ISSN 1916-6958

2010; Volume 1 (Number 1):
Pages 3-13

REVIEW ARTICLE

Mitochondria
as a target for novel chemotherapeutic agents based on phenolic acids

Teresa L. SERAFIMa, Maria P.
MARQUESb, Fernanda M. BORGESb,c, Paulo J. OLIVEIRAa,*

aCenter for Neurosciences and Cell Biology,
Department of Zoology, and bResearch Unit ‘Química-Física
Molecular’, University of Coimbra; and cOrganic Chemistry
Department, Pharmacy School,  University
of Porto, Portugal

*Corresponding author:
[email-masked]

ABSTRACT

Every day, new molecules with antineoplastic potential are
discovered. Unfortunately, most of these molecules lack cell-type specificity,
and are unable to kill tumor cells any more efficiently than normal cells.
Also, a high number of those molecules are very toxic. In order to design more
effective chemotherapeutic drugs, it is important to understand the interaction
between novel molecules and biological systems. Certain cellular components are
particularly relevant in the context of specific targeting and mechanisms of
action. Mitochondria are not only the major source of cell energy but are also
important in the control of processes that culminate in apoptotic cell death.
Particular aspects of mitochondrial physiology (e.g. the negative transmembrane
electric potential) facilitate selective targeting by anti-cancer molecules.
Such mitochondria-specific drugs are referred to as ‘mitocans’. Among potential
mitocans, phenolic acids are attractive candidates. Plant-derived phenolic
compounds are widely consumed in a normal diet, especially in fruits and
vegetables. Besides their antioxidant properties, phenolic acids have been
reported to display antiproliferative activity by promoting selective induction
of tumor cell apoptosis. In some cases, the molecule acts by triggering the
mitochondrial pathway for apoptosis. Here we review the potential role of
several phenolic acids and derivatives as anti-cancer agents, highlighting the
role of mitochondria as a primary subcellular target for this class of
compounds.  The present review intends to
raise awareness for this promising direction of research.

Key words

Antineoplastic drugs; Antioxidants; Hydroxybenzoic acids;
Mitochondria

RÉSUMÉ

Des nouvelles
molécules anticancéreuses potentielles sont découvertes quasi journellement.
Malheureusement la plupart de ces molécules n’exercent pas de spécificité
cellulaire et ne tuent pas plus les cellules tumorales que les cellules
normales. Un grand nombre de ces molécules sont par ailleurs toxiques. Dans le
but de développer des molécules anticancéreuses plus efficaces il est dès lors
important de comprendre l’interaction entre de nouvelles molécules et les
systèmes biologiques. Certains composants cellulaires sont particulièrement
appropriés comme cibles spécifiques pour leurs mécanismes d’action. Les
mitochondries sont la source principale d'énergie cellulaire, mais sont aussi
importantes dans le contrôle des processus qui aboutissent à la mort cellulaire
par apoptose. Certaines caractéristiques physiologiques des mitochondries,
comme leur potentiel de membrane, permettent une attaque spécifique des
mitochondries par des molécules anticancéreuses appelées «mitocans». Les acides
phénoliques sont candidats mitocans très intéressants. Les composés phénoliques
naturels sont abondamment présents dans l’alimentation, en particulier dans les
fruits et les légumes. Outre leurs propriétés antioxydantes il a été dans
certains cas démontré qu’ils possédaient des propriétés antiproliférative par
promotion d’une apoptose sélective par mécanisme mitochondrial. Dans cet
article nous revoyons le rôle potentiel de certains acides phénoliques et de
leurs dérivés comme agents anticancéreux, en insistant sur le rôle des
mitochondries comme cible subcellulaire pour cette classe de composés. Cette
revue de la littérature souligne l’importance de cette nouvelle voie
d’investigation.

