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Seminars in Cancer Biology 17 (2007) 370–376

Review

Oxidative breakage of cellular DNA by plant polyphenols: A putativemechanism for anticancer properties

S.M. Hadi ∗, Showket H. Bhat 1, Asfar S. Azmi 1,2, Sarmad Hanif, Uzma Shamim, M.F. UllahDepartment of Biochemistry, Faculty of Life Sciences, A.M.U., Aligarh 202002, UP, India

bstract

Plant polyphenols are important components of human diet and a number of them are considered to possess chemopreventive and therapeuticroperties against cancer. They are recognized as naturally occurring antioxidants but also act as prooxidants catalyzing DNA degradation in theresence 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 aernary 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), whoseeoxidation generates a variety of reactive oxygen species (ROS). We have further confirmed that the polyphenol–Cu(II) system is indeed capable ofausing DNA degradation in cells such as lymphocytes. We have also shown that polyphenols alone (in the absence of added copper) are also capablef causing DNA breakage in cells. Neocuproine (a Cu(I) sequestering agent) inhibits such DNA degradation. It also inhibits the oxidative stressenerated in lymphocytes indicating that the cellular DNA breakage involves the generation of Cu(I) and formation of ROS. It is well establishedhat tissue, cellular and serum copper levels are considerably elevated in various malignancies. Therefore, cancer cells may be more subject tolectron transfer between copper ions and polyphenols to generate ROS. Thus, our results are in support of our hypothesis that anticancer mechanismf plant polyphenols involves mobilization of endogenous copper possibly chromatin bound copper and the consequent prooxidant action.

2007 Elsevier Ltd. All rights reserved.

eywords: Plant polyphenols; Endogenous copper; Prooxidant; Anticancer; Apoptosis; Reactive oxygen species (ROS)

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3702. Oxidative DNA cleavage by plant polyphenols in vitro in the presence of copper ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3713. Polyphenol–Cu(II) mediated DNA breakage in human peripheral lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3724. Evidence for the prooxidant action of plant polyphenols as an important mechanism for their anticancer properties . . . . . . . . . . . . . . . . 3725. Oxidative DNA breakage by plant polyphenols in human peripheral lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3736. Putative mobilization of endogenous copper by plant polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373

7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374

Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375. . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Plant-derived polyphenolic compounds that include flavo-oids, tannins, curcuminoids, gallocatechins, stilbenes such as

∗ Corresponding author. Tel.: +91 571 2700741; fax: +91 571 2706002.E-mail address: smhadi@vsnl.com (S.M. Hadi).

1 These authors contributed equally.2 Present address: Department of Pathology, Karmanos Cancer Institute,ayne State University School of Medicine, Detroit, MI 48201, USA.

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044-579X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcancer.2007.04.002

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375

esveratrol, anthocyanidins such as delphinidin possess a wideange of pharmacological properties the mechanisms of whichave been the subject of considerable interest (Fig. 1). They areecognized as naturally occurring antioxidants and have beenmplicated as antiviral and antitumor compounds [1,2]. In recentears, a number of reports have appeared which have shown

hat gallocatechins found in green tea and which include tan-ic acid, gallic acid, epigallocatechin, epicatechin-3-gallate andpigallocatechin-3-gallate (EGCG) induce apoptosis in variousancer cell lines [3,4]. Similarly curcumin [5] from the spice

S.M. Hadi et al. / Seminars in Cancer Biology 17 (2007) 370–376 371

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Fig. 1. Structures of (a) gallic acid, (b) syringic acid, (c) curcumin, (d)

urmeric and resveratrol [6] which is found in grapes and redine have also been shown to be inducers of apoptosis in cancer

ells. The consumption of green tea is considered to reduce theisk of various cancers such as that of bladder, prostate, esopha-us and stomach [4]. Of particular interest is the observation thatGCG was found to induce internucleosomal DNA fragmenta-

