Seminars in Cancer Biology 17 (2007) 370 376
Review
Oxidative breakage of cellular DNA by plant polyphenols: A putative
mechanism for anticancer properties
" 1 1,2
S.M. Hadi , Showket H. Bhat , Asfar S. Azmi , Sarmad Hanif, Uzma Shamim, M.F. Ullah
Department of Biochemistry, Faculty of Life Sciences, A.M.U., Aligarh 202002, UP, India
Abstract
Plant polyphenols are important components of human diet and a number of them are considered to possess chemopreventive and therapeutic
properties against cancer. They are recognized as naturally occurring antioxidants but also act as prooxidants catalyzing DNA degradation in the
presence of transition metal ions such as copper. We have shown that several of these compounds are able to bind both DNA and Cu(II) forming a
ternary complex. A redox reaction of the polyphenols and Cu(II) in the ternary complex may occur leading to the reduction of Cu(II) to Cu(I), whose
reoxidation generates a variety of reactive oxygen species (ROS). We have further confirmed that the polyphenol Cu(II) system is indeed capable of
causing DNA degradation in cells such as lymphocytes. We have also shown that polyphenols alone (in the absence of added copper) are also capable
of causing DNA breakage in cells. Neocuproine (a Cu(I) sequestering agent) inhibits such DNA degradation. It also inhibits the oxidative stress
generated in lymphocytes indicating that the cellular DNA breakage involves the generation of Cu(I) and formation of ROS. It is well established
that tissue, cellular and serum copper levels are considerably elevated in various malignancies. Therefore, cancer cells may be more subject to
electron transfer between copper ions and polyphenols to generate ROS. Thus, our results are in support of our hypothesis that anticancer mechanism
of plant polyphenols involves mobilization of endogenous copper possibly chromatin bound copper and the consequent prooxidant action.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: Plant polyphenols; Endogenous copper; Prooxidant; Anticancer; Apoptosis; Reactive oxygen species (ROS)
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
2. Oxidative DNA cleavage by plant polyphenols in vitro in the presence of copper ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
3. Polyphenol Cu(II) mediated DNA breakage in human peripheral lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
4. Evidence for the prooxidant action of plant polyphenols as an important mechanism for their anticancer properties . . . . . . . . . . . . . . . . 372
5. Oxidative DNA breakage by plant polyphenols in human peripheral lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
6. Putative mobilization of endogenous copper by plant polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
1. Introduction
resveratrol, anthocyanidins such as delphinidin possess a wide
range of pharmacological properties the mechanisms of which
Plant-derived polyphenolic compounds that include flavo- have been the subject of considerable interest (Fig. 1). They are
noids, tannins, curcuminoids, gallocatechins, stilbenes such as
recognized as naturally occurring antioxidants and have been
implicated as antiviral and antitumor compounds [1,2]. In recent
years, a number of reports have appeared which have shown
"
Corresponding author. Tel.: [phone]; fax: [phone].
that gallocatechins found in green tea and which include tan-
E-mail address: [e-mail] (S.M. Hadi).
nic acid, gallic acid, epigallocatechin, epicatechin-3-gallate and
1
These authors contributed equally.
2
epigallocatechin-3-gallate (EGCG) induce apoptosis in various
Present address: Department of Pathology, Karmanos Cancer Institute,
Wayne State University School of Medicine, Detroit, MI 48201, USA. cancer cell lines [3,4]. Similarly curcumin [5] from the spice
1044-579X/$  see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.semcancer.2007.04.002
S.M. Hadi et al. / Seminars in Cancer Biology 17 (2007) 370 376 371
Fig. 1. Structures of (a) gallic acid, (b) syringic acid, (c) curcumin, (d) resveratrol, (e) delphinidin, (f) epigallocatechin-3-gallate, and (g) quercetin.
turmeric and resveratrol [6] which is found in grapes and red even at relatively high concentrations. Further they may also act
wine have also been shown to be inducers of apoptosis in cancer as lead compounds for the synthesis and development of novel
cells. The consumption of green tea is considered to reduce the anticancer drugs.