Mots clés

Médicaments
anticancéreuses;  Antioxydants ;
Acides hydro-xybenzoïques; Mitochondries

RESUMEN

Todos los
días se descubren nuevas moléculas con actividad antineoplásica. Sin embargo,
la mayoría de ellas carecen de especificad celular, lo que se traduce en una
incapacidad para eliminar a las células tumorales en forma más eficiente que a
las células normales. Un número importante de estas moléculas también son
altamente tóxicas. En el diseño de fármacos quimioterapéuticos más efectivos es
importante entender la interacción entre las nuevas moléculas y los sistemas
biológicos. Algunos componentes celulares son especialmente relevantes en el
contexto de su especificidad y mecanismo de acción. Las mitocondrias, además de
ser la principal fuente de energía celular, también son importantes en el
control de la apoptosis. Algunos aspectos fisiológicos de las mitocondrias,
p.ej. el potencial eléctrico de membrana negativo, podrían ser un blanco
selectivo molecular de algunos anticancerígenos. Estos fármacos
antimitocon-driales específicos son citados en la literatura como ‘mitocanos’.
Los ácidos fenólicos son candidatos atractivos para actuar como mitocanos. Los
compuestos fenólicos derivados de plantas son ampliamente consumidos en la
dieta diaria, especialmente en las frutas y los vegetales. Además de su
actividad antioxidante, los ácidos fenólicos han mostrado actividad antiproliferativa
promoviendo la inducción selectiva de la apoptosis de las células tumorales; en
algunos casos, activando la vía mitocondrial de la apoptosis. En este
manuscrito hemos revisamos el papel potencial de varios ácidos fenólicos  y sus derivados como agentes anticancerígenos,
enfatizando el papel de las mitocondrias como el principal blanco subcelular de
esta clase de compuestos. Esta revisión intenta dirigir la atención a esta prometedora
línea de investigación.

Palabras clave

Medicamentos
antineoplásicos; Antioxidantes; Ácidos hidroxibenzoicos; Mitocondrias

INTRODUCTION

Significant progress has been made in cancer
research in recent decades. Profound changes are occurring in several new experimental
approaches currently under pursuit [1], including genetic strategies, recombinant biology,
irradiation, and anticancer chemotherapy. Despite improved pre-clinical data,
some cancers are not easy to control while others display a high rate of
remission. Chemotherapy has been one of the strategies with a higher success
rate, particularly when based on natural compounds. The use of
phytochemical-type molecules has emerged as a promising and pragmatic clinical
approach: besides reducing cancer risk, these molecules constitute a wide
family of natural compounds with a considerable range of important properties
such as low toxicity, low cost, and effectiveness when orally administrated [2].

Among phytochemicals, phenolic compounds
[such as phenolic acids, stilbenes, curcuminoids, vanilloids, chalcones, and
flavonoids (Figure 1)], are one of the most numerous
and ubiquitous groups of plant metabolites, and an integral part of the human
diet. Phenolic acids and derivatives are a particular phenolic class known to
display a wide variety of biological functions: besides their high antioxidant
capacity, phenolic acids have shown remarkable modulating properties in the
carcinogenic process. In this context, phenolic derivatives have been under
intense scrutiny, in some cases having a higher biological activity than their
parent compounds [2].

The determination of cellular target(s) for
these natural compounds, as well as the characterization of the corresponding
molecular mechanisms of action, can lead to the design of new and more
effective agents based on the chemical structure of the parent (lead) compound.
One possible experimental strategy is the development of molecules capable of
triggering the mitochondrial pathway for apoptosis. Targeting particular
aspects of mitochondrial physiology may provide an effective strategy to
selectively trigger apoptosis in cancer cells. In fact, there are several
differences between normal and transformed cells concerning the main routes for
ATP production: although neoplastic cells are mostly dependent on glycolysis
for ATP generation, mitochondria are usually functional, with other roles on
cellular physiology besides ATP production [3]. Mitochondria also participate in the regulation of
intracellular calcium and iron concentrations, as well as in several
biosynthetic pathways. Also, production of reactive oxygen species (ROS), which
might be harmful if excessively produced, and more importantly, the regulation
of cell death pathways, are major roles of mitochondria in cancer cells.
Finding a strategy to destabilize mitochondrial homeostasis can lead to
compromised integrity and to the elimination of malignant cells [4].

