Impaired Expression and Function of Cancer-Related Enzymes by

Anthocyans: An Update

Roberto Bei1,*, Camilla Palumbo2, Laura Masuelli2, Mario Turriziani3,

Giovanni Vanni Frajese4, Giovanni Li Volti5, Michele Malaguarnera5

and FabioGalvano5

1Department of Experimental Medicine and Biochemical Sciences,

University “Tor Vergata”, Rome, Italy; 2Department of Experimental

Medicine, University “Sapienza”, Rome, Italy; 3Department of

Internal Medicine, University “Tor Vergata”, Rome, Italy; 4Faculty

of Motor and Adaptive Sciences, University of Cassino, Cassino,

Italy; 5Department of Biological Chemistry, Medical Chemistry and

Molecular Biology, University of Catania, Catania, Italy

*Address correspondence to this author at Department of

Experimental Medicine and Biochemical Sciences, University “Tor

Vergata”, Via Montpellier 1, 00133 Rome, Italy; Tel:+39-06-

72596514; Fax:+39-06-72596506; E-mail: bei@med.uniroma2.it

1

Abstract

Anthocyans (ACNs), i.e. anthocyanins and anthocyanidins, belong to

a widespread group of plant constituents, collectively known as

flavonoids, which occur in the western diet at relatively high

concentrations. ACNs display a variety of pharmacological

properties which make them potential anti-inflammatory and anti-

cancer agents. In addition to their ability to scavenge reactive

oxygen species, ACNs can affect the functions of enzymes involved

in DNA damage and in cancer-related signaling pathways. The

antiproliferative, proapoptotic and antiangiogenic effects of ACNs

rely on the inhibition of signaling by tyrosine kinase growth

factor receptors (EGFR/ErBs, c-Met, VEGFRs, Met receptor, PDGFRs)

as well as on the impairment of cAMP phosphodiesterase, proteasome

chymotrypsin–like, ornithine decarboxylase and glyoxalase I

activity. ACNs interfere also with cancer cell invasion by

lowering the expression of the urokinase-type plasminogen

activator and matrix metalloproteinases. Further, these compounds

have been found to affect the transcriptional activity of NF-B by

inhibiting the IκB kinase complex and histone acetyltransferases,

the inhibition of NF-B being closely linked with the

downregulation of COX-2 expression. Finally, ACNs are regarded as

2

multi-target kinase inhibitors due to their ability to bind and

inhibit a number of signaling kinases, such as Raf, MEK, MAPKK4,

PI3-K and Fyn. This review will provide an update on the effects

of ACNs on the expression and function of enzymes involved in

cancer development and progression, and will discuss the

preventive/therapeutic potential of these compounds against human

cancers.

3

1. ENZYMES AND SIGNALING PATHWAYS IN CANCER DEVELOPMENT AND

PROGRESSION: AN OVERVIEW

Carcinogenesis, a multi-step process triggered by cumulative

genetic alterations, drives the progressive transformation of a

normal cells into a cancer cell. The development of a malignant

tumor consists of: a) tumor cell initiation, regarded as an

irreversible short term process caused by DNA damage and

mutations, b) tumor promotion, considered a reversible long term

process in which genetically modified cells are stimulated to grow

and finally, c) tumor progression, in which genetically unstable

cancer cells gain a more aggressive phenotype and eventually

acquire the ability to invade and metastasize [1, 2] (Fig. 1).

The outcome of DNA damage induced by the exposure to chemical

carcinogens, radiant energy or biological agents is conditioned by

the activity of DNA repair enzymes. Further, most chemical

carcinogens require metabolic activation by xenobiotic

metabolizing enzymes in order to exert their effects [1, 3]. DNA

mutations can lead to the overexpression or hyperactivation of

genes which support cell survival and proliferation (oncogenes)

4

or, viceversa, to the loss of expression or functional inactivation

of genes which negatively affect cell proliferation or are able to

induce cell death (tumor suppressor genes). As a consequence,

while growth and survival of normal cells is regulated by

exogenous stimuli, cells bearing mutations in oncogenes and/or

tumor suppressor genes can proliferate in the absence of exogenous

signals and become unresponsive to negative regulators of growth

and survival [1]. For instance, the overexpression of growth

factors and/or their receptors can cause the constant activation

of downstream signaling pathways leading to cell growth and

survival [1]. Besides, growth factor receptors and the downstream

signal transduction effectors or transcription factors can be

directly activated by mutational events [1].

Upon ligand binding, growth factor receptors of the tyrosine

kinase superfamily [epidermal growth factor receptor (EGFR),

platelet derived growth factor receptors (PDGFRs), vascular

endothelial growth factor receptors (VEGFs), fibroblast growth

factor receptors (FGFRs), etc.] undergo dimerization and trans-

phosphorylation. These events lead to the activation of the

receptor tyrosine kinase which then activates by phosphorylation

the downstream effector enzymes phospholipase C gamma (PLCγ),

5

phosphatidylinositol 3-kinase (PI3K) and Src, as well as the

adaptor proteins Grb2 and Shc, which contain multiple protein

interaction motifs able to recruit and activate the Ras GTPase [4,

5]. PLCγ activity results in the formation of diacylglycerol,

which triggers the serine-threonine kinase protein kinase C (PKC),

that in turn activates various transcription factors. PI3K

activates the kinase Akt (protein kinase B), which is involved in

cell proliferation and inhibition of apoptosis [4]. Ras proteins

directly interact with the Raf kinases converting them from

inactive to active enzymes. Upon activation, Raf kinases start a

phosphorylation cascade, which triggers the classical mitogen-

activated protein kinases (MAPKs) signaling pathway: Raf activates

the MAP kinase kinase 1 (MEK1 or MAPKK1), which in turn activates

the extracellular signal-regulated kinases 1 and 2 (ERK1/2). The

ERKs translocate to the nucleus and activate several transcription

factors, including activator protein-1 (AP-1), c-Myc, etc. [4-8].

The MAPK/ERK pathway represents one of the major signaling

cascades regulating cell proliferation. Growth factors, cytokines

and cellular stress can also activate different MAPK cascades via

other MEKs/MAPKKs isoforms, which phosphorylate and activate the

c-Jun amino-terminal kinases (JNK1/2/3), p38 and ERK5. JNK and p38

6

kinases mainly regulate cell differentiation and apoptosis [4, 5,

9, 10]. Given the established role of abnormal MAPKs activation in

tumor cell proliferation, survival and metastasis, many efforts

are currently made to identify drugs which inhibit their signaling

pathway [5]. The principal tyrosine kinase receptor-mediated

signaling pathways are illustrated in Fig. (2), Panel A. As for

the signaling pathways initiated by receptors without intrinsic

tyrosine kinase activity, these involve the recruitment of kinases

such as JAK (janus kinase), able to activate cytoplasmic

transcription factors named STATs (signal transducers and

activators of transcription), which translocate to the nucleus and

mediate gene transcription. Non-tyrosine kinase receptors include

growth factor as well as cytokine receptors [11].

The transcription factor NF-κB is involved in several aspects of

cancerogenesis such as cell transformation, tumor cell

proliferation, invasion, metastatization and neoangiogenesis [12-

14]. NF-B is a homodimeric or heterodimeric transcription which

can be composed by five subunits (RelA/p65, c-Rel, RelB, NF-

κB1/p50, and NF-κB2/p52). It is normally sequestered in an

inactive state in the cytoplasm by the inhibitors of NF-κBs

(IκBs). Activation of the IκB kinase (IKK) complex, which

7

comprises two catalytic (IKKα and IKKβ) and one regulatory

(IKKγ/NEMO) subunit, leads to phosphorylation of the IκBs,

targeting them for ubiquitination and proteasomal degradation. The

degradation of IκBs allows NF-B to translocate to the nucleus,

where it activates several genes involved in inflammation, cell

growth and invasivity [15]. In fact, the activation of NF-B

represents an important link between inflammation and

tumorigenesis and is triggered by growth factors, cytokines and

multiple stress stimuli [12-16] (Fig. 2, Panel B).

Other signal transduction pathways regulating cell proliferation

and survival involve the 3', 5'-cyclic monophosphate (cAMP) as

second messenger. It is synthesized by adenylyl cyclase from ATP

in response to a wide range of signals, for instance upon

activation of G-protein-coupled receptors (GPCR), and is

hydrolyzed by cyclic nucleotide phosphodiesterases (PDEs) to

adenosine 5’-monophosphate [17, 18]. Remarkably, depending on the

cell type, cAMP can either stimulate or inhibit cell growth and

survival [18-20]. Still, fast proliferating cancer cells are

frequently characterized by low levels of cAMP and overexpression

of PDEs [21]. Although this cyclic nucleotide may activate

different proteins, its best known mode of action is through

8

binding to cAMP-dependent protein kinases (PKA). PKA activity can

result either in the stimulation of B-Raf or in the inhibition of

Raf-1, thus modulating the activation of MAPKs. Besides, the PKA-

dependent cAMP signaling for proliferation involves activation of

the transcription factor cAMP response element (CRE)-binding

protein (CREB) [18-20] (Fig. 2, Panel C).

