation;
induction of apoptosis
[45,70]
Combination of polyphenols
with therapeutic drugs
HT-29, mouse colon 26 Inhibition of cell growth and proliferation;
induction of apoptosis
[70,71]
In certain cases, opposing results have been obtained because the studies were carried out in different cell types. In addition, the chemopreventive mechanismwill
also depend on the concentration and time of treatment with the polyphenol(s) and/or therapeutic drug.
80 J.R. Arajo et al. / Nutrition Research 31 (2011) 7787
protein kinase C, protein tyrosine kinases [56], and cyclic
adenosine monophosphatesensitive casein kinases [46]);
(c) reduction of inflammatory metabolites formation through
inhibition of Cox-2 activity and expression [58], lipoxy-
genase and iNOS activities, and eicosanoid biosynthesis
[18]; (d) interaction with type II estrogen receptors (ER)
[16]; (e) down-regulation of expression of the cell cycle
genes cell division cycle 6 (CDC6), CDK4, and cyclin D1
[46,54]; (f) up-regulation of the tumor-suppressor genes
breast cancer type 2 susceptibility protein and mucin 2 and
down-regulation of oncogene Ras [54] and survivin
expression [55]; (g) down-regulation of the -catenin
pathway [18,55]; (h) rearrangement of cytoskeleton actin
microfilaments [54] and tubulin microtubules [100]; and (i)
inhibition of P-glycoprotein, a membrane transporter that
extrudes chemotherapeutic drugs [101].
Besides inhibiting proliferation, quercetin has been
reported to strongly induce apoptosis in poorly differentiated
SW480 cells [55] and in well-differentiated T84, VACO-235,
[52] HT-29 [44], and Caco-2 cells [47,57,59] (Table 1).
Quercetin-induced apoptosis has been demonstrated to be
associated with the following: (1) DNA strand breakage
[51,52]; (2) up-regulation of caspases 3 [52], 7, and 9 and
Bax; (3) proteolitic cleavage of nuclear enzyme poly
(adenosine diphosphate [ADP]ribose) polymerase; (4)
release of cytochrome C; (5) down-regulation of Bcl-xL
and Bcl-2 proteins [12]; (6) down-regulation of protein kinase
B pathway [58], extracellular signalregulated kinase (ERK)
[53], and human EGFR 2 and 3 signaling [44]; and (7)
inhibition of heat shock protein synthesis [99] and protea-
some activity [102]. An interesting fact is that quercetin can
also induce CRC cell death through autophagy, a caspase-
independent type of cell death distinct fromapoptosis [54]. In
relation to rutin's effect on CRC cellular apoptosis, studies
are scarce and contradictory [44]. Rutin may induce [47,59]
or have no effect on apoptosis [44] (Table 1).
In CRC cell lines, quercetin is also capable of reducing
cell migration, a prerequisite for cell invasion and one of the
most important tumorigenic properties [54] (Table 1).
Interestingly enough, inhibition of cell proliferation by
quercetin showed, in some cases, a biphasic response: at low
concentrations (0.5-5 mol/L), cell proliferation was mod-
estly decreased by quercetin, [57,59] or even slightly
increased [56,59], but at high concentrations (10 mol/L),
cell proliferation showed a profound decrease. This biphasic
effect on cell proliferation, generally called growth hormesis,
might be explained by several facts. First, at low concentra-
tions, quercetin might behave like a phytoestrogen acting as
an ER agonist [103], eventually inducing activation of
intracellular MAPK pathways (ERK and c-Jun NH
2
-terminal
kinases) and leading to expression of survival genes (c-Fos
and c-Jun) [12,22], but at higher concentrations, quercetin
may lead to inhibition of cell proliferation and activation of
apoptosis [12], which are particularly seen in ER-positive
CRC cell lines [22,56,104]. Second, regulatory overcorrec-
tions by proliferation control mechanisms in response to low,
growth-inhibiting concentrations of quercetin might occur
[56]. Third, quercetin may target different phases of cell cycle
according to the concentrations used, probably blocking S
phase at low concentrations and G
1
phase at higher
concentrations [57]. Finally, because the balance between
the antioxidant and the pro-oxidant activity of quercetin is
concentration dependent [56], high concentrations of quer-
cetin may be oxidized, thus generating reactive oxygen
species (superoxide radicals and H
2
O
2
), which induce
cytotoxicity and mutagenic alterations in cell culture systems
[49,53,56,58]. On the other hand, low concentrations of
quercetin might behave as a powerful antioxidant and
antimutagenic stimulus, effectively decreasing oxidative
stress [53,56].