Figure 1

Examples of phenolic systems that can be
used to investigate potential chemotherapeutic properties by inducing the mitochondrial
pathway for apoptosis.

CHEMISTRY OF PHENOLIC ACIDS AND DERIVATIVES

Structural diversity is one of the main
characteristics of phenolic systems, which constitute one of the most numerous
and ubiquitous families of plant metabolites. Phenolic molecules display an
impressive array of structures (more than 8,000 compounds have been described
to date) classified into 10 different groups according to their chemical
characteristics [5].

Apart from variations in the carbon skeleton,
these compounds differ in the hydroxylation pattern of the phenolic ring, in
the presence of alkyl ester or amide side chains, in the occurrence of stereoisomers,
in the tendency for oligomerization in condensed phases, or in the conjugation
to amines and sugars [6]. The biological activity of polyphenols is known to
be largely by this structural wealth as much as by their antioxidant potency.
In fact, their biochemical properties have been shown to be highly
structure-dependent [7]: bioactivity varies considerably upon minor
structural modifications, since these often induce significant conformational
changes [8]. The bioavailability of phenolic compounds is
directly related to their lipophilicity, which, in turn, is strongly influenced
by chemical and structural preferences.

Several structure-activity relationship
(SAR’s) studies carried out on promising anticancer phenols [9] have shown that geometrical preferences are mainly
determined by electrostatic factors, as well as by the formation of both intra-
and intermolecular hydrogen bonds. Consequently, the most stable geometries
display a clear preference for planarity. In the case of hydroxycinnamates, the
presence of an ethylenic spacer in the carbon chain allows the formation of a
conjugated system, strongly stabilized through p-electron delocalization.

An E-orientation of the aromatic ring and the
C=O group relative to the carbon chain double bond was verified to be essential
for the cytotoxic activity of caffeates and their derivatives [10]. The well known ability of phenolic compounds to form
dimeric structures through OH•••O and/or CH•••O close contacts [11] should be taken into account, since their
conformation and, consequently, their chemical properties vary considerably
upon dimerization.

Furthermore, the biological activity of these
systems has been reported to be affected by the introduction of alkyl groups in
the carboxylic moiety of the molecule (ester or amide formation) [12]. Several reports describe a higher growth-inhibition
and cytotoxic activity of phenolic esters relative to their acid counterparts [13]. Regarding the phenolic aromatic ring substitution
pattern, trihydroxylated phenolic acids and esters were found to display a
greater cytotoxicity, as well as antiproliferative properties, in comparison
with their dihydroxylated analogues [14]. Preliminary studies on the antioxidant activity of
trihydroxy- and dihydroxyphenolic acids showed that the former reveal a
significantly higher activity [15]. Concerning the relevance of the degree of saturation
of the carbon chain, it has been described that the presence of a double bond
in cinnamates is associated with increased cell viability as opposed to the
saturated compounds [16], which may be partially related to the increased
antioxidant ability of the unsaturated phenols [17].

The biological behavior of phenolic systems
may also be affected by their conjugation with other biomolecules (e.g.
polyamines and glycosides), either by intracellular metabolic processes, or
even by in vivo absorption and transport mechanisms. Since in vivo biological
efficacy of phenolic compounds is influenced by bioavailability, knowledge of
these types of structural factors is considered crucial for predicting the
absorption versus concentration and metabolic profile.

On the other hand, a thorough understanding at
the molecular level of the physicochemical properties (such as partition coefficients
or redox behavior) and structural preferences including hydrogen-bonding motif
of phenolic systems is essential for establishing the
structure-property-activity relationships, allowing for optimization as
antineoplastic agents with selective targeting.