In addition to growth and survival signals, the tumor

microenvironment furnishes molecules which sustain the production

of new vessels and recruitment of inflammatory cells. The

formation of new vessels, termed neoangiogensis, is a critical

phase in cancer progression and is promoted by different growth

factors, including VEGF, basic FGF (bFGF) and PDGF [22]. Some

neoangiogenic factors are stored in the extracellular matrix and

can be released and activated by matrix metalloproteinases (MMPs).

In addition, the extracellular matrix-degrading activity of these

enzymes favours the migration and spreading of cancer cells [22].

Finally, chronic inflammation appears to have a versatile function

in the carcinogenesis process. Indeed, a long-lasting inflammation

can contribute to cancer initiation through the production of

reactive oxygen and nitrogen species (ROS and RNS) with DNA-

damaging properties. Moreover it can participate in cancer

9

promotion and progression by increasing the availability of

mediators (growth factors, cytokines, chemokines, prostaglandins)

which contribute to the growth of initiated cells and to

neoangiogenesis [23, 24].

2. ANTHOCYANS

Cancer chemoprevention aims at arresting, inhibiting, reversing or

delaying the carcinogenesis process in normal and/or high risk

populations through the administration of synthetic molecules or

dietary compounds [25]. The anti-cancer properties of

chemopreventive agents rely on several mechanisms which can affect

both cancer development and progression by modulating different

signaling pathways. Polyphenols are among the most promising

dietary compounds with high chemopreventive potential [26, 27].

Among polyphenols, anthocyans (ACNs) [from Greek: (anthos) =

flower + (kyanos) = blue] are a group of pigments belonging to the

very large and widespread class of plant constituents collectively

known as flavonoids. ACNs include anthocyanins and anthocyanidins.

Chemically, anthocyanins are water-soluble glycosides or

acylglycosides of anthocyanidins [28, 29]. Anthocyanidins are

10

polyhydroxy and polymethoxy derivates of 2-phenylbenzopyrylium or

flavylium salts and contain two benzoyl rings (A and B) separated

by a heterocyclic (C) ring [29-31]. Anthocyanidins are unstable to

light and poorly soluble in water, and hence they do not usually

occur in their free state [32, 33]. More than 550 ACNs have been

identified to date, with molecular weights ranging from 400 to

1200 as a consequence of the differences related to the number of

hydroxyl groups, the degree of methylation of these hydroxyl

groups, the nature, number, and location of sugars attached to the

molecule, the number and nature of aliphatic or aromatic acids

attached to the sugars in the molecule [29, 32]. On the other

hand, only six anthocyanidins, i.e. cyanidin, delphinidin,

petunidin, peonidin, pelargonidin, and malvidin, are widespread in

plants (Fig. 3), commonly being bonded to sugars such as glucose,

galactose, arabinose, rhamnose and xylose in the form of mono-,

di- or trisaccharides [28, 31]. Cyanidin and cyanidin-3-glucoside

are the most widespread ACNs [32].

ACNs confer to plants, leaves and fruits different pigmentations

ranging from purple red to blue, depending on the substitution

pattern of the B ring of the aglycone, the pattern of

glycosylation, the degree and nature of esterification of the

11

sugars with aliphatic or aromatic acids, and, above all, the pH

[28, 31]. The ability to form flavylium cations distinguishes ACNs

from other classes of flavonoids. At pHs lower than 2 ACNs exist

primarily in the form of the red (R1 = O-sugar) or yellow (R1 = H)

flavylium cation. As the pH increases a rapid proton loss occurs

and a series of equilibria take place leading to colourless

compounds via hydration of the flavylium cation or its

tautomerization followed by isomerization [31]. Thus, depending on

the pH the biological properties of ACNs are likely to vary, which

complicates the definition of the pharmacological effects of these

compounds [31].

The marked daily intake of ACNs (180 to 215 mg/day in the United

States), which is much higher than the intake (23 mg/day)

estimated for other flavonoids including quercetin, kaempferol,

myricetin, apigenin and luteolin, makes these compounds of great

nutritional interest [28]. The most important food sources of ACNs

are berry fruits (Vaccinium and Rubus spp.), cherries, red grapes

and currant, red wines, blood oranges, the black varieties of

soybean, rice and beans and the red varieties of onions, potato

and cabbage [28, 30]. In addition, due to their intense

coloration, ACNs are frequently used as food additives [29].

12

In the last years a great attention was given to the possible

protection exerted by natural antioxidants present in dietary

plants. ACNs are included in the list of natural compounds known

to work as powerful antioxidants, have been reported to have

positive effects in the treatment of various diseases and are

prescribed as folk medicines in many countries [30]. Indeed,

because of their electron deficiency ACNs are very reactive

towards reactive oxygen species (ROS), which are largely

demonstrated to be involved in carcinogenesis as well as in the

pathogenesis of many chronic degenerative diseases [29, 30, 34].

However, it is current opinion that the ACN antioxidant properties

per se cannot thoroughly explain their beneficial effects and that

additional biochemical mechanisms are likely triggered by these

compounds, for instance the modulation of enzymes expression and

activity [29].

This review will provide an update on the effects of ACNs on the

expression and function of enzymes involved in cancer development

and progression (Table 1) and will discuss the

preventive/therapeutic potential of these compounds against human

cancers [35] (Fig. 1).

13

3. ANTHOCYANS’ EFFECTS ON ENZYMES INVOLVED IN DNA DAMAGE OR

MAINTENANCE OF GENOME INTEGRITY

3.1. Inhibition of cytochrome P450 (CYP)

In a paper by Gasiorowski et al. it was reported that ACNs dose-

dependently inhibited the mutagenic activity of the microsomal

oxidoreductase-activated promutagens benzo[a]pyrene and 2-amino

fluorene in in vitro assays [36]. The authors proposed that the

antimutagenic effect of ACNs was exerted through both their free

radicals scavenging action and the inhibition of enzymes

activating promutagens to DNA-reacting derivatives [36]. The

cytochrome P450 (CYP) superfamily encompasses a large group of

drug-metabolizing enzymes involved in the biotransformation of

carcinogens to either biologically inactive metabolites or to

electrophilic metabolites that bind to DNA producing

carcinogenicity [37]. Several flavonoids have been shown to

modulate CYP enzymes expression and/or activity [37, 38]. CYP3A4,

the most abundant CYP isoenzyme in human liver and

gastrointestinal tract, participates in the metabolic activation

of many environmental carcinogens [37, 39]. Dreiseitel et al.

demonstrated that CYP3A4 activity is dose-dependently inhibited by

14

anthocyanidins, anthocyanins and the ACN precursors procyanidins

B1 and B2, which are also common constituents of many foods [38].

Interestingly, the number of sugar moieties of the tested ACN

compounds was found to predict a decline in the inhibitory effect

on CYP3A4 [38] (Table 1). Besides its role in the metabolic

activation of procarcinogens, CYP3A4 is also involved in the

biotransformation of several anticancer drugs, resulting either in

drug inactivation (e.g. irinotecan, docetaxel), or in the

production of more potent drug metabolites (e.g. from

cyclophosphamide and isofosfamide) [39, 40]. Accordingly,

inhibition of CYP3A4 by ACNs might bear relevance both in cancer

prevention and in modulating, either positively or negatively, the

efficacy of specific anticancer treatments [39, 40].

More recently, anthocyanins and anthocyanidins have also been

reported to act as moderate inhibitors of CYP2C19 and weak

inhibitors of CYP2D6. These two CYP isoforms are involved in the

metabolic activation of some anticancer drugs, such as

cyclophosphamide and tamoxifen, but play a minor role in the

activation of procarcinogens [39, 41, 42]. CYP2C19 and CYP2D6 are

expressed in hepatocytes, intestinal epithelial cells and other

15

extrahepatic cell types, although at lower levels as compared to

CYP3A4 [39, 43].

3.2. Inhibition of topoisomerase activity

The superstructure of the DNA is affected by processes such as DNA

replication, transcription, recombination, repair, chromatin

remodelling, chromosome condensation and segregation.

Topoisomerases regulate DNA supercoiling by introducing temporary

single- (topoisomerase I) or double-strand breaks (topoisomerase

II, isoforms and ) in the phosphodiester DNA backbone. After

the release of the tortional stress in the DNA molecule, the

cleaved strand/strands of the DNA are religated. DNA cleavage by

topoisomerases is accompanied by the formation of a transient

phosphodiester bond between a tyrosine residue in the protein and

one of the ends of the broken strand, the covalent DNA-

topoisomerase intermediate being called the cleavable complex [44,

45].

Mutant or abnormal topoisomerase activities cause defects in

chromosome recombination and segregation, affect genome integrity

and can result in cell death or neoplastic transformation [44, 46,

47]. Several anticancer agents are topoisomerase poisons that

16

generate cytotoxic lesions by binding and stabilizing the

cleavable complex, thus impairing the religation of the DNA

strand/s [44, 48]. This can cause aberrant recombination events

and lead to aneuploidy by interfering with chromosome segregation.

In fact, topoisomerase-targeted anticancer drugs act as potent

carcinogens themselves [48]. In this regard, cancer treatment with

topoisomerase II poisons has been found to be sporadically

associated with the development of human leukemias with

chromosomal translocations involving the MLL (mixed lineage

leukemia) gene at chromosome band 11q23 [49]. Further, the

consumption during pregnancy of foods rich of substances acting as

topoisomerase II poisons increases more than three-fold the risk

of developing infant leukemias with MLL translocations [50].