6. Myricetin and chrysin
Little attention has been paid to the study of the
chemopreventive properties of the flavonol myricetin
[50,60,61,105] and of the flavone chrysin against CRC
[18,50,62,106].
Myricetin (3,3,4,5,5,7-hexahydroxyflavone) is structur-
ally similar to quercetin [105] and occurs in significant
amounts in berries, tea, and red wine [105]. Studies have
shown that myricetin has antiproliferative properties (Table 1):
it is cytotoxic to HT-29 cells due to an auto-oxidation process;
induces growth inhibition and apoptosis in Caco-2 cells due
to stimulation of apopain activity [50] and, in SW480, T84,
and VACO-35 cells, due to inhibition of EGFR kinases [61];
and has potent antimetastatic properties because it inhibits
matrix metalloproteinase isoform 2 activity and expression in
various CRC cell lines [60].
Chrysin (5,7-dihydroxyflavone), present at high levels in
honey and propolis [62], exerts growth-inhibitory and
antiproliferative effects in CRC cells [47,50,59,62] (Table 1)
through induction of cell cycle arrest, particularly at the G
2
/M
phase [62], induction of CDK inhibitors p21
WAF1/CIP1
[16]
and anti-inflammatory effects due to inhibition of Cox-2
expression [18], suppression of NF-B and TNF- activation
[18,106], inhibition of iNOS, prostaglandin E2, and cytokines
(such as interleukin 6) production, inhibition of mieloperox-
idase activity [106], activation of PPAR- transcription factor
[107], and inhibition of proteasome activity [108]; and
induction of apoptosis [47,59,107], probably through activa-
tion of caspase 3 and inactivation of Akt (protein kinase B)
[107,109].
7. Catechin, epicatechin, and EGCG
Flavan-3-ols, also known as catechins, are mainly found
in green and black tea and in red wine [11]. Green tea
consumption has been associated with many health benefits
including prevention of cancer and inflammatory diseases
[65,86,110]. Although epidemiological studies have not
yielded a clear positive correlation between tea consumption
81 J.R. Arajo et al. / Nutrition Research 31 (2011) 7787
and cancer-risk reduction, there is no doubt that tea
polyphenols have promising chemopreventive effects in
CRC cell models [45]. These effects can be attributed to
some of its flavan-3-ols constituents (1 L of green tea
infusion contains 1 g of flavan-3-ols) [13], mainly EGCG,
the most abundant one [6,111], and the less common
epigallocatechin, epicatechin, and catechin [66]. Epigallo-
catechin-3-gallate has been found to inhibit tumorigenesis in
many animal models of CRC [16,17,65] and in studies using
CRC cell lines [58,66,70,110]. In CRC cell lines, EGCG
may induce (1) inhibition of neoplastic transformation,
growth, and proliferation [33,64,69]; (2) induction of cell
cycle arrest, particularly in the G
0
/G
1
phase [70]; (3)
induction of apoptosis [33,47,59,65]; and (4) inhibition of
cell invasion and angiogenesis [33,67] (Table 1). These
effects may be achieved through the modulation of a wide
range of signaling molecules and their pathways, including
inhibition of growth factorrelated cell signaling pathways
(inhibition of EGFR activation; down-regulation of insulin-
like growth factors [IGFs] 1 and 2 [16,110], IGF receptor 1,
IGF binding protein 3 [12], human EGFR 2 [66],
transforming growth factor [70], and platelet-derived
growth factor; inhibition of platelet-derived growth factor
receptor activation; down-regulation of fibroblast growth
factor and vascular endothelial growth factor [VEGF];
inhibition of VEGF receptors 1 and 2 activation; and
down-regulation of hypoxia-inducible factor 1a [12]); down-