Phenolic derivatives aimed for
chemopreventive or chemotherapeutic applications can thus be rationally
designed, through tailored chemical changes, in light of the structure-activity
relationships or structure-property-activity relationships. Benzoic and
cinnamic acids, as well as the phenolic flavonoids, have been extensively used
as templates for this purpose, as the chemical modification of the lead
phenolic compound is an important research area in medicinal chemistry.

APOPTOSIS

Transformed cells in cancer tissue are
normally characterized by excessive proliferation, accompanied by the loss of
control of cell processes. Activation of cell death by chemotherapy is beneficial
if occurring in pre-neoplastic or tumor cells, but it may result in toxicity
when taking place in normal cells. Of obvious clinical interest, some phenolic
compounds were found to selectively induce time- and dose-dependent apoptosis
only in immortalized (malignant) cells [18].

Apoptosis is a programmed cell death
mechanism, finely regulated and genetically encoded. A typical phenotype
consists in a progressive shrinkage of cells and formation of apoptotic bodies,
as well as nuclear (chromatin condensation and nuclear fragmentation) and
plasma membrane (e.g. phosphatidylserine exposure) alterations. Apoptosis can
be monitored through the evaluation of several markers such as decreased
mitochondrial membrane potential (mitochondrial depolarization), enhanced
mitochondrial release of cytochrome c, increased caspase activation, chromatin
condensation, or cleavage of Poli-ADP-ribose polymerase [19], among others. Apoptosis develops in different
stages: 1) an initiation phase, which depends on the nature of the lethal
signal; 2) a decision phase, characterized by equilibrium between pro- and
anti-apoptotic molecules levels and 3) a common degradation phase, when the
cell ceases its function [20]. However, cells that are deficient in cell death
processes may not exactly follow this sequence. Programmed cell death can
follow two major and well characterized routes, the extrinsic and the intrinsic
pathway. The former is a death receptor-dependent pathway, consisting in the
regulation of death receptors located on the cell surface. The intrinsic route
is mitochondrial-dependent and includes the release of apoptotic signals from
the mitochondrial inter-membrane space [21].

In the extrinsic pathway, a cell death signal
is transduced within cells, either by the activation of the receptor-mediated
process (absence of the corresponding ligands), or by the ligand-induced
activation of receptors such as TNFR1, Fas, CD95/Fas (the receptor of
CD95L/FasL) and of the tumor necrosis factor-a (TNF-a) receptor-1 [22]. In some cases, activated TNF receptors have been
demonstrated to promote a different signalosome through NF-kB activation,
leading to a release of inflammatory cytokines and to anti-apoptosis responses.
TNF can also target angiogenesis, inducing tumor necrosis, overall having an
antiproliferative activity [23]. If coupled to pro-apoptotic processes, death-inducible
signaling complexes (DISC) are formed and cause activation of pro-caspase-8.It
has been demonstrated that caspase-8 can activate pro-caspase-3, which then
cleaves target proteins [21].

Also, in several cell types, caspase-8 first
cleaves Bid (a Bcl-2 family protein) which, in turn, is directed to
mitochondria or induces translocation, oligomerization and insertion of Bax
and/or Bak, another Bcl-2 family member. Mitochondrial permeabilization follows,
which leads to the release of several proteins from the mitochondrial
inter-membrane space, including cytochrome c. A cytosolic complex is formed in
the presence of dATP, comprising cytochrome c, the apoptosis activating
factor-1 (Apaf-1) and pro-caspase-9, resulting in caspase-9 activation, which
triggers caspase-3 activity and signal amplification [22].

The intrinsic pathway is also characterized
by the initial role of subcellular compartments such as the nucleus, lysosomes,
the endoplasmic reticulum, or the cytosol itself, originating death-promoting
stimuli that will act (directly or indirectly) on mitochondria.
Permeabilization of the mitochondrial outer membrane (MOM) can occur, leading,
as described above, to the release of intermembrane space proteins, including
caspase activators such as cytochrome c, Omi/HtrA2 (Omi stress-regulated
endoprotease/High temperature requirement protein A 2) and Smac/Diablo (second
mitochondria-derived activator), as well as caspase-independent death effectors
such as the apoptosis-inducing factor (AIF) and endonuclease G. AIF,
endonuclease G and HtrA2/Omi are then directly translocated to the nucleus,
triggering chromatin condensation and the appearance of high-molecular weight
chromatin fragments [19].