Several studies have been performed to investigate whether ACNs

may act as topoisomerase poisons, since such property would

represent a serious limitation to their use as chemopreventive

agents [45, 51-53]. Fridrich et al. reported that oligomeric

procyanidins inhibited the catalytic activity of topoisomerases I

and II in cell free systems starting at a concentration of 0.5 M

[45]. According to Habermeyer et al., delphinidin and cyanidin

strongly inhibited the catalytic activity of human topoisomerases

17

I and II at concentrations in the low micromolar range [52] (Table

1). Of note, the effects reported for delphinidin and cyanidin,

bearing vicinal hydroxyl groups at the B-ring, were not induced by

peonidin, pelargonidin and malvidin. Indeed, vicinal hydroxyl

groups at the B ring appear to be required for efficient

topoisomerase targeting by flavonoids [45, 52]. On the other hand,

other authors reported that ACNs inhibited the activity of

topoisomerase I only when present at relatively high

concentrations (>50 M) [54]. In any case, delphinidin and

cyanidin appeared to act as topoisomerase catalytic inhibitors

rather than affecting the stability of the cleavable complex [52,

53, 55]. Moreover, at concentrations of 1-10 M they prevented the

stabilization of the cleavable complex by the topoisomerase I

poison camptothecin, thus inducing a protective effect against the

DNA-damaging effects of this drug [52]. Delphinidin (at 10 M)

was also found to exert a similar protective effect toward the

DNA-damaging properties of the toposoisomerase II inhibitors

doxorubicin and etoposide [53]. In addition, the ability of

cyanidin-3-glucoside and cyanidin (100 M) to decrease DNA damages

induced by camptothecin, and the ability of cyanidin (100 M) to

decrease DNA damages induced by doxorubicin were also reported

18

[55]. These protective effects of ACNs may be beneficial, since

compounds acting as topoisomerase poisons are normally introduced

with the diet, but may also compromise the therapeutic efficacy of

anticancer drugs with topoisomerase poison activity [52, 53].

Still, Habermeyer et al. also demonstrated that anthocyanidins can

cause DNA strand breaks, but only when used at high concentrations

(50 M) [52]. In this regard, we have recently demonstrated that

at doses ≥25 M cyanidin-3-glucopyranoside, as well as cyanidin

chloride, induce DNA fragmentation in human colon carcinoma cells

[56]. However, the DNA-damaging properties of ACNs have recently

been challenged. Indeed, it has been demonstrated by different

authors that in the presence of catalase, which suppresses the

accumulation of hydroxide peroxide in cell culture medium or in in

vitro assays solutions, ACNs do not cause DNA strand breaks when used

at concentrations up to 100 M [54, 55].

4. INHIBITION OF CANCER CELL GROWTH AND SURVIVAL BY ANTHOCYANS

The ability of ACNs to inhibit cancer cell growth and survival is

documented by a large number of preclinical studies [28, 57-75].

19

As reported and discussed below, the molecular mechanisms involved

in these biological effects of ACNs encompass the modulation of a

wide array of enzymes, many of which participate in signal

transduction pathways (Fig. 2) (Table 1). Depending of their

specific structure, ACNs can affect different signaling elements.

4.1. Inhibition of receptor tyrosine kinase activity

Impaired activity of tyrosine kinase receptors associated with

that of downstream signaling effectors has often been reported

upon cell treatment with ACNs (Fig. 2, Panel A). These effects of

ACNs appear to rely on the direct inhibition of the receptor

catalytic activity [76-79].

4.1.1. EGFR receptor family

Epidermal growth factor receptor (EGFR) family members, including

EGFR, ErbB2 (Neu, HER2), ErbB3 and ErbB4 play a critical role in

cancer development [80]. Aberrant activation of EGFR family

members, in particular EGFR, has been shown to be frequently

involved in malignant transformation and in cancer cell

survival/proliferation as a consequence of receptor

overexpression, mutation or autocrine stimulation [77, 78, 80].

20

Among ACNs, cyanidin and delphinidin, bearing vicinal hydroxyl

groups at the B ring, have been found to act as potent inhibitors

of the kinase activity of EGFR, while the methoxylated analogue

malvidin has been reported to be much less effective [66, 76, 79].

As a general rule, the presence of vicinal hydroxyl groups at the

B ring rather than metoxy groups appears to be required for

inhibition of a broad panel of receptor tyrosine kinases,

including EGFR family members as well as VEGFR family members

[79].

Meiers et al. firstly reported that cyanidin and delphinidin

inhibited the tyrosine kinase activity of EGFR in cell-free kinase

assays (their IC50 values being, respectively, 0.8 and 1.3 M),

while malvidin, carrying methoxy groups at the B ring, did not

affect enzyme activity. The glycoside cyanidin-3-galactoside was

inactive as well. Moreover, cyanidin and delphinidin were more

potent than malvidin in inhibiting the growth of vulva carcinoma

cells overexpressing the EGFR, while cyanidin-3-galactoside had no

effect on cell growth at concentrations up to 100 M. Of note, the

anthocyanidin concentration range for EGFR inhibition in cell-free

assays was more than an order of magnitude lower as compared to

that required for cell growth inhibition, probably as a result of

21

cellular pharmacokinetics [76]. The delphinidin-induced inhibition

of EGFR and downstream signaling pathways was also reported by

Afaq et al. In their study, delphinidin treatment (5-40 μM) of

breast carcinoma cells produced a dose-dependent decrease of the

constitutive as well as EGF-induced phosphorylation of specific

EGFR tyrosine residues (sites 1068, 1045 and 845), without

affecting total EGFR levels [81]. Impairment of EGFR signaling by

delphinidin was reported to involve the inhibition of the EGF-

induced activation of PI3K, Akt and MAPKs (ERK1/2, JNK1/2 and

p38). These molecular events were associated with the reduction of

EGF-induced cell invasion as well as with growth inhibition and

induction of apoptosis [81] (Fig. 2, Panel A). Fridrich et al.

provided evidence that delphinidin, but not malvidin, can suppress

the phosphorylation of both EGFR and ErbB2 in human carcinoma cell

lines, with IC50 values in the range of 50-70 µM [82]. Recently,

Teller et al. corroborated and expanded these findings. In fact,

their studies confirmed the ability of anthocyanin-rich mixtures

(i.e. mirtocyan from bilberries and oenocyanin from grapes) and

purified delphinidin to inhibit the kinase activity of EGFR and

ErbB2 in cell-free assays as well as to decrease the ligand-

induced autophosphosphorylation of EGFR, ErbB2 and ErbB3 in intact

22

cells [78, 79]. The IC50 values for the delphinidin-induced

inhibition of EGFR, ErbB2 and ErbB3 autophosphorylation were,

respectively, 72, 51 and less than 0.1 M. Thus, delphinidin

showed a preference for the suppression of ErbB3 activity.

Moreover, delphinidin also reduced the protein levels of ErbB3,

but not those of EGFR and ErbB2. Worthy of note, ErbB3 is a major

upstream activator of the PI3K pathway, which in turn plays a key

role in cell survival [79].

4.1.2 c-Met Receptor

Hepatocyte growth factor (HGF) and its receptor tyrosine kinase,

named c-Met, signal multiple biological activities, including cell

proliferation, survival and motility [83, 84]. Activation of c-Met

oncogenic signaling relies on abnormal autocrine/paracrine HGF

production as well as on receptor mutation or overexpression [83].

The transmembrane Met β-chain contains the kinase domain and a

multifunctional docking site which, upon tyrosine phosphorylation,

recruits transducer and adaptor molecules that couple c-Met

activation to several signaling pathways [85, 86] (Fig. 2, Panel

A). In a study performed using immortalized human breast

epithelial MCF-10A cells, Syed et al. reported that delphinidin (5–

23

40 μM) inhibited the HGF-induced phosphorylation of c-Met and the

activation of its two major downstream pathways, i.e. the

Ras/MAPK/ERK and the PI3K/Akt pathways [86]. Total c-Met protein

levels were also reduced by delphinidin. Furthermore, delphinidin

treatment resulted in a decrease of HGF-induced phosphorylation of

IKKα/β and their target IκBα, in the reduced activation and

nuclear translocation of NF-κB/p65 and in the inhibition of NF-κB

promoter activity [86].

4.2. Impairment of cAMP phosphodiesterase (PDE) activity

Cyclic nucleotide phosphodiesterases (PDEs) are cAMP- and cGMP-

hydrolyzing enzymes ubiquitously distributed in mammalian tissues.

PDEs are key enzymes in the maintenance of cAMP homeostasis and

play an important role in the regulation of cAMP signaling

cascades [87]. Depending on the cell type and stimulating ligand,

cAMP can either inhibit or stimulate MAPK signaling and cell

proliferation/survival [18-20]. On the other hand, low levels of

cAMP are frequently found in fast proliferating cancer cells in

association with PDE overexpression and PDE inhibitors have been

reported to induce apoptosis and cell cycle arrest in a broad

24

spectrum of tumor cells [21, 88]. Accordingly, PDEs are currently

under investigation as potential targets for cancer therapy [21].