risk of various cancers such as that of bladder, prostate, esopha-
gus and stomach [4]. Of particular interest is the observation that 2. Oxidative DNA cleavage by plant polyphenols in vitro
EGCG was found to induce internucleosomal DNA fragmenta- in the presence of copper ions
tion in cancer cell lines such as human epidermoid carcinoma
cells, human carcinoma keratinocytes, human prostate carci- Studies in our laboratory have shown that a number of plant
noma cells, mouse lymphoma cells but not in normal human polyphenols such as flavonoids [8], tannic acid and its structural
epidermal keratinocytes [4]. Similarly gallic acid showed cyto- constituent gallic acid [9], curcumin [10], gallocatechins [11]
toxicity for a number of tumor cell lines but primary cultured and resveratrol [12] cause oxidative strand breakage in DNA
rat hepatocytes and macrophages were found to be refractory to either alone or in the presence of transition metal ions such
the cytotoxic effect [3]. Resveratrol also was shown to induce as copper. Recent studies by Liu and co-workers [13] demon-
apoptotic cell death in HL60 human leukemia cell lines but not strated that resveratrol as well as its certain synthetic analogs
in normal peripheral blood lymphocytes [6]. The hallmark of namely 3,4,4 -trihydroxy-trans-stilbene, 3,4-dihydroxy-trans-
apoptosis is internucleosomal DNA fragmentation, which dis- stilbene, 3,4,5-trihydroxy-trans-stilbene, which are generally
tinguishes it from necrosis. Other changes such as shrinkage effective antioxidants, can switch to prooxidants in the presence
of cells, membrane blebbing and the dissociation of the nucleus of Cu(II) to induce DNA damage. Copper is an important metal
into chromatoid bodies also occur. It is to be noted that most clin- ion present in chromatin and is closely associated with DNA
ically used anticancer drugs can activate late events of apoptosis bases particularly guanine [14,15]. It is also one of the most
(DNA degradation and morphological changes) and there are redox active of the various metal ions present in cells. We have
differences in essential signalling pathways between pharmaco- also shown that the flavonoid quercetin [16] and curcumin [10]
logical cell death and physiological induction of programmed are capable of binding to DNA and copper. Evidence deduced in
cell death [7]. Based on our own observations and those of our laboratory has shown that polyphenols such as the flavonoid
others we propose a mechanism of DNA fragmentation in can- quercetin and the stilbene resveratrol can not only bind copper
cer cells by plant polyphenolics that involves mobilization of ions but also catalyze their redox cycling [12]. In the case of
intracellular copper. Studies on chemopreventive and therapeu- quercetin a mechanism was proposed which involved the for-
tic plant-derived phytonutrients assume significance in view of mation of a ternary complex of DNA quercetin Cu(II) [17,16].
the fact that such compounds exhibit negligible or low toxicity A redox reaction of the compound and Cu(II) in the ternary com-
372 S.M. Hadi et al. / Seminars in Cancer Biology 17 (2007) 370 376
derived polyphenolics rather than their antioxidant activity may
be an important mechanism for their anticancer and apoptosis
inducing properties:
(1) Structure activity studies carried out in our laboratory with
gallic acid (a structural constituent of tannic acid) indicate
that if two of the three hydroxyl groups are methylated
(syringic acid), the DNA degrading capacity decreases
sharply [9]. The results correlate with those of Inoue et al.
[3] who showed that modification of hydroxyl groups, such
as that resulting in the formation of syringic acid, abolishes
the apoptotic activity of gallic acid.
(2) Apoptotic DNA fragmentation properties of several anti-
cancer drugs [21,22] and -radiation [23] are considered
to be mediated by ROS. It may also be mentioned that
doxorubicin induced apoptosis in human osteocarcinoma
Fig. 2. Involvement of a ternary complex of quercetin, DNA and Cu(II)/Cu(I)
Saos-2 cells is mediated by ROS and is independent of
in the generation of active oxygen species. The oxidized forms of quercetin
p-53 [24]. Interestingly, certain properties of polyphenolic
(represented as quercetin*) are not necessarily identical.
compounds, such as binding and cleavage of DNA and the
generation of ROS in the presence of transition metal ions
plex may occur leading to the reduction of Cu(II) to Cu(I), whose
[16] are similar to those of certain known anticancer drugs
reoxidation generates a variety of ROS (Fig. 2). Most of the phar-
[25].
macological properties of plant polyphenols are considered to
(3) Fe3+ and Cu2+ are the most redox-active of the metal ions
reflect their ability to scavenge endogenously generated oxygen
in living cells. Wolfe et al. [26] have proposed that a copper
radicals or those free radicals formed by various xenobiotics,
mediated Fenton reaction, generating site-specific hydroxyl
radiation etc. However, some data in the literature suggests that
radicals, is capable of inducing apoptosis in thymocytes.