regulation of survival signaling pathways components such
as MAPK (c-Jun NH
2
-terminal kinases, ERK-1 and ERK-2,
p38, and activating protein 1 transcription factor), signal
transducer and activator of transcription, phosphatidyl-
inositol-3-kinase, Akt [12], c-Fos [70], c-Jun, early growth
response protein 1, and, very important, NF-B [12];
modulation of cell cycle regulators, such as inhibition of
cyclin D1, p21, p27, pRb, CDK-2, CDK-4, and CDK-6 [12]
and activation of p16 and retinoic acid receptor [45];
modulation of apoptosis regulators, such as down-regulation
of the proteins Bcl-2, Bcl-xL, Bid, inhibitor of apoptosis
protein 2, X-linked inhibitor of apoptosis, and myeloid cell
leukemia 1 [12], release of apoptogenic cytochrome C
[12,45], second mitochondria-derived activator of caspase/
direct inhibitor of apoptosis-binding protein with low pI and
apoptosis-inducing factor [12], up-regulation of Bax, Bad,
caspases 3, 7, 8, and 9, and p53 [12,45], and proteolytic
cleavage of poly (ADP-ribose) polymerase [12,91,110].
Other mechanisms involved in EGCG anticarcinogenic
effect include attenuation of the inflammatory response
through inhibition of Cox and lipoxygenase expression and
activity [71]; inhibition of arachidonic acid metabolism
[6,69]; reduction of expression and secretion of the
chemokines interleukin 8, TNF-, macrophage inflammatory
protein 2 and 3, and melanoma growthstimulating activity
and [68]; and inhibition of expression and activity of
DNA topoisomerase I [72], matrix metalloproteinases
(particularly matrix metalloproteinase isoforms 2 and 9)
[66,110], and telomerase [112,113]. An interesting point is
that concentrations of EGCG necessary for inhibition of
activity of these enzymes (IC
50
= 0.5-20 mol/L) [86,110]
are lower than those necessary for inhibition of CRC cell
growth (IC
50
= 10-90 mol/L) [110]. Other enzymes
inhibited by EGCG include DNA methyltransferase [63]
(which reactivates the expression of methylated-silenced
genes such as the growth-inhibitory p16
INK4a
gene [70]),
chymotryptic subunit of the proteasome [63,102],
teleocidin-induced protein kinase C, 12-O-tetradecanoyl-
phorbol-13-acetateinduced epidermal ornithine decarbo-
xylase and urokinase [72]. Finally, EGCG may exert a pro-
oxidant effect because it may be oxidized generating
superoxide radicals and H
2
O
2
[33,86], which may, in turn,
activate 5-adenosine monophosphateactivated protein
kinase, down-regulate VEGF and the glucose transporter 1
[12], and inactivate EGFR [86], inducing apoptosis [33].
However, it is not clear whether EGCG oxidation-induced
effects also occur inside animal tissues because these are
usually under lower O
2
partial pressure (b40 mm Hg) than
cell culture systems (152 mm Hg) [86].
A matter of debate about EGCG is whether it binds to
membrane receptors and/or needs cellular uptake to exert its
chemopreventive effects on cancer cells [70]. A 67-kd
laminin surface receptor has been identified for EGCG [114]
being responsible for its interaction with lipid rafts, which
could eventually result in alterations of membrane fluidity
and enzymatic activity of membrane-anchored proteins,
culminating in various downstream effects [63]. However,
intestinal absorption of EGCG through passive nonionic
diffusion of the undissociated form has also been demon-
strated [115]. After transport, EGCG can be converted to
aromatic short-chain fatty acid derivatives, particularly
phenylacetate, phenylbutyrate, and phenylpropionate
[66,112,116], that have been demonstrated to exert potent
anticarcinogenic effects in CRC cells [117,118].