ion in cancer cell lines such as human epidermoid carcinomaells, human carcinoma keratinocytes, human prostate carci-oma cells, mouse lymphoma cells but not in normal humanpidermal keratinocytes [4]. Similarly gallic acid showed cyto-oxicity for a number of tumor cell lines but primary culturedat hepatocytes and macrophages were found to be refractory tohe cytotoxic effect [3]. Resveratrol also was shown to inducepoptotic cell death in HL60 human leukemia cell lines but notn normal peripheral blood lymphocytes [6]. The hallmark ofpoptosis is internucleosomal DNA fragmentation, which dis-inguishes it from necrosis. Other changes such as shrinkagef cells, membrane blebbing and the dissociation of the nucleusnto chromatoid bodies also occur. It is to be noted that most clin-cally used anticancer drugs can activate late events of apoptosisDNA degradation and morphological changes) and there areifferences in essential signalling pathways between pharmaco-ogical cell death and physiological induction of programmedell death [7]. Based on our own observations and those ofthers we propose a mechanism of DNA fragmentation in can-

er cells by plant polyphenolics that involves mobilization ofntracellular copper. Studies on chemopreventive and therapeu-ic plant-derived phytonutrients assume significance in view ofhe fact that such compounds exhibit negligible or low toxicity

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atrol, (e) delphinidin, (f) epigallocatechin-3-gallate, and (g) quercetin.

ven at relatively high concentrations. Further they may also acts lead compounds for the synthesis and development of novelnticancer drugs.

. Oxidative DNA cleavage by plant polyphenols in vitron the presence of copper ions

Studies in our laboratory have shown that a number of plantolyphenols such as flavonoids [8], tannic acid and its structuralonstituent gallic acid [9], curcumin [10], gallocatechins [11]nd resveratrol [12] cause oxidative strand breakage in DNAither alone or in the presence of transition metal ions suchs copper. Recent studies by Liu and co-workers [13] demon-trated that resveratrol as well as its certain synthetic analogsamely 3,4,4′-trihydroxy-trans-stilbene, 3,4-dihydroxy-trans-tilbene, 3,4,5-trihydroxy-trans-stilbene, which are generallyffective antioxidants, can switch to prooxidants in the presencef Cu(II) to induce DNA damage. Copper is an important metalon present in chromatin and is closely associated with DNAases particularly guanine [14,15]. It is also one of the mostedox active of the various metal ions present in cells. We havelso shown that the flavonoid quercetin [16] and curcumin [10]re capable of binding to DNA and copper. Evidence deduced inur laboratory has shown that polyphenols such as the flavonoiduercetin and the stilbene resveratrol can not only bind copper

ons but also catalyze their redox cycling [12]. In the case ofuercetin a mechanism was proposed which involved the for-ation of a ternary complex of DNA–quercetin–Cu(II) [17,16].redox reaction of the compound and Cu(II) in the ternary com-

372 S.M. Hadi et al. / Seminars in Cance

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ig. 2. Involvement of a ternary complex of quercetin, DNA and Cu(II)/Cu(I)n the generation of active oxygen species. The oxidized forms of quercetinrepresented as quercetin*) are not necessarily identical.

lex may occur leading to the reduction of Cu(II) to Cu(I), whoseeoxidation generates a variety of ROS (Fig. 2). Most of the phar-acological properties of plant polyphenols are considered to

eflect their ability to scavenge endogenously generated oxygenadicals or those free radicals formed by various xenobiotics,adiation etc. However, some data in the literature suggests thathe antioxidant properties of the polyphenolic compounds mayot fully account for their chemopreventive effects [18,19]. Mostlant polyphenols possess both antioxidant as well as prooxidantroperties [3,8] and we have proposed that the prooxidant actionf plant polyphenolics may be an important mechanism of theirnticancer and apoptosis inducing properties [19]. Such a mech-nism for the cytotoxic action of these compounds against cancerells would involve mobilization of endogenous copper ions andhe consequent prooxidant action.

. Polyphenol–Cu(II) mediated DNA breakage inuman peripheral lymphocytes

Using a cellular system of lymphocytes isolated from humaneripheral blood and alkaline single cell gel electrophoresiscomet assay), we have confirmed that polyphenol–Cu(II) sys-em is indeed capable of causing DNA degradation in cells suchs lymphocytes [20]. Further, the DNA degradation of lympho-ytes is inhibited by scavengers of ROS and neocuproine, au(I) specific sequestering agent. Also, similar to the in vitro

esults, trans-stilbene which does not have any hydroxyl groups inactive in the lymphocyte system. These findings demon-trate that the polyphenol–Cu(II) system for DNA breakage ishysiologically feasible and could be of biological significance.