Also, as previously referred, cytochrome c
promotes activation of caspase-9, while Omi/HtrA2 and Smac/Diablo indirectly
favor the caspase cascade by antagonizing the activity of the inhibitor of
apoptosis proteins (IAPs), an endogenous caspase inhibitor. Besides IAP
activation, another mechanism capable of rescuing cells from death involves
Bcl-2 and Bcl-xL, which act by preventing the permeabilization of MOM and the
release of proteins from the inter-membrane space [24]. Interestingly, studies have demonstrated that Bcl-2
proteins are strongly expressed in human breast cancer cells; in particular,
Bcl-xL expression confers resistance to chemotherapy-induced apoptosis [25].

It has also been described that the intrinsic
pathway for apoptosis can be triggered by the induction of the mitochondrial
permeability transition (MPT), which involves the formation of protein pores
that span the inner and outer mitochondrial membranes, resulting in
mitochondrial depolarization, oxidative stress, and in some cases in outer
membrane rupture due to matrix expansion, allowing pro-apoptotic proteins to
escape to the cytosol [26]. The degree of MPT induction among the mitochondrial
network, and the consequent decrease of mitochondrial ATP production,
determines if apoptosis can be redirected to necrosis [27].

MITOCHONDRIA AS CHEMOTHERAPEUTIC TARGET

The discovery that mitochondria play a
critical role in the process of cell death promoted a radical change in cell
death research. Several oxidative stress-related diseases, in particular
cancer, involve the understanding of mitochondrial-mediated apoptosis [3].

Mitochondria are thus potential targets for
anti-cancer therapy. Different processes are suggested to interfere with vital
mitochondrial functions in cancer cells. The vast majority of conventional
anti-cancer drugs indirectly exploit the activation of intrinsic pathways in
order to exert their cytotoxic action by using multiple activation routes (e.g.
p53 or death receptors) [3]. Conventional anticancer agents, as well as numerous
dietary components and micronutrients, have considerable potential for
hindering in vivo deleterious oxidative processes and for inhibiting
carcinogenesis through induction of apoptosis in neoplastic cells, in some
cases via mitochondria-mediated pathways [28].

As discussed in the previous section, MOM
permeabilization is generally regarded as a crucial control point in the
commitment of a cell to apoptosis. During normal physiology, the MOM is considered
freely permeable to solutes and small metabolites (approximately 5 kDa) due to
the presence of the abundant protein voltage-dependent anion channel (VDAC),
which allows the diffusion of such molecules through the MOM [29]. MOM rupture due to excessive osmotic influx can be
lethal, not only by releasing caspase-activating molecules and
caspase-independent death effectors, but also by initiating metabolic failure
of mitochondria. For example, phenols such as gallic acid down-regulate Bcl-xL
protein and activate Bak, Bax and Bad proteins, causing the release of mitochondrial
cytochrome c to the cytosol, through MOM permeabilization [30].

Furthermore, oncogene products modulate MOM
permeability by regulating Bcl-2 family proteins, which modulate the
bioenergetic metabolite flux and putative components of the MPT pore. In this regard,
phenolic acids may act through similar mechanisms. Several authors have
reported that CAPE (caffeic acid phenyl ester)
is cytotoxic against tumors, but not against normal cells, the mechanism
involving mitochondria through altered Bcl-2 protein expression [31]. A number of other experimental chemotherapeutic
agents have the opposite effect, acting on mitochondrial lipids and proteins
and inhibiting MPT pores opening through direct binding to certain protein
components [32].

A general rule is that antioxidants act as
MPT inhibitors, decreasing or hindering the oxidative stress responsible for
oxidation of proteins of the pore complex, and is a prime cause for the consequent
inner membrane permeabilization [33].