Similarly to quercetin, EGCG shows a biphasic response
on CRC cell proliferation [12,59,112,119]. This issue might
be explained by several facts: (1) at low concentrations,
EGCG activates MAPK pathways, eliciting survival of CRC
cells, whereas at high concentrations, it activates the caspase
pathway, leading to apoptosis [12,119]; (2) overcorrection
mechanisms in the presence of low, growth-inhibiting
concentrations of EGCG [56]; (3) modulation of different
phases of the cell cycle in response to different concentra-
tions of EGCG [57]; and (4) the balance between its pro-
oxidant and antioxidant activity [33,86,119].
The flavan-3-ols with a galloyl moiety in their structure,
with EGCG being the most representative one, have a more
pronounced chemopreventive effect against CRC cells [69]
compared to those without a galloyl moiety, such as
catechin and epicatechin. These 2 flavan-3-ols do not
display [73] or display only a moderate growthinhibitory
[12,70,72] and apoptotic effect [12,70] (Table 1). Indeed,
growth inhibition and apoptosis of CRC cells were found
only with high concentrations (100 mol/L) of catechin
and epicatechin [72]. Because of the magnitude of these
82 J.R. Arajo et al. / Nutrition Research 31 (2011) 7787
concentrations, generation of H
2
O
2
may be the explanation
for these effects [69].
8. Resveratrol
The phytoalexin resveratrol (3,5,4-trihydroxy-trans-
stilbene) is found largely in grape products [21,120], red
wine being its major source for human consumption
[17,21,120], followed by peanuts, blueberries, and cranber-
ries [7]. Its antitumor activity was first recognized in 1997
when it was shown to block all 3 major stages of
carcinogenesis induced by the polynuclear aromatic hydro-
carbon dimethylbenz(a)anthracene [120]. More recently, its
antitumor activity has been confirmed in human cell lines
[7,17,35,43,78,79,90] and animal models of CRC [121,122].
In CRC cell lines, resveratrol (a) showed growth-inhibitory
activity [17,43,79]; (b) induced differentiation [120]; (c)
arrested cell proliferation [79-81] and neoplastic transfor-
mation [7,75]; (d) arrested the cell cycle in the S, G
1
/S
[43,52,79,81], G
2
/M [52,57,81] or, less frequently, in S/G
2
phase of cell cycle [81]; (e) induced apoptosis [7,79,80]; and
(f) presented antiangiogenic, anti-invasion, and antimeta-
static characteristics [43] (Table 1). Concentrations required
for resveratrol-induced apoptosis (100-200 mol/L) are
usually higher than those required to induce growth
inhibition and cell cycle arrest (10-100 mol/L) [79].
However, at lower concentrations (b1-10 mol/L), resver-
atrol targets multiple intracellular pathways responsible for
the aforementioned effects, including (1) inhibition of Cox-1
and Cox-2 [78,120], hydroperoxidase [120], and iNOS [7]
activities [18]; (2) inhibition of matrix metalloproteinase
isoform 2 and 9 expressions [7]; and (3) down-regulation of
DNA polymerase, ribonucleotide reductase, ornithine decar-
boxylase [79], and telomerase [35,77]. Resveratrol was also
found to (4) down-regulate CDKs 2, 4, 6, and 7 and p34cdc2
[57,79,81,123] and their activators (cyclins A, D1, D2, and E
and -catenin [57,79,81]), while up-regulating their inhibi-
tors, tumor suppressor p53 transcription factor, and their
responsive genes (p21, p21
WAF1
, p27, p27
KIP1
, p300/cyclic
adenosine monophosphate response element binding protein
(CREB), apoptotic peptidase activating factor 1, Bak, Bax,
PUMA, Noxa, and Bim), and Rb/E2F cell cycle pathway
[7,35,43]. Still, other effects include (5) down-regulation of
Bcl-2, Bcl-xL, myeloid cell leukemia 1, X-linked inhibitor of
apoptosis, and interleukin 6 [7,43,76] and decrease of
survivin (inhibitor of apoptosis protein [IAP] family
member) levels [46] and (6) up-regulation of poly (ADP-
ribose) polymerase [16,35], caspase 3 and caspase 9 [45,81],
Fas ligands [74], ceramide biosynthesis [16,35], proteasome
activity [102], and cytochrome C release [7,43,45].