Marko et al. proved that malvidin, bearing methoxy substituents in

the 3´- and 5´-position (B ring), inhibited cAMP hydrolysis (IC50

value of 23 µM) in lysates of a human colon cancer cell line.

Further, they observed that inhibition of PDE activity by

anthocyanidis was affected by their substitution pattern. Indeed,

the PDE-inhibitory properties of anthocyanidins lacking methoxy

groups and/or bearing vicinal hydroxyl groups at the B ring were

much lower as compared to those of malvidin [66] (Table 1).

Interestingly, the opposite structure-activity relationship was

reported for the inhibition of the EGFR by anthocyanidins [66].

According to these authors, inhibition of PDEs by malvidin was

associated with a significant reduction of colon cancer cell

growth. However, the relevance of PDE inhibition for the

antiproliferative properties of malvidin in the same cancer cell

line was questioned by other authors [17].

4.3. Inhibition of ornithine decarboxylase (ODC) expression and

activity

25

Ornithine decarboxylase (ODC), which decarboxylates ornithine to

form putrescine, is the first and rate-limiting enzyme in

polyamine biosynthesis. An increased ODC activity has been

demonstrated to play a significant role in tumor development and

progression [89]. Indeed, high polyamine levels are associated

with decreased apoptosis as well as with increased cell

proliferation and expression of genes promoting tumor invasion and

metastatization [89]. Although the inhibition of polyamine

biosynthesis appears to be generally ineffective as an anticancer

strategy, it could have significant potential in cancer

chemoprevention [89].

Bomser et al. reported that anthocyanidins extracts from fruits of

four Vaccinium species (lowbush blueberry, bilberry, cranberry and

lingonberry) were either inactive or relatively weak inhibitors of

ODC activity induced by the tumor promoter phorbol 12-myristate

13-acetate (TPA) in mouse epidermal cells [90]. More recently,

Afaq et al. demonstrated that the topical application of

anthocyanin- and hydrolysable tannin-rich pomegranate fruit

extracts prior to that of TPA resulted in a significant inhibition

of TPA-induced epidermal ODC activity and protein expression in a

mouse model of skin carcinogenesis. These effects were associated

26

with a substantial reduction of both incidence and burden of TPA-

induced skin tumors [91]. Thole et al. provided evidence that

fractions from elderberry extracts containing proanthocyanidins

and other phenolic compounds inhibited ODC activity in vitro [92]. On

the whole, these findings suggest that ACNs may have ODC

inhibitory properties. However, further studies performed with

purified ACN fractions or individual ACNs are needed to clearly

establish whether these compounds can interfere with ODC

expression/activity and polyamine biosynthesis.

4.4. Inhibition of proteasome chymotrypsin-like activity

The proteasome consists of a complex of enzymes involved in the

degradation of intracellular proteins, especially short-lived

proteins involved in the regulation of cell proliferation and

programmed cell death [93, 94]. In fact, proteasome inhibition has

been shown to increase the levels of proteins that can antagonize

cell survival and uncontrolled proliferation, including cyclins,

cyclin-dependent kinase inhibitors, the tumor suppressor p53, the

proapoptotic Bax protein and the NF-κB inhibitor I-κB [94].

Transformed cells show a higher sensitivity to growth and survival

inhibition by proteasome inhibitors as compared to non-transformed

27

cells [93, 94]. Dreiseitel et al. tested 17 ACNs and found that

anthocyanins and their aglycons inhibited proteasome activity in a

concentration dependent manner, with IC50 values between 7.8 and

32.4 µM [95] (Table 1). Experiments were performed using the

chymotryptic site of the proteasome in HL-60 cells. Among the 17

ACNs under investigation, the anthocyanidins kaempferidinidin,

pelargonidin, and peonidin were the most potent inhibitors, while

malvidin, pelargonidin-3,5-diglucoside, and delphinidin were the

least potent inhibitors [95]. When the compounds were grouped

according to the presence or absence of a sugar moiety, no trend

was seen with regard to proteasome inhibition, the mean IC50 values

of anthocyanins (IC50 = 17.5 ± 6.6 μM) and anthocyanidins (IC50 =

18.7 ± 11.0 μM) showing only a marginal difference. Instead, the

ACNs’ inhibitory potency appeared associated with their

substitution-pattern. Indeed, the most effective inhibitors

carried only one or two, hydroxyl-or methoxyl-substituents on the

B-ring [95].

4.5. Impairment of cyclin-dependent kinase activity

Cell cycle progression is driven by cyclin-dependent kinases

(CDKs), whose catalytic activity is dependent on cyclin binding

28

and is negatively regulated by CDK inhibitors, such as p27kip1 and

p21WAF1/Cip1 [67, 68]. Several findings indicate that ACNs can impair

the activity of CDKs by affecting the expression levels of

regulatory proteins. Indeed, as reported by Malik et al., the

exposure of colon cancer cells to an anthocyanin-rich extract

resulted in cell cycle arrest at the G1/G0 and G2/M phases,

increased expression of p27kip1 and p21WAF1/Cip1 genes, decreased

expression of cyclin A and B genes and 60% cell growth inhibition

[69]. Similarly, Wu et al. described an increase in the expression

of p21WAF1, associated with inhibition of cell proliferation, upon

treatment of colon cancer cells with anthocyanin-rich berry

extracts [68]. On the other hand, according to Chen and

colleagues, the cell cycle inhibitory effects of anthocyanins can

also rely on inhibition of CDKs expression. Indeed, they reported

that treatment of breast cancer cells with peonidin-3-glucoside or

cyanidin-3-glucoside resulted in a strong reduction of cell growth

via G2/M arrest associated with the down-regulation of both CDKs

and cyclin protein levels. In particular, peonidin-3-glucoside

induced the down-regulation of CDK-1, CDK-2, cyclin B1, and cyclin

E, whereas cyanidin-3-glucoside decreased CDK-1, CDK-2, cyclin B1

and cyclin D1 protein levels [96].

29

4.6. Inhibition of glyoxalase I (GLO I) activity

The glyoxalase system is the main metabolic pathway for the

detoxification of methylglyoxal, a side-product of glycolysis and

highly reactive dicarbonyl compound whose accumulation inhibits

cell growth and promotes apoptosis [75, 97, 98]. The glyoxalase

system is comprised of two enzymes, glyoxalase I (GLO I) and

glyoxalase II (GLO II). Of these, GLO I catalyzes the rate-

limiting step in methylglyoxal detoxification. An abnormal

expression or high activity of GLO I has been described in many

tumors, including colon, prostate and lung tumors. Indeed, the

increased activity of GLO I appears to support the viability of

tumor cells with high glycolytic rates and to promote multidrug

resistance, the latter finding suggesting that mechanisms of

toxicity of antitumour agents may involve the accumulation of

methylglyoxal at cytotoxic levels. Accordingly, GLO I is regarded

as a potential molecular target for tumor therapy [75, 97, 98].

Having reported that natural flavonoid compounds structurally

related to anthocyanidins effectively inhibited human GLO I [97],

Takasawa and colleagues tested the activity of delphinidin,

cyanidin and pelargonidin in in vitro GLO I assays performed with

30

recombinant human GLO I. The results of their study confirmed that

the three anthocyanidins shared the ability to inhibit GLO I,

albeit with different potency [75]. In fact, the IC50 values of

delphinidin, cyanidin and pelargonidin were calculated to be 1.9,

11.7 and 16.4 M, respectively. Thus, delphinidin had the most

potent inhibitory effects. In the same study, the authors provided

evidence that among the three compounds under investigation,

delphinidin only was able to reduce the viability of human

leukemic cells, and suggested that the accumulation of cytotoxic

methylglyoxal due to GLO I inhibition may contribute to

delphinidin cytotoxic effects. The analysis of the structure-

activity relationships of the tested compounds indicated that the

hydroxy groups on the B ring, especially at the R1 position,

greatly contribute to the inhibitory potency of anthocyanidins on

GLO I activity. Based on these results, the authors speculated

that the structure of delphinidin could represent a valuable

scaffold to design specific inhibitors of the human GLO I [75].

5. ANGIOGENESIS INHIBITION BY ANTHOCYANS

31

ACNs have been implicated in the antiangiogenic effects of mixed

berry extracts [99]. ACNs-rich extracts from black raspberries

were found to reduce the development of N-nitrosomethylbenzylamine

(NMBA)-induced esophageal tumors in rats, via the inhibition of

angiogenesis and cell proliferation, as well as through the

induction of apoptosis [100]. Among ANCs, delphinidin has been

repeatedly reported to exhibit antiangiogenic properties both in

vitro and in vivo [101-103]. Other anthocyanidins with reported

antiangiogenic activity include cyanidin and malvidin [104]. In

addition, delphinidin-3-(p-coumaroylrutinoside)-5-glucoside

(nasunin), an anthocyanin isolated from eggplant peels, was found

to inhibit microvessel outgrowth in a rat aortic ring assay

through the suppression of endothelial cell proliferation, without

affecting endothelial cells migration and tube formation [105].

The molecular events involved in the inhibition of angiogenesis by

ACNs are described below. These include the impairment of the

activity of VEGFRs and PDGFRs. In fact, these receptors are among

the major players in tumor angiogenesis and their inhibition has

been shown to induce the regression of well established tumors

[106, 107]. The antiangiogenic effects of ACNs can further rely on

32

the inhibition of matrix metalloproteinases (MMPs), which also

play a key role in tumor cell invasion and metastatization [108].