the antioxidant properties of the polyphenolic compounds may
In a study with thiol-containing compounds, apoptosis was
not fully account for their chemopreventive effects [18,19]. Most
induced in different cell lines when either free copper or
plant polyphenols possess both antioxidant as well as prooxidant
ceruloplasmin (a copper binding protein) was added; such
properties [3,8] and we have proposed that the prooxidant action
activity was not observed, however, when either free iron
of plant polyphenolics may be an important mechanism of their
or the iron-containing serum protein transferrin was added
anticancer and apoptosis inducing properties [19]. Such a mech-
[27]. Most of the copper present in human plasma is associ-
anism for the cytotoxic action of these compounds against cancer
ated with ceruloplasmin, which has six tightly held copper
cells would involve mobilization of endogenous copper ions and
atoms and a seventh, easily mobilized one [28]. In another
the consequent prooxidant action.
study supporting these observations, copper was found to
enhance the apoptosis-inducing activity of polyphenolic
3. Polyphenol Cu(II) mediated DNA breakage in
antioxidants, whereas iron was inhibitory [29]. Although
human peripheral lymphocytes
iron is considerably more abundant in biological systems,
the major ions in the nucleus are copper and zinc [15]. Fur-
Using a cellular system of lymphocytes isolated from human
ther, although in general, tumors are considered to contain
peripheral blood and alkaline single cell gel electrophoresis
less total iron and have less iron saturation in ferritin than
(comet assay), we have confirmed that polyphenol Cu(II) sys-
do normal cells that is not always the case [30]. As already
tem is indeed capable of causing DNA degradation in cells such
mentioned copper ions occur naturally in chromatin and
as lymphocytes [20]. Further, the DNA degradation of lympho-
can be mobilized by metal chelating agents. Burkitt et al.
cytes is inhibited by scavengers of ROS and neocuproine, a
[31] suggested that the internucleosomal DNA fragmenta-
Cu(I) specific sequestering agent. Also, similar to the in vitro
tion might be caused not only by endonuclease but also by
results, trans-stilbene which does not have any hydroxyl group
metal-chelating agents such as 1,10-phenanthroline (OP),
is inactive in the lymphocyte system. These findings demon-
which promotes the redox activity of endogenous copper
strate that the polyphenol Cu(II) system for DNA breakage is
ions and the resulting production of hydroxyl radicals. Thus,
physiologically feasible and could be of biological significance.
the internucleosomal DNA  laddering often used as an
indicator of apoptosis may also reflect DNA fragmentation
4. Evidence for the prooxidant action of plant by non-enzymatic processes. Several reports indicate that
polyphenols as an important mechanism for their serum [32,33], tissue [34] and intracellular copper levels
anticancer properties in cancer cells [35] are significantly increased in various
malignancies. Indeed, such levels have been described as
We give below several lines of indirect evidence in literature, a sensitive index of disease activity of several hematologic
which strongly suggest that the prooxidant action of plant- and non-hematologic malignancies [36].
S.M. Hadi et al. / Seminars in Cancer Biology 17 (2007) 370 376 373
(4) A comparison of the properties of complexes formed nuclear fragmentation and internucleosomal DNA cleavage
between plant polyphenolics and Cu2+ and Fe3+ should in human myelogenous leukemia cell lines [41]. The apopto-
indicate which of these two metal ions could lead to DNA sis inducing activity of ascorbic acid has been ascribed to its
fragmentation in the nucleus when complexed. Not much prooxidant action and is inhibited by catalase, antioxidants
is known about the properties of such complexes. However, like N-acetylcysteine and GSH, Ca2+ and Fe3+ depletion but
considerable information is available about OP chelation stimulated by H2O2, Cu2+, and iron chelators [42].
of copper and iron ions. Burkitt et al. [31] cited several (7) It has recently been shown that the polyphenol curcumin
reasons why Cu2+ rather than Fe3+ may be responsible for mediated apoptosis of HL60 cells is closely related to an
OP-stimulated internucleosomal DNA fragmentation in iso- increase in the concentrations of ROS possibly generated
lated nuclei. For example, the cumulative affinity constants through the reduction of transition metals in cells [43].