Resveratrol is also capable of modulating MAPK
transduction pathways (it up-regulates c-Jun, NH
2
-terminal
kinases 1 and 2, and p38 but down-regulates ERK-1 and
ERK-2 [124]); down-regulates Src tyrosine kinase [7,43],
focal adhesion kinase, protein kinase C isoform and ,
protein kinase B, kappa B kinase inhibitor [74], and the
checkpoint kinases ataxia telangiectasia mutated and ataxia
telangiectasia and Rad3 related [43]; down-regulates the
transcriptional factors early growth response protein 1,
activating protein 1 transcription factor [7], c-Jun, c-MYC, c-
Fos [43], and p65 NF-B subunit [7]; and inhibits epidermal
growth factor, VEGF [16,43], and EGFR activation [45].
Some authors argue that resveratrol has high-lipid solubility,
so its major targets might be membrane components such as
tyrosine kinases [74].
By exerting an antiestrogenic action [16], resveratrol can
potentially inhibit estrogen-induced tumoral proliferation and
transformation [7]. Nevertheless, it must be taken into
account that depending on its concentration, cell type, and
ERs expression, this phytoestrogen [7] can act as an
antagonist and/or agonist [75] of both - and -ERs [7,35].
9. Xanthohumol
Xanthohumol is the most abundant prenylated chalcone
in hops extract [125], beer being its major source of dietary
consumption [82]. Xanthohumol has been described as a
broad-spectrum chemopreventive agent [83,126,127] in
various cell- and enzyme-based in vitro bioassays
[45,78,82-84,87,126,127], but only a few were performed
in CRC cell lines (HT-29, HCT-116, CCL 220.1 [82,83], and
Caco-2 cells [84]) (Table 1). In enzymatic assays, xantho-
humol was able (a) to modulate the activity of several
enzymes involved in carcinogen metabolism, namely, by
inhibiting CYP1A, CYP1B1, CYP1A2, CYP3A4, and
CYP2E1 and by inducing NAD(P)H:quinone reductase; (b)
to scavenge reactive oxygen species production, including
hydroxyl, peroxyl [78], and superoxide anions [78,83]; and
(c) to decrease inflammation by inhibiting Cox-1 and Cox-2
activity [45], nitric oxide production [45,78], and TNF-
secretion [126]. In the assays performed in cancer cells,
xanthohumol induces apoptosis [82,83,87], cell cycle arrest,
particularly in S, S/G
2
, or at the end of G
1
(subG
1
peak)
phase [82,83], and terminal differentiation [83,126], and
inhibits cell growth and proliferation [82,83] (through DNA
polymerase activity inhibition) [126], invasiveness [87],
and angiogenisis [78,128]. In addition, xanthohumol also
demonstrates potent antiestrogenic properties, without
possessing intrinsic estrogenic potential [45,126], and
inhibits P-glycoprotein [82].
At the transcriptional level, chemopreventive properties
of xanthohumol include down-regulation of p53/p53
negative regulator Mdm2 network and of Bcl-2 [83];
inhibition of NF-B activity; up-regulation of Bax [82,87]
and of caspases 3, 7, 8, and 9; and induction of poly (ADP-
ribose) polymerase cleavage [83].