5.1. VEGFRs

VEGF is a key signaling molecule controlling the proliferation and

migration of endothelial cells, and is regarded as the main

angiogenic growth factor [106]. VEGF is a ligand for a family of

three tyrosine kinase receptors, VEGFR-1, VEGFR-2, and VEGFR-3

[106]. The biological effects of VEGF are mainly transduced by

VEGFR-2 [109].

Favot et al. reported that delphinidin inhibited the VEGF-induced

migration and proliferation of human umbilical vein endothelial

cells (HUVECs). These effects were associated with the blockade of

cells in the G0/G1 phase of the cell cycle, with the reversal of

the VEGF-induced decrease in expression of the CDKs inhibitors

p27kip1 and p21WAF1/Cip1, and with the inhibition of the VEGF-induced

increase of cyclin D1 and cyclin A [103]. Similar findings

regarding the inhibition of VEGF-induced endothelial cell

proliferation by delphinidin were obtained by Martin and

colleagues [110]. Moreover, according to Matsunaga et al.,

delphinidin, cyanidin and malvidin impaired VEGF-induced tube

33

formation in co-cultures of HUVECs and fibroblasts, their effect

being significant at concentrations of 3-10 M [104]. The results

of these studies clearly indicate that anthocyanidins can

counteract the angiogenic effects of VEGF.

Lamy et al. demonstrated for the first time that the inhibitory

effects of delphinidin on angiogenesis involve the inhibition of

VEGFR-2 signaling. Indeed, they investigated the antiangiogenic

properties of anthocyanidins, including cyanidin, delphinidin,

malvidin, pelargonidin, peonidin and petunidin and found that

among the tested compounds delphinidin was the most potent

angiogenic inhibitor in endothelial cells tube formation assays,

its IC50 being 9.5 M [101]. Next, they showed that delphinidin was

able to reduce the VEGF-induced chemotactic motility of

endothelial cells and their differentiation into capillary-like

tubular structures. Last, these authors provided evidence that

delphinidin inhibited the VEGF-induced tyrosine phosphorylation of

VEGFR-2 (IC50: 2 M) in HUVECs, thus impairing the phosphorylation

and activation of ERK-1/2. The authors associated the

antiangiogenic properties of delphinidin with the presence of

three hydroxyl groups at the B-ring and a free hydroxyl group at

position 3. Indeed, the presence of sugar residues at this

34

position, as in delphinidin-3-glucopyranoside, abolished the

antiangiogenic effects of delphinidin [101].

The results provided by Teller et al. further indicate that the

antiangiogenic properties of ACNs actually involve the direct

inhibition of VEGFRs catalytic activity [78, 79]. Indeed, they

demonstrated that anthocyanin-rich mixtures and purified

delphinidin inhibited the activity of VEGFR-2 and VEGFR-3 kinase

domains in cell-free assays. The inhibitory effect of delphinidin

in these assays was evident at concentrations ≥ 1 M. A decrease

of the ligand-induced autophosphosphorylation of VEGFR-2 and

VEGFR-3 in intact cells was also described upon delphinidin

treatment: at 50 M delphinidin, the phosphorylation of VEGFR-2

was suppressed to about 50%, whereas that of VEGFR-3 was

completely abolished [79]. Remarkably, VEGFR-3 plays an important

role in angiogenesis as well as in tumor-induced

lymphangiogenesis, a process that is deeply involved in tumor

dissemination. As reported above, the presence of vicinal hydroxyl

groups at the B ring, rather than metoxy groups, is a critical

determinant for the inhibitory activity of ACNs on receptor

tyrosine kinases, including VEGFRs [79].

35

Besides, other studies indicate that ACNs can exert antiangiogenic

effects also by downregulating the expression of both VEGFR-2 and

VEGF [111-113].

5.2. PDGFRs

PDGFs and their tyrosine kinase receptors (PDGFR- and PDGFR-β)

play an important role in tumor growth, metastasis and

angiogenesis, and are implicated in the pathogenesis of different

cancers [107].

Smooth muscle cells actively participate in different aspects of

angiogenesis. Indeed, smooth muscle cells, triggered by PDGF-

expressing endothelial cells, are recruited to stabilize new

vessels and induced to release VEGF [102, 111]. Lamy et al. reported

that delphinidin dramatically reduced the phosphorylation of

PDGFR-β induced by PDGF-BB in smooth muscle cells [102]. The

inhibitory effect of delphinidin was dose-dependent (IC50: about 10

M) and accompanied by the inhibition of ERK-1/2 phosphorylation.

These molecular events were correlated with the reduction of PDGF-

induced smooth muscle cell migration, and with the impaired

formation of capillary-like structures in co-cultures of

endothelial and smooth muscle cells stimulated by PDGF and/or

36

different growth factors. Furthermore, the authors demonstrated

that delphinidin was able to provoke the destabilization and

regression of established vessels. Finally, ACNs-rich extracts

were shown to inhibit FGF-2 and PDGF-BB-induced angiogenesis in in

vivo assays [102].

Oak et al. investigated the effect of anthocyanidins on the release

of VEGF induced by PDGF-AB in vascular smooth muscle cells. The

PDGF-AB-induced expression of VEGF is known to be predominantly

mediated by the p38 MAPK and JNK pathways, and to some extent by

the ERK1/2 pathway. In the study by Oak and colleagues,

anthocyanidins presenting a hydroxyl residue at position 3’, i.e.

delphinidin and cyanidin, markedly reduced the PDGF-AB-induced

phosphorylation of p38 MAPK and JNK, without affecting that of

ERK1/2, and effectively prevented the expression of VEGF [111].

The inhibitory effects of delphinidin and cyanidin were observed

at concentrations of 10-30 M. These findings provide additional

evidence that ACNs can interfere with PDGF signaling.

6. INHIBITION OF CELL INVASION AND MIGRATION BY ANTHOCYANS:

IMPAIRMENT OF METALLOPROTEINASES (MMPs) AND PLASMIN ACTIVITY

37

The degradation of the extracellular matrix (ECM) is a

prerequisite for cancer cell invasion, migration and

metastatization, as well as for tumor angiogenesis. Such

degradation is accomplished by an array of proteases, including

metalloproteinases (MMPs), serine and cysteine proteinases [22,

114, 115]. The proteolytic degradation of the ECM creates the

paths for invading cells and promotes migration by allowing the

cells to alternate between adhesive and non-adhesive states [116,

117]. Moreover, ECM fragments have been found to promote tumor

cell motility [118]. ECM degradation can also lead to the

mobilization of growth and angiogenic factors [116, 117].

MMPs play a major role in ECM degradation and their gene

expression and catalytic activation are strictly controlled [115-

117]. Negative regulation of MMP activity relies on the tissue

inhibitors of MMPs (TIMPs-1/4) [22, 115, 116]. Increased levels of

MMPs, in particular MMP-2 and MMP-9, are frequently found in human

cancers and correlate with invasive/metastatic behavior and poor

prognosis [114, 115, 117]. ECM proteolysis is also performed by

the plasminogen activator/plasmin system in which the conversion

of plasminogen to plasmin is realized by two types of plasminogen

activators (PAs): the urokinase-type (u-PA) and tissue-type (t-PA)

38

plasminogen activators. In turn, PAs activity is monitored by the

plasminogen activator inhibitors (PAIs). Remarkably, plasmin can

also activate MMPs [114].

The transcription of MMPs and u-PA genes is regulated by upstream

sequences, including motifs corresponding to NF-κB and AP-1

binding sites, thus linking the activation of these transcription

factors with the proteolytic degradation of the ECM [71, 114,

119]. Impairment of AP-1 activity by ACNs can result from their

ability to inhibit different signal transduction pathways which

trigger the MAPK cascades, including pathways activated by growth

factors, cytokines and stress stimuli [8, 9, 71, 114, 119] (Fig.

2, Panel A). Moreover, as it will be discussed further on in this

review, ACNs have been shown to effectively inhibit the activation

of NF-κB (Fig. 2, Panel B).