In contrast to the MOM, the inner membrane is
impermeable to the majority of ions, including protons. Membrane selectively is
important for maintaining the proton gradient that is required for oxidative
phosphorylation [21]. It is at the inner mitochondrial membrane that ATP
synthesis occurs, through electron transfer coupled to proton ejection. Some
potential anticancer molecules can also inhibit the respiratory chain,
promoting mitochondrial failure and the release of proteins from the
inter-membrane space, increasing the generation of reactive oxygen species
(ROS) and decreasing ATP generation. Ultimately, cell death occurs through
mitochondrial failure [3].

The selective accumulation of promising anticancer
molecules inside the mitochondria of tumor cells, rather than normal cells, is
a key point in the design of novel molecules tested in new pharmacological
strategies [34]. Several reports indicate that mitochondria have a
higher transmembrane electric potential in tumor cells than in normal,
non-neoplastic cells [35]. This difference has been used to induce a selective
accumulation of specific molecules in the mitochondria of tumor cells,
explaining its selective toxicity. We have reported a similar mechanism for the
alkaloid berberine, which was found to inhibit proliferation in a murine
metastatic melanoma cell line through a mitochondrial mechanism, mostly due to
enhanced mitochondrial accumulation [36].

BIOLOGICAL
ACTIVITY OF PHENOLIC ACIDS AND DERIVATIVES

Although naturally occurring phenolic acids
have been the subject of numerous studies in cells, information on the
molecular basis of their mechanisms of action at the sub-cellular level (e.g.
mitochondria) is still somewhat scarce [37]. Phenolic compounds are usually associated with a
beneficial effect on carcinogenesis principally due to their antioxidant
activity, which mainly occurs by a free radical scavenging process [38].

Other pathways have been suggested, including
the modulation of specific enzymes and signal transduction processes leading to
cell proliferation arrest, cell cycle regulation, inhibition of DNA synthesis,
or inhibition of angiogenesis and metastasis [39].

Phenolic acids can be subdivided into two
major groups, hydroxybenzoic acids and hydroxycinnamic acids. Hydroxybenzoic
acid derivatives include p-hydroxybenzoic, protocatechuic, vannilic, syringic,
and gallic acid. These derivatives can be found in plants in free or bound
forms, either as structural components of lignins and hydrolyzable tannins, or
as a sugar derivatives and organic acids in plant foods. Hydroxycinnamic acids
include p-coumaric, caffeic, ferulic, and sinapic acids. Like hydroxybenzoic
acids, these molecules are present in the bound form, linked by ester bonds to
components of the plant cell-wall such as cellulose, lignin and proteins [40].

Gallic acid and its esters are presently used
as antioxidant additives in both food and pharmaceutical industry – propyl
gallate (E-310) and octyl gallate (E-311) – since they are known to protect
against oxidative damage induced by reactive oxygen, nitrogen and sulphur
species [41]. Gallic acid derivatives have been described to cause
apoptosis in tumor cell lines, to inhibit lymphocyte proliferation and to
inhibit protein tyrosinase kinase (PTK’s) activity [42].

Among hydroxycinnamic acids, caffeic acid is
one of the most relevant and potent antioxidants found in the human diet,
causing a significantly decrease in in vitro oxidative damage to DNA [43]. It has been demonstrated that the antioxidative
capacity of caffeic acid attenuates the proliferation of Vascular Smooth Muscle
Cells (VSMC) induced by angiotensin II stimulation, through a mechanism that
involves inhibiting generation of reactive oxygen species generation and
partially blockade of the JAK/STAT and the Ras/Raf-1/ERK1/2 cascades [44]. As potential anti-carcinogenic agent, caffeic acid
inhibits cell growth during S-phase, causing further apoptosis in MCF-7 and
T47D human breast cancer cells by activating pro-apoptotic proteins such as Bax
and caspases [43] [45]. Alternatively, caffeic acid can also promote
apoptosis in a number of cancer cell lines by inhibiting the NF-kB pathway [45].