10. Combination of polyphenols
Rather than a single compound, combinations of poly-
phenols may target overlapping and complementary phases
83 J.R. Arajo et al. / Nutrition Research 31 (2011) 7787
of the carcinogenic process [7,21,45], thus increasing the
efficacy and potency of the chemopreventive effect [7,45,62].
For instance, a synergistic effect when EGCG is used in
combination with other flavan-3-ols, such as epicatechin
[45,70], toward inhibition of cell growth and proliferation
and induction of apoptosis in CRC cells, has been
documented [70,71]. The same synergic effect occurs when
EGCG is combined with therapeutic drugs, such as
nonsteroidal anti-inflammatory drugs (sulindac and tamox-
ifen) [71] or with drugs used in chemotherapy (5-fluorouracil,
oxaliplatin, or paclitaxel) [70]. In other cell types, resveratrol
and quercetin interact in more than an additive manner in the
activation of caspase 3 and cytochrome C release, inducing
apoptosis [129]; in inhibition of cell growth and DNA
synthesis [130]; and in arrest of cells in G
0
/G
1
and S phase of
cell cycle [52].
11. Polyphenols and BT: impact in CRC
The short-chain fatty acid BT is one of the main end
products of bacterial fermentation of dietary fiber within the
human colon [131]. Butyrate plays a key role in colonic
epithelium homeostasis because it is the main energy source
for colonocytes [132] and exhibits various anticarcinogenic
properties [26,118,133]. The ability of BT to exert these
effects depends on its intracellular concentrations, which
requires its absorption into colonocytes [134]. Recently, our
group demonstrated that long-term exposure to quercetin,
EGCG, rutin, and chrysin increased the uptake of BT and
that long-term exposure to chrysin increased the messenger
RNA expression levels of its transporter monocarboxylate
transporter 1 (H
+
-coupled monocarboxylate transporter 1;
MCT1) in human adenocarcinoma Caco-2 cells [47,59]. This
effect on BT uptake may constitute an additional mechanism
by which polyphenols might protect against CRC.
12. Conclusion
Polyphenols are promising chemopreventive agents for
CRC management because they restore normal cell growth
by modulating proliferation, apoptosis, angiogenesis, metas-
tasis, and inflammation and by targeting multiple molecular
and biochemical pathways implicated in tumor development.
However, caution is mandatory when attempting to extrap-
olate observations obtained in CRC cell line studies to
humans because none of these experimental features have
been proved to occur among humans yet [12]. Indeed,
variables such as the CRC cell line used; the concentrations
of polyphenol tested (which are usually much higher than
those achievable in the human body [17,135]); the stability
and/or oxidation of polyphenols, which are redox-sensitive
compounds; and the time of cell exposure to the polyphenol
as well as other factors should be considered when
extrapolating cell culture experimental results [135].
Despite the evidence provided by cell culture studies
presented in this article, the molecular mechanisms respon-
sible for the anticarcinogenic effect of polyphenols,
particularly at physiologic doses, are not completely clarified
[12,17,85]. To overcome this issue, investigation on this
subject should focus more on (a) studies about the combined
chemopreventive effects of different polyphenols and of
polyphenols and therapeutic drugs in cell culture and animal
models and clinical trials [12]; (b) studies about polyphenol
bioavailability (eg, intestinal absorption and metabolism) in
animal models, to better elucidate the chemopreventive
properties of its intestinal metabolites (particularly the
conjugated ones) [10,78]; and (c) further clinical studies in
human subjects to fully confirm and quantify the bioavail-
ability, safety, efficacy, and chemopreventive properties of
polyphenols [18].
Acknowledgment
This work was supported by Fundao para a Cincia e a
Tecnologia and Programa Cincia, Tecnologia e Inovao do
Quadro Comunitrio de Apoio (PTDC/SAU-FCF/67805/
2006) and iBesa (Instituto de Bebidas e Sade)/UNICER
bebidas SA.
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