Nagase et al., early reported a slight inhibitory effect on MMPs

activity upon treatment of human fibrosarcoma cells with

delphinidin [60]. According to Huang et al., mulberry ACNs inhibited

mouse melanoma cells motility in vitro and metastatization in vivo

through the impairment of ECM enzymatic degradation. Indeed,

melanoma cells treated with non-toxic concentrations of mulberry

ACNs showed a reduced activity of both MMP-2 (68%) and MMP-9 (55%)

39

by gelatine zymography assay [70]. The observed reduction of MMP

activity was supposed to result from the downregulation of MMP-2

and MMP-9 gene expression via the ACNs-mediated suppression of NF-

κB and AP-1 activation [70]. Chen et al. found that non-toxic doses

of cyanidin-3-glucoside and cyanidin-3-rutinoside (25-100 M)

inhibited the invasive behaviour of a highly metastatic squamous

lung carcinoma cell line. Both compounds were found to reduce MMP-

2 and u-PA mRNA levels, whereas cyanidin-3-glucoside was found to

increase TIMP-2 and PAI mRNA levels [71]. The transcriptional

downregulation of MMP-2 and u-PA was also suggested to be involved

in the inhibition of motility and/or invasion induced by cyanidin-

3-glucoside and peonidin-3-glucoside (100 M) in various tumor

cell lines [114]. Similar results were obtained in two other

studies: a mixture on ACNs isolated from meoru fruits, and mainly

composed of glucoside and biglycoside forms of anthocyanins, was

found to reduce hepatoma and colon carcinoma cell invasion and to

inhibit the constitutive and TNF-induced expression of MMP-2 and

MMP-9, as well as the constitutive and TNF-induced activation of

NF-κB [119, 120]. Finally, non-toxic doses of peonidin-3-glucoside

(10-40 M) were demonstrated to reduce motility, invasion, mRNA

levels of MMP-2, MMP-9 and u-PA, and AP-1 activity in lung cancer

40

cells [121]. Anthocyanins have also been reported to inhibit the

secretion of the collagenase MMP-1 [122]. Remarkably,

administration of anthocyanin-rich extracts from black rice to

nude mice bearing breast cancer cell xenografts significantly

suppressed tumor growth, angiogenesis and expression of MMP-2,

MMP-9 and u-PA in tumor tissues, indicating that the ability of

ACNs to impair the expression of ECM degrading enzymes can occur

also in vivo [123]. On the whole, these results indicate that the

impairment of MMP and u-PA activity by ACNs results, at least in

part, from a reduced expression of these enzymes caused by the

inhibition of upstream signaling pathways leading to the

activation of AP-1 and NF- κB.

On the other hand, in a minor number of studies it has been

reported that ACNs can also interfere with the catalytic activity

of ECM degrading enzymes. Santos et al., demonstrated that

blackcurrant extract (at 50 g/mL) and its major anthocyanins,

i.e. cyanidin-3-glucoside, cyanidin-3-rutinoside and delphinidin-

3-rutinoside (at 6.25 to 50 g/mL), significantly inhibited the

activity of recombinant MMP-1 and MMP-9 in in vitro assays [124].

Lamy et al., provided evidence that ACNs can interfere with the

catalytic activity of u-PA as well [125]. In their study,

41

cyanidin, delphinidin and petunidin were found to act as potent

inhibitors of glioblastoma cell migration, with delphinidin

exhibiting the highest inhibitory potency (IC50: 7.0 M).

Conversely, delphinidin-3-glucoside, peonidin, pelargonidin and

malvidin were much less potent inhibitors of migration, suggesting

that the presence of vicinal hydroxyl groups at the B ring and of

a free hydroxyl group at position 3’ is required for efficient

inhibition of cell migration. Delphinidin was also found to reduce

matrigel invasion by glioblastoma cells (IC50: 9.5 M).

Interestingly, the anthocyanidins with inhibitory effects on

glioma cell migration/invasion, were able to reduce the uPA-

dependent conversion of plasminogen to plasmin (at 5-25 M) in in

vitro assays performed using purified uPA and plasminogen, thus

demonstrating that ACNs can actually impair the activity of u-PA

[125].

7. CANCER-RELATED INFLAMMATION AND ANTHOCYANS

Autocrine and paracrine mechanisms involving inflammatory

mediators are known to play a wide range of effects in cancer

development and progression [24, 126, 127].

42

7.1 NF-κB inhibition at the crossroad of the anti-cancer and anti-

inflammatory properties of anthocyans: impairment of IκB kinase

complex activity

The persistent activation of NF-κB has been associated with

several aspects of oncogenesis. Indeed, this transcription factor

can promote cell transformation, tumor cell survival,

proliferation, invasion, metastatization and also neoangiogenesis

[12-14]. Accordingly, the blockade of the transcriptional activity

of NF-κB is supposed to counteract cancer development at multiple

levels [12-14]. The persistent activation of NF-B in cancer cells

is sustained by the high concentration of inflammatory mediators

within the tumor microenvironment. ROS have been identified as

second messengers leading to the activation of NF-κB as well. [12-

14, 24, 128]. The “canonical” NF-κB signaling pathway, closely

linked with inflammation and production of tumor necrosis factor

(TNF), interleukin-1 (IL-1), IL-6, prostaglandin E2 (PGE2), etc.,

relies on NEMO, activation of IKK, IκB degradation and nuclear

translocation of NF-κB dimers [15] (Fig. 2, Panel B).

Interestingly, the anti-cancer activities of many anti-

inflammatory drugs are thought to be, at least partly, related to

the inhibition of NF-κB [129]. Indeed, inhibition of NF-κB has

43

been demonstrated to arrest tumor cell growth and to induce

apoptosis [12-14, 129, 130].

That ACNs can impair the constitutive and/or induced activity of

NF-κB is supported by multiple evidence [10, 62-65, 86, 100, 112,

120, 131-138]. Although the molecular mechanism through which ACNs

interfere with NF-κB are not completely understood, it is without

doubt that these compounds can prevent the degradation of the NF-

κB inhibitor IκB by inhibiting the activity of the IκB kinase

complex IKK [62-64, 86, 120, 132-134, 136, 137] (Fig. 2, Panel B).

For instance, Hafeez et al. demonstrated that delphinidin inhibited

the growth of human prostate cancer cell lines, with IC50 values

ranging from 50 to 90 M, but not of normal human prostate

epithelial cells. Further, caspase-dependent apoptosis was induced

by delphinidin in prostate cancer cells at doses of 30 to 180 M.

These effects were associated with the decreased phosphorylation

of the IKK regulatory subunit IKK/NEMO and of its substrate IκBα.

Moreover, delphinidin treatment also reduced phosphorylation of

RelA/p65 at Ser536 and NF-κB1/p50 at Ser529, nuclear translocation of

RelA/p65, and NF-κB DNA-binding activity [62, 63]. These findings

indicate that impairment of NF-κB signaling by delphinidin was

mediated by the inhibition of the upstream kinase IKK which

44

phosphorylates and targets for degradation IkB [62, 63]. Results

similar to those reported above were obtained by Yun et al. in human

colon cancer cells: delphinidin treatment resulted in decreased

viability (with an IC50 value of 110 M), induction of apoptosis

(at doses of 30 to 240 M), and impairment of NF-κB activity

through the inhibition of the upstream kinase IKK. In this study

the delphinidin-induced inhibition of IKK was found to involve the

IKK catalytic subunit of the complex [64].

Inhibition of NF-κB activity has also been observed in studies

performed using different purified ACN compounds. The

lipopolysaccharide (LPS)-induced phosphorylation of IκBα and

nuclear translocation of NF-κB were reported to be inhibited by

cyanidin-3-glucoside and cyanidin in mouse leukemic macrophage-

like cells [134], and by cyanidin-3-glucoside in a human

monocyte/macrophage cell line [136]. Cyanidin-3-glucoside and

cyanidin were also identified as good inhibitors of NF-κB activity

induced by the mutagenic and highly carcinogenic benzo[a]pyrene-

7,8-diol-9,10-epoxide (B[a]PDE) [131, 138]. Further, pretreatment

with cyanidin-3-glucoside was reported to impair TPA- and UVB-

induced NF-κB activity, as well as AP-1 activity, in a mouse

epidermal cell model of skin carcinogenesis [65]. As for in vivo

45

studies, delphinidin administration to mice carrying prostate

cancer cell tumor xenografts was found to reduce to a significant

extent tumor growth and NF-κB protein levels in tumor tissues

[62]. In addition, suppression of NMBA-induced, rat esophageal

tumor development by anthocyanins was associated to the reduction

of NF-κB p50 and COX-2 expression at the tumor level [100].

Collectively, these findings suggest that ACNs can affect both NF-

κB expression and activity in tumor cells.

7.2 Impairment of NF-κB activity via the inhibition of histone

acetyltransferases (HATs)

To achieve its full biological activity, NF-κB must undergo a

variety of post-translational modifications, including

acetylation. In particular, acetylation at different lysine

residues in the RelA/p65 subunit modulates distinct functions of

NF-κB, including transcriptional activation, DNA binding, and

assembly with its inhibitor IκB [139, 140]. NF-κB acetylation is

accomplished by different histone acetyltransferases (HATs).

Indeed, HATs have been reported to acetylate a variety of non-

histone substrates, such as transcription factors, co-activators,

nuclear transport proteins, structural proteins and cell cycle

46

regulators [139, 141]. Interestingly, increased levels of

acetylated NF-κB have been found in human bone metastatic breast

cancer cells as compared to parental invasive breast cancer cells,

thus suggesting a relationship between the post-translational

modification of this transcription factor and the tumor metastatic

phenotype [142].

Seong et al. performed in vitro acetylation assays using recombinant

HAT enzymes and demonstrated that delphinidin (10-100 M) acted as

a specific inhibitor of their activity. In particular, delphinidin

was found to affect the HAT activity of p300/CBP transcriptional

coactivators. Further, the authors investigated the effects of

delphinidin on NF-κB function in TNF-stimulated human rheumatoid

arthritis synovial cells and reported that treatment with

delphinidin inhibited RelA/p65 acetylation. The delphinidin-

induced hypoacetylation was accompanied by cytosolic accumulation

of p65 and suppression of the TNF-induced expression of NF-κB

target genes, including IL-6, COX-2 and IL-1 [143].