. Evidence for the prooxidant action of plantolyphenols as an important mechanism for their

nticancer properties

We give below several lines of indirect evidence in literature,hich strongly suggest that the prooxidant action of plant-

r Biology 17 (2007) 370–376

erived polyphenolics rather than their antioxidant activity maye an important mechanism for their anticancer and apoptosisnducing properties:

1) Structure activity studies carried out in our laboratory withgallic acid (a structural constituent of tannic acid) indicatethat if two of the three hydroxyl groups are methylated(syringic acid), the DNA degrading capacity decreasessharply [9]. The results correlate with those of Inoue et al.[3] who showed that modification of hydroxyl groups, suchas that resulting in the formation of syringic acid, abolishesthe apoptotic activity of gallic acid.

2) Apoptotic DNA fragmentation properties of several anti-cancer drugs [21,22] and �-radiation [23] are consideredto be mediated by ROS. It may also be mentioned thatdoxorubicin induced apoptosis in human osteocarcinomaSaos-2 cells is mediated by ROS and is independent ofp-53 [24]. Interestingly, certain properties of polyphenoliccompounds, such as binding and cleavage of DNA and thegeneration of ROS in the presence of transition metal ions[16] are similar to those of certain known anticancer drugs[25].

3) Fe3+ and Cu2+ are the most redox-active of the metal ionsin living cells. Wolfe et al. [26] have proposed that a coppermediated Fenton reaction, generating site-specific hydroxylradicals, is capable of inducing apoptosis in thymocytes.In a study with thiol-containing compounds, apoptosis wasinduced in different cell lines when either free copper orceruloplasmin (a copper binding protein) was added; suchactivity was not observed, however, when either free ironor the iron-containing serum protein transferrin was added[27]. Most of the copper present in human plasma is associ-ated with ceruloplasmin, which has six tightly held copperatoms and a seventh, easily mobilized one [28]. In anotherstudy supporting these observations, copper was found toenhance the apoptosis-inducing activity of polyphenolicantioxidants, whereas iron was inhibitory [29]. Althoughiron is considerably more abundant in biological systems,the major ions in the nucleus are copper and zinc [15]. Fur-ther, although in general, tumors are considered to containless total iron and have less iron saturation in ferritin thando normal cells that is not always the case [30]. As alreadymentioned copper ions occur naturally in chromatin andcan be mobilized by metal chelating agents. Burkitt et al.[31] suggested that the internucleosomal DNA fragmenta-tion might be caused not only by endonuclease but also bymetal-chelating agents such as 1,10-phenanthroline (OP),which promotes the redox activity of endogenous copperions and the resulting production of hydroxyl radicals. Thus,the internucleosomal DNA “laddering” often used as anindicator of apoptosis may also reflect DNA fragmentationby non-enzymatic processes. Several reports indicate thatserum [32,33], tissue [34] and intracellular copper levels

in cancer cells [35] are significantly increased in variousmalignancies. Indeed, such levels have been described asa sensitive index of disease activity of several hematologicand non-hematologic malignancies [36].

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rDcthe results of an experiment where three progressively increas-ing concentrations of neocuproine were tested on resveratrolinduced DNA breakage in lymphocytes. A progressive decreasein the tail length as a function of increasing neocuproine con-

Table 1Effect of scavengers of active oxygen species on resveratrol induced lymphocyteDNA breakage

Dose Tail length (�m) Inhibition (%)

Untreated 1.22 ± 0.08a –Resveratrol (200 �M) 20.84 ± 1.28 –Resveratrol + SOD (100 �g/ml) 7.05 ± 0.25* 66

S.M. Hadi et al. / Seminars in

4) A comparison of the properties of complexes formedbetween plant polyphenolics and Cu2+ and Fe3+ shouldindicate which of these two metal ions could lead to DNAfragmentation in the nucleus when complexed. Not muchis known about the properties of such complexes. However,considerable information is available about OP chelationof copper and iron ions. Burkitt et al. [31] cited severalreasons why Cu2+ rather than Fe3+ may be responsible forOP-stimulated internucleosomal DNA fragmentation in iso-lated nuclei. For example, the cumulative affinity constants(�3 in 0.1 M salt) for chelation of various metal ions by OPare in the order Cu2+ ≈ Fe2+ > Zn2+ > Fe3+. The complexformed between