Interestingly, caffeic acid is mentioned in
some studies as a chemosensitizer, having a synergistic effect with doxorubicin
treatment on MCF-7 human breast cancer cells [43].Caffeic acid phenyl ester (CAPE) is a well-known
phenolic acid derivative with promising potential, possessing antioxidant,
anti-inflammatory, anti-viral, immunostimulatory, anti-angiogenic,
anti-invasive, and anti-metastatic activities [46]. CAPE, together with
other phenolic acid phenethyl esters, also possesses anti-cancer activity
against some human cancer cells [47]. In human ME180 cervical cancer cells, CAPE promotes apoptosis mediated by down-regulation of
Mcl-1 gene expression and activation of caspase-8 [46].

Moreover, CAPE
was also reported to decrease cell viability in human U937 myeloid leukemic
cells, in a dose and time-dependent manner. The apoptotic action of CAPE is mitochondrial-mediated and is accompanied by a
decrease in Bcl-2 expression, increase of Bax expression, release of cytochrome
c, activation/cleavage of caspase-3, and PARP cleavage [31].

Ferulic acid (4-hydroxy-3-methoxycinnamic
acid), is a cinnamic acid structurally related to caffeic acid, which is
present in rice bran and other plants.
Food processing, such as thermal processing, pasteurization,
fermentation, and freezing, contributes to the release of ferulic acid [40], which is rapidly absorbed and excreted as glucuronides
or sulphates in men. Radical scavenging activity experiments showed that
ferulic acid has antioxidant activity and that its esterification leads to a
modification of its efficacy [48]. Ferulic acid derivatives present in the diet possess
interesting biological effects including chemoprevention of colon and tongue
cancers. One particular example is 3-(4'-geranyloxy-3'-methoxyphenyl)-2-trans-propenoic
acid and its derivatives [49].

Curcumin is a phenolic derivative present in
plants and is formed by two ferulic acids linked by a methylene group, in a
diketone structure. The compound is the active ingredient of turmeric (Curcuma
longa) and displays anti-inflammatory, antioxidative and anticarcinogenic
properties, with an immense potential in cancer chemotherapy because of its
effect in cell growth regulatory mechanisms. Analysis of its structure revealed
that the presence of the beta-diketone moiety and phenolic groups in the
structure have a direct contribution to its antioxidant behavior [50]. Curcumin exhibits growth inhibitory activity against
prostate, colon and breast cancer [51]. Some studies evidence a selective growth-inhibiting
effect of curcumin on transformed cell lines, as compared to non-transformed
cell lines [52]. It was also previously demonstrated that the
anticarcinogenic action of curcumin in colon have the involvement of the
phenolic groups and the conjugated bonds in the central seven carbon chain [53].

Curcumin also affects various cell cycle
proteins and checkpoints, leading to the down-regulation of some cyclins and
cyclin-dependent kinases, as well as to up-regulation of cdk inhibitors, and to
inhibition of DNA synthesis [54]. Of special relevance is the fact that curcumin can
also induce apoptosis by targeting mitochondria, affecting p53-related
signaling and blocking NF-kB activation, in a similar fashion to caffeic acid [50]. Prostate cancer cells can be sensitized with
curcumin to TRAIL treatment, inducing both the extrinsic and the intrinsic
pathways for apoptosis, which suggests that curcumin is a beneficial adjunct
against chemoresistance to standard therapeutic drugs [54].

Phenolic compounds in general have been
reported to affect nitric oxide (NO) production and its activity on physiologic
functions. The phenolic acid 3,4-dihydroxy-phenylacetic acid (PAA) decreases
the activity of NO synthase and consequently the possible production of
endogenous reactive nitrogen species, besides arresting cells in the S-phase
and inducing apoptosis of T47D breast cancer cells. The induction of apoptosis
in this cell line occurs via FKHRL1 (FOXO3a) kinase pathway, while in MCF-7, a different breast cancer
cell line, apoptosis is activated by NO via p53-associated pathways [45].