Worthy of note, HATs are chromatin-modifying enzymes which play an

important role in the control of gene expression. These enzymes,

and p300/CBP in particular, have been implicated in growth and

survival of some types of cancer, and molecules with HAT

47

inhibitory properties are currently regarded as potential

pharmacological tools for tumor therapy in specific contexts [144-

147]. In addition to delphinidin, the small number of HAT

inhibitors identified to date includes different polyphenols [144,

146, 147].

7.3 Inhibition of cyclooxygenase-2 (COX-2) expression and

lipoxygenase (LOX) activity

Cyclooxygenases (COXs), are key enzymes responsible for the

biosynthesis of prostaglandins (PGs) and thromboxanes (TXs) from

arachidonic acid. These enzymes and their products play a pivotal

role in inflammation and tumor progression [148]. COXs exist in

two isoforms: COX-1 and COX-2. While COX-1 is constitutively

expressed, the expression of COX-2 is highly induced by growth

factors, proinflammatory cytokines and tumor promoters. COX-2

appears to be dysregulated in many types of cancer, including

colon, pancreas, lung, gastric, breast, prostate and head and neck

cancer, and the COX-2-derived PGE2 is the main prostaglandin found

in human tumors [148]. PGE2, by stimulating their cognate

receptors, known as EP receptors, promotes tumor progression by

48

inducing cell proliferation, migration, invasion, angiogenesis and

by inhibiting apoptosis [148]. Of note, a complex interplay occurs

between PGE2 and growth factors signaling. In fact, PGE2 can

transactivate the EGFR. Moreover, it can induce the production of

angiogenic growth factors, including VEGF and bFGF, which, in turn

stimulate COX-2 expression. Signaling by PGE2 receptors further

involves the activation of the PI3K/Akt survival pathway and of

the Ras/MAPK pathway, which is also able to upregulate COX-2

expression, and increases the transcriptional activity of NF-B,

whose target genes include COX-2 [12-14, 148, 149]. Indeed, COX-2

expression is primarily regulated by NF-B, AP-1 and their upstream

kinases, MAPKs [150]. In addition to PGE2, also TXA2 has been

implicated in oncogenesis, mainly as a promoter of angiogenesis

[148, 151].

Seeram et al. provided evidence that ACNs from raspberries and sweet

cherries (125 g/mL) inhibited COX-2 and COX-1 activity by 47% and

45%, respectively. The aglycone cyanidin was shown to have

superior inhibitory activity than its glycosides. In addition, the

inhibitory potency of the ACNs was found to increase with the

decreasing number of sugar residues attached to the anthocyanidin

moiety, as established for cyanidin-3-rutinoside compared to

49

cyanidin-3-glucosylrutinoside [152]. On the other hand, inhibition

of COX-2 expression by ACNs has been demonstrated in many studies.

Tsoyi et al. reported that anthocyanin extracts from black soybean

seed coats, containing cyanidin-3-glucoside (72%), delphinidin-3-

glucoside (20%) and petunidin-3-glucoside (6%), inhibited UVB-

induced COX-2 expression (at 50-100 g/mL) and PGE2 production (at

10-100 g/mL) in human keratinocytes by reducing the activation of

NF-B through the prevention of IκBα phosphorylation [133]. The

results of this study also suggested that the anthocyanin-induced

inhibition of the PI3K/Akt pathway triggered by UVB irradiation

may be involved in the observed downregulation of COX-2 expression

[133]. Kwon et al. analyzed the molecular events underlying the

inhibitory activity of peonidin on TPA-induced COX-2 expression

and mouse epidermal cell transformation [153]. Based on the

evidence that ERK activation is required for both TPA-induced

transformation and COX-2 expression, they investigated whether

peonidin could affect the phosphorylation of ERK induced by TPA.

Peonidin (5-20 M) was found to effectively downregulate both the

TPA-induced phosphorylation of ERK and the expression of COX-2

[153]. Similarly, Ding et al. demonstrated that pretreatment with

cyanidin-3-glucoside (10-40 M) inhibited the UVB- and TPA-induced

50

transactivation of NF-B and AP-1 as well as the expression of COX-

2 in mouse epidermal cells [65]. The ability of ACNs to reduce the

expression of COX-2, either basal or induced by different stimuli

(LPS, UVB), through the inhibition of MAPK pathways and/or NF-B

activation has been confirmed in a series of other studies

performed using various cell types [134, 154-156]. The ortho-

dihydroxyphenyl structure of anthocyanidins on the B ring appears

to be important for efficient inhibition of COX-2 expression

[154].

Lipoxygenases (LOXs), the other class of arachidonic acid

processing enzymes, have also been implicated in cancer onset and

progression [157]. LOX-derived products such as

hydroxyeicosatetraenoic acids (HETEs) have been found to promote

cell proliferation and inhibit apoptosis [157, 158]. According to

Knaup et al., LOX activity can be inhibited by a spectrum of ACNs

[159]. In fact, they performed in vitro assays using extracts of

human neutrophil granulocytes as a source of 5-LOX, and

demonstrated that delphinidin-3-glucoside and delphinidin-3-

galactoside inhibit the activity of 5-LOX with IC values of 2.15

and 6.9 M, respectively. Besides, they provided additional

insights into the mechanisms and structure-activity relationships

51

for the ACNs-induced inhibition of LOX enzymes using soybean LOX-1

as a model. According to their results, a prototypical

anthocyanidin (peonidin) appeared to act as an uncompetitive

inhibitor of soybean lipoxygenase-1, i.e. an inhibitor that binds

exclusively to the enzyme-substrate complex. Finally, in soybean

LOX-1 assays the IC50 values of the unmethylated ACNs, were

significantly lower than those of the methylated ACNs [159].

7.4 Impairment of NF-B and AP-1 activation and downregulation of

COX-2 expression as a result of the direct binding/inhibition of

signaling kinases by ACNs

In the last years, several signaling kinases have been identified

as direct targets of ACNs. Binding and inhibition of these kinases

by ACNs has been linked to the impairment of NF-B and AP-1

transactivation and to downregulation of COX-2 expression [10,

138, 150, 156, 160].

The signaling kinases directly targeted by ACNs have been

identified in different studies performed using the tumor-

promotion sensitive mouse skin epidermal cells JB6 P+. Kang et al.

investigated the mechanisms responsible for the inhibitory effects

of delphinidin on TPA-induced transformation of JB6 P+ cells [150].

52

The TPA-induced transformation of these cells was associated with

the activation of the MEK/ERK pathway, transactivation of NF-B and

AP-1, upregulation of COX-2 and increased production of PGE2.

Treatment of JB6 P+ with delphinidin (5-20 M) was shown to inhibit

all of the above reported molecular events and to result in a

marked reduction (43%) of neoplastic transformation. Both

neoplastic transformation and COX-2 expression were previously

reported to be abolished by the pharmacological inhibition or

dominant-negative knockout of MEK [150]. Accordingly, in the

attempt to find the direct molecular targets of delphinidin, Kang

and colleagues investigated the effects of this anthocyanidin on

the activity of MEK1 and its upstream kinase Raf1 in in vitro kinase

assays. It was thus found that delphinidin at 10 M blocked Raf1

and MEK1 activities by 67% and 64%, respectively, while it had

minor effects on other kinases such as ERK2 or JNK1. It was

further demonstrated that delphinidin directly binds Raf1 or MEK1

non-competitively with ATP. Inhibition of Raf1 and MEK1 by

delphinidin was also obtained in ex vivo kinase assays. Therefore,

these findings linked the blockade of the Raf/MEK/ERK pathway,

induced by the direct binding of delphinidin to Raf1 and MEK1, to

53

the inhibition of NF-B and AP-1 transactivation and COX-2

expression [150].

JB6 P+ cells are also widely used to investigate the mechanisms of

UVB-induced skin cancerogenesis [10]. COX-2 plays a pivotal role

in UV-related skin carcinogenesis. Indeed, UVB irradiation leads

to the activation of NF-B and AP-1, and to the consequent

upregulation of COX-2 expression. The major pathways that are

known to mediate UVB-induced biological responses are the MAPKs

and PI3K signaling pathways [10]. Delphinidin treatment (5-20 M)

of JB6 P+ cells was found to inhibit the transactivation of AP-1

and NF-KB, the expression of COX-2 and the synthesis of PGE2

induced by UVB irradiation. Further, delphinidin suppressed the

UVB-induced phosphorylation of JNKs, p38 and Akt. In vitro and ex vivo

kinase assays were thus performed to evaluate whether delphinidin

could inhibit the activity of JNKs-, p38- and Akt-upstream

kinases, i.e. MAPKK4 (or MEK4), MAPKK6 (or MEK6) and PI3K. The

assays demonstrated the ability of delphinidin (10-20 M) to

inhibit MAPKK4 and PI3-K, but not MAPKK6. Besides, it was shown

that delphinidin directly binds MAPKK4 and PI3K in an ATP-

competitive manner. These findings indicated that delphinidin

inhibits UVB-induced COX-2 expression by blocking the MAPKK4 and

54

PI3K pathways, and subsequently suppressing AP-1 and NF-B

activities [10]. Using similar approaches, cyanidin (5-20 M) was

shown to suppress UVB-induced COX-2 expression in JB6 P+ cells by

directly targeting Raf-1, MEK1 and MAPKK4 [156]. ATP was found to

compete with cyanidin for MAPKK4 binding but not for MEK1 or Raf-1

binding [156]. Remarkably, the UVB-induced expression of COX-2 was

impaired in JB6 P+ cells transfected with MAPKK4 siRNAs or with a

dominant-negative ERK-2 mutant, demonstrating that the

upregulation of COX-2 induced by UVB irradiation is actually

mediated by the MAPKK4 and Raf/MEK/ERK pathways [156].