( 3 in 0.1 M salt) for chelation of various metal ions by OP
5. Oxidative DNA breakage by plant polyphenols in
are in the order Cu2+ H" Fe2+ >Zn2+ >Fe3+. The complex
formed between OP and Cu2+ has a redox potential (Eć% human peripheral lymphocytes
for Cu2+/Cu+ = 0.17 V) that favors redox cycling, whereas
Using the comet assay our laboratory has shown that plant
that for Fe3+/Fe2+ is 1.1 V, presumably because of stabiliza-
polyphenols cause DNA breakage in isolated human peripheral
tion in the ferrous state. Copper is also shown to be present
lymphocytes [44]. Photographs of comets seen on treatment with
in chromosomes as Cu+ ions because of stabilization in the
different concentrations of resveratrol are shown in Fig. 3. At
presence of DNA. This overcomes the need for the reduction
50 and 100 M concentrations resveratrol did not damage the
of Cu2+ to Cu+ and can directly generate the hydroxyl rad-
lymphocyte DNA to any significant extent whereas at 200 M
icals. Finally, copper and zinc are major metal ions present
concentration a comet with a tail indicative of DNA breakage
naturally in chromosomes [15]. Because most polypheno-
was observed. The results demonstrate that resveratrol alone
lics are also polycyclic compounds similar in size to OP,
is capable of DNA breakage in lymphocytes. We have further
conceivably their metal binding properties are also similar.
shown that several other polyphenols such as gallic acid, EGCG
(5) Evidence suggests that the antioxidant properties of
and piceatannol are also able to catalyze DNA breakage in lym-
polyphenolics may not fully account for their chemopre-
phocytes. Table 1 gives the results of an experiment where three
ventive effects. For example, it was shown that although
scavengers of ROS have been tested namely, SOD and catalase,
ellagic acid is an antioxidant ten times more potent than
which remove superoxide and H2O2, respectively and thiourea,
tannic acid, the latter was more effective in inhibiting skin
which is a scavenger of several ROS. All three cause signif-
tumor promotion by 12-O-tetradecanoyl phorbol-13 acetate
icant inhibition of DNA breakage as evidenced by decreased
than the former [18]. It was suggested that the antioxi-
tail lengths. We concluded that superoxide anion and H2O2 are
dant effects of these polyphenols might be essential but not
essential components in the pathway that leads to the forma-
sufficient for their antitumor promotion. In any case ROS
tion of hydroxyl radical and other species which would be the
scavenging properties of plant polyphenols may account
proximal DNA cleaving agents.
for their chemopreventive effects but not for any thera-
peutic action against cancer cells [22]. Expression of the
6. Putative mobilization of endogenous copper by plant
bcl-2 proto-oncogene, which blocks apoptosis, decreases
polyphenols
cellular production of ROS [37], whereas the coadministra-
tion of antioxidant enzymes such as superoxide dismutase
In a previous study [20] we have shown that the
(SOD) and catalase prevents curcumin mediated apoptosis
resveratrol Cu(II) mediated DNA degradation of lymphocyte
in human leukemia cells [5]. Further it has been shown that
DNA is inhibited by neocuproine which is a Cu(I) specific
the programmed cell death induced by curcumin in human
chelating agent and is membrane permeable [45]. Fig. 4 gives
leukemic T-lymphocytes is independent of the involvement
the results of an experiment where three progressively increas-
of mitochondria and caspases suggesting the existence of
ing concentrations of neocuproine were tested on resveratrol
pathways other than the  classical ones [38]. Caspases are
induced DNA breakage in lymphocytes. A progressive decrease
essential for both Fas and mitochondria mediated apop-
in the tail length as a function of increasing neocuproine con-
tosis. However, inhibition of caspases or the use of cells
with defective apoptosis machinery has demonstrated that
Table 1
alternative types of programmed cell death could occur as Effect of scavengers of active oxygen species on resveratrol induced lymphocyte
DNA breakage
well and such alternative death mechanisms are divided into
 apoptosis-like and  necrosis-like [39].