A second molecule, either a classic
anti-cancer molecule (doxorubicin, taxol, cis-platin, etc) or an alternative
molecule (a second phenolic acid or another natural phenolic compound), will be
able to trigger signaling pathways that will ultimately end up affecting the
already destabilized mitochondrial network. The outcome will be the release of
mitochondrial proteins including cytochrome c or the apoptosis-inducing factor
(AIF). These proteins are involved in the intrinsic pathway of apoptosis and
the cascade of events will cause tumor cell death. It is noteworthy to point
out that one advantageous aspect of mitochondrial physiology is that the
transmembrane electric gradient (negative inside) can be used to drive the
accumulation of lipophilic cations. Also, it is possible that at least part of
the mitochondrial transmembrane electric gradient is maintained during the
process of drug-induced cell killing, thus allowing mitochondrial ATP to be
used during apoptosis. A complete collapse of the cell energy processes
(mitochondrial oxidative phosphorylation and glycolysis) can drive the process
of cell death from apoptosis to necrosis

FUTURE
PERSPECTIVES

The efficacy of many clinically used
chemotherapeutic drugs is often compromised by acquired chemoresistance by
cancer cells. In fact, many cancer cells undergo mutations or loss of proteins
that are intimately involved in cell death (e.g. p53 and Bcl-2 proteins).
Phenolic derivatives present in fruits and vegetables, have long been
recognized to display antioxidant and antiproliferative activities, thus being
promising chemopreventive/chemotherapeutic compounds.

Exploiting the effect of these
bio-antioxidants on cellular processes will lead to new chemopreventive and
chemotherapeutic phenolic-based strategies that will hopefully be non-toxic to
healthy cells. In order to overcome chemoresistance, mitochondria are an
attractive biological target due to their major role in bioenergetics and vital
signaling in mammalian cell. One new approach suggested is the use of compounds
that specifically interact with the MOM, contributing to the release of
pro-apoptotic proteins contained in mitochondria. Secondly, since the
electrochemical gradient is apparently higher in mitochondria from cancer
cells, lipophilic cations can be preferentially accumulated in the
mitochondrial matrix [20].

Figure 2

A
proposed strategy in which the co-treatment of tumor cells with both a
phenolic acid or derivative and a second molecule results in toxicity for the
cancer cell. By altering the structure of the molecule (including modifying
the polarization/charges), it is possible to direct the phenolic acid or
derivative to mitochondria in order to create an initial destabilization in
the mitochondrial membranes.

These strategies rely on the condition that
derivatives from phenolic acids are small and positively charged, although
neutral molecules can also, in theory, accumulate in mitochondrial membranes. A
third method is based on the synergistic action of two or more compounds, the
first of which is used to sensitize mitochondria to undergo failure and trigger
apoptosis, if and when a second or a third molecule is added to the cell (Figure 2).

Although the presence of a positive moiety
does not guarantee that the molecules will be accumulated by mitochondria, it
is likely that once inside the cell lipophilic cations will tend to accumulate
within organelles with a negatively-charged environment. Thus, the secret for
success is to rationally design and synthesize phenolic acid derivatives that
can be selectively accumulated by mitochondria in tumor cells, in such a way
that the threshold for cytotoxicity exerted by a second molecule is
significantly lowered (Figure 2). The mechanism of
action results in a novel pharmacological strategy, coupling high efficacy
against neoplastic cells to a low toxicity towards non-tumor cells. The
advantage of using phenolic acids which are normally present in human diet is
that the toxicity threshold is already understood

AUTHORS’ PARTICIPATION

All authors participated in the composition
of the text, figures and review of the manuscript.

ACKNOWLEDGEMENTS

This work was supported by FCT (Portugal):
Project POCTI/QUI/55631/2004 (co-financed by the European Community fund FEDER)
and PhD scholarship SFRH/BD/38067/2007 (T.L.S.).

CONFLICT OF INTERESTS/DISCLAIMERS

PJO is member of the Editorial Board of the
journal. No other conflicts of interest exist.

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