TNF is a potent inducer of COX-2 expression. The expression of

COX-2 induced by TNF in JB6 P+ cells was also strongly inhibited

by delphinidin (10-40 M) [160]. TNF signaling has been shown to

involve, among the others, the Fyn kinase. Fyn is a member of the

Src family of non-receptor tyrosine kinases (SFKs). SFKs are known

to be associated with growth factor receptors, hormone receptors

and cytokine receptors and to transduce signals which promote cell

growth, differentiation, survival, adhesion and migration.

Accordingly, the abnormal activation of SFKs, including Fyn, is

involved in tumorigenesis [160]. Delphinidin was shown to inhibit

Fyn kinase activity in in vitro assays and to directly bind to Fyn

55

non-competitively with ATP. The involvement of Fyn kinase in the

induction of COX-2 expression by TNF was demonstrated by using a

specific Fyn kinase inhibitor as well as by tranfection with Fyn

siRNAs. Thus, Fyn kinase was identified as one of the direct

molecular targets of delphinidin for the suppression of TNF-

induced COX-2 expression [160]. Unpublished observations indicate

that delphinidin can inhibit also Src kinase, which is highly

homologous to Fyn [160]. The ability of ACNs to bind and inhibit

Fyn kinase was confirmed in a recent study aimed at investigating

the mechanisms responsible for the inhibitory effect of cyanidin-

3-glucoside (5-20 M) on the B[a]PDE-induced expression of COX-2

in JB6 P+ cells [138]. Cyanidin-3-glucoside was shown to bind Fyn

kinase non-competitively with ATP and to inhibit its activity.

Moreover, by blocking Fyn activity through a pharmacological

inhibitor, it was demonstrated that Fyn kinase regulates the

B[a]PDE-induced expression of COX-2 by activating MAPKs, AP-1 and

NF-B [138].

Collectively, the reported studies indicate that ACNs can act as

multi-target kinase inhibitors and substantiate their potential as

chemopreventive agents.

56

8. ANTHOCYANS BIOAVAILABILITY AND PREVENTIVE/THERAPEUTIC POTENTIAL

AGAINST HUMAN CANCERS

The first requirement for a dietary compound to have an in vivo

effect is that it must enter the blood circulation and reach the

tissues, in the native or metabolized form, in a dose sufficient

to exert a biological activity. In this regard, the whole body of

ACNs in vivo studies provides a quite striking evidence that the

bioavailability of ACNs is poor [31]. This conclusion is based on

the recovery of negligible amounts of intact ACNs and of some of

their metabolites (i.e. glucuronidated and methylated derivatives)

in urine and plasma. In fact, the concentrations of ACNs which

have been measured in human plasma so far after single or repeated

intake of ACN-rich food or food extracts are in the nanomolar

range, accounting for less than 1% of the ingested dose [31, 161-

170]. Of note, the metabolic fate of the remaining, most abundant

fraction of the ingested ACNs is unclear. According to these

findings, the evidence for ACNs activity on enzymes and related

signaling pathways has been mainly obtained using ACNs

concentrations higher than those which appear to be achievable in

vivo after oral consumption. On the other hand, the poor

bioavailability of dietary ACNs is in apparent contrast with the

57

reported physiological effects of these compounds. Indeed, ACNs,

predominantly in the form of mixtures, have demonstrated cancer

chemopreventive properties in animal models of breast, skin,

esophageal, lung, oral and gastrointestinal carcinogenesis [171].

In addition, some clinical evidence supporting the cancer

chemopreventive efficacy of ACNs comes from human studies in which

the effects of ACN-rich fruits or berry preparations have been

investigated in individuals at high risk of developing cancer

[166, 171].

A possible explanation to reconcile this apparent paradox is that

yet unidentified ACN metabolites and/or breakdown products

maintain to some extent the biological properties of the native

compounds but reach cells and tissues in much higher amounts. The

definition of the metabolic fate of ingested ACNs is of pivotal

importance in this respect.

At acidic pH (< 2), ACNs primarily exist as stable flavylium

cations. Therefore, the acidity of the gastric content should

constitute a stabilizing environment for these molecules. The

rapid appearance (1 h after ingestion) of intact ACNs in plasma

could result in part from absorption through the gastric wall

[30]. However, gastric absorption of ACNs appears quantitatively

58

negligible, and the metabolic fate of ACNs in the intestinal tract

is of much greater importance. In this regard, the example of

cyanidin-glucosides is illustrative. At physiological pH, as it

occurs in the small-large intestine, cyanidin and cyanidin

glucosides have been reported to convert to protocatechuic (PCA)

and other hydroxybenzoic acids [172]. That such transformation

actually occurs in vivo is suggested by a recent study showing that

after ingestion of blood orange juice, which contains an high

amount of cyanidin-glucosides, the serum maximal concentration of

cyanidin-3-glucoside (measured at 0.5 h) was about 2 nM, while

that of PCA (measured at 2 h) was about 500 nM [161]. In this

study PCA was also identified as the major human metabolite of

cyanidin glucosides, accounting for about 73% of the ingested ACNs

[161]. Also considering its marked antioxidant properties, it is

realistic to presume that PCA is the missing human metabolite of

cyanidin-glucosides, which effectively reaches the tissues in a

dose sufficient to exert biological effects, although the

occurrence of other unknown biologically active metabolites cannot

be excluded [172, 173]. Besides, the gut microflora plays a

critical role in the biotransformation of ACNs, leading to the

formation of diverse stable metabolites (i.e. phenolic acids) and

59

breakdown compounds in both the small and large intestine [161].

On the whole, the health benefits associated with ACN-rich foods

may be explained by a rapid, but quantitatively negligible,

gastric absorption of the ACNs intact form, followed by a slow

release of ACN metabolites through the gut into the bloodstream,

which may actually provide a chronic defence against body

oxidative processes [161, 173].

The hypothesis that ACN metabolites may be responsible for the

healthy benefits associated with the consumption of ACN-rich

foods, arises important concerns on the scientific value of in vitro

experiments used to test the biological effects of ACNs,

considered exclusively in their native form [173]. Nonetheless, it

should be considered that native ACNs may actually exert

biological functions in the gastrointestinal tract, prior to their

metabolization and absorption [79, 174]. Further, the great body

of studies aimed at defining the biological effects of ACNs in vitro

are of critical importance for the development of future ACN-based

drugs with enhanced bioavailability and efficacy [75, 77]. Still,

according to the reported considerations, it is not adequate to

predict the health properties of ACNs by considering exclusively

their native structures [173].

60

Thus, in order to assess the preventive/therapeutic properties of

dietary ACNs toward cancer, a great deal of work is still

necessary. First, studies should be performed on ACNs metabolism

in animal and human models, with the aim to identify the putative

metabolites and/or breakdown compounds which effectively reach the

tissues at doses sufficient to exert an effect. Once identified,

these compounds need to be thoroughly investigated to verify

whether they lose, retain or magnify the biological effects of the

native compounds. Epidemiological studies evaluating the

relationship between ACN-rich food consumption and incidence of

given tumours may also provide important information. Finally,

future studies are also necessary to fully define the

pharmacological profile of ACNs, including information on

absorption, distribution, metabolism and excretion of ACNs

administered by various routes (oral, intraperitoneal,

intravenous, intrathecal).

In conclusion, the regulatory abilities of ACNs toward enzymes

involved in cancer development and progression emphasize that ACNs

represent a very promising group of compounds with a fascinating

preventive/therapeutic potential (Table 1). A rigorous application

of evidence-based medical rules to the studies on dietary

61

compounds will increase the quality of the research in this field

and avoid the generation of false myths about miraculous foods.

62

ACKNOWLEDGEMENTS

The authors’ work was partly supported by PRIN 2009 (R.B). The

authors do not have conflict of interest. We wish to thank Barbara

Bulgarini for her help in manuscript editing.

63

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Figure legends

Fig. (1). Cancer development and progression: potential

preventive/therapeutic effects of anthocyans. The multi-step

carcinogenesis process includes tumor initiation, promotion and

progression. As indicated in the figure, anthocyans can interfere

with multiple events involved in this process. ACN : anthocyans ;

T : cancer cells; ECM : extracellular matrix; MMPs :

metalloproteinases.

Fig. (2). Impaired expression and function of signaling pathway

enzymes by anthocyans. Panel A: receptor tyrosine kinases (RTKs)

mediated-signaling. Panel B: the “canonical” signaling pathway

which leads to NF-κB activation. Panel C: 3',5'-cyclic

monophosphate (cAMP) signaling pathway. The molecular targets

inhibited by anthocyans (ACN) are indicated.

Figure 3. Chemical structure of the most common anthocyanidins.

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