Dose Tail length ( m) Inhibition (%)
(6) Ascorbic acid is an essential micronutrient and is considered
Untreated 1.22 Ä… 0.08a 
to have an antioxidant function in living systems. However,
Resveratrol (200 M) 20.84 Ä… 1.28 
it may also act as a prooxidant and site specific DNA cleav-
Resveratrol + SOD (100 g/ml) 7.05 Ä… 0.25* 66
age by ascorbic acid in the presence of Cu(II) has been Resveratrol + catalase (100 g/ml) 8.86 Ä… 0.29* 57
Resveratrol + thiourea (1 mM) 11.93 Ä… 1.01* 45
described [40]. Relatively high concentrations of ascor-
a
bic acid are able to induce apoptosis in various tumor cell
All values represent S.E.M. of three independent experiments.
*
lines. Consequently, it has been shown to induce cell death, P <0.05 when compared to control.
374 S.M. Hadi et al. / Seminars in Cancer Biology 17 (2007) 370 376
Fig. 3. Alkaline single cell gel electrophoresis (comet assay) of human peripheral lymphocytes showing comets (100×) after treatment with different concentrations
of resveratrol: (A) untreated, (B) 50 M, (C) 100 M and (D) 200 M.
centration was seen. From the results we concluded that the
DNA breakage by the polyphenol involves endogenous copper
ions and that Cu(I) is an essential intermediate that leads to
DNA breakage. We presume that the lymphocyte DNA break-
age is the result of the generation of hydroxyl radicals and
other ROS in situ. Oxygen radical damage to deoxyribose or
DNA is considered to give rise to thiobarbituric acid reactive
substance (TBARS) [46,47]. We therefore determined the for-
mation of TBARS in lymphocytes as a measure of oxidative
stress with increasing concentrations of resveratrol. The effect
of preincubating the cells with neocuproine and thiourea was
also studied. Results given in Fig. 5 show that there is a dose
dependent increase in the formation of TBARS in lymphocytes.
However, when the cells were preincubated with neocuproine
and thiourea there was a considerable decrease in the rate of
formation of TBARS by resveratrol. These results indicate that
both DNA breakage and oxidative stress in cells is inhibited by
Cu(I) chelation and scavenging of ROS. Similar results were
obtained with caffeic acid [48], a major polyphenolic compo- Fig. 5. Effect of preincubation of lymphocytes with neocuproine and thiourea on
TBARS generated by increasing concentrations of resveratrol. Resveratrol alone
nent of coffee. Thus, it can be concluded that the formation of
( ); resveratrol + neocuproine (1 mM) (©); resveratrol + thiourea (1 mM) ( ).
ROS by polyphenols in lymphocytes involves their interaction
The isolated cells (1 × 105) suspended in RPMI 1640 were preincubated with the
with intracellular copper as well as its reduction to Cu(I). ć%
indicated concentrations of neocuproine and thiourea for 30 min at 37 C. After
It is well known that polyphenols autooxidize in cell cul-
pelleting the cells were washed twice with PBS (Ca2+ and Mg2+ free) before
resuspension in RPMI and further incubation for 1 h in the presence of increasing
ture media to generate H2O2 and quinones that can enter cells
resveratrol concentrations. Viability of lymphocytes after preincubation with
causing damage to various molecules [49 51]. This may lead to
neocuproine and thiourea was more than 90%. Values reported are Ä…S.E.M. of
three independent experiments.
extracellular production of ROS that could account for lympho-
cyte DNA breakage. However, this does not appear to be the case
in our system since we have previously shown that no lympho-
cyte DNA breakage is observed on preincubating the cells with
resveratrol alone up to a concentration of 50 M. DNA breakage
could only be seen after incubating the pre-treated cells further
in the presence of Cu(II) [20]. Further, we could not detect any
H2O2 formation on incubating resveratrol (up to a concentration
of 300 M) in the suspension medium (RPMI) [44].
7. Conclusions
Most studies on anticancer mechanisms of plant polyphenols
invoke the induction of cell cycle arrest at S/G2 phase transition
brought about by an increase in cyclins A and E and inactivation
of cdc 2. Other mechanisms have also been proposed [52]. In
Fig. 4. Effect of increasing concentrations of neocuproine on resveratrol induced
view of the above findings in our laboratory and those of oth-
DNA breakage in human lymphocytes. Values reported are Ä…S.E.M. of three
independent experiments. ers in literature we suggest that the conclusion of our studies is
S.M. Hadi et al. / Seminars in Cancer Biology 17 (2007) 370 376 375
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Acknowledgment
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FEBS Lett 2005;579:3131 5.
The authors acknowledge the financial assistance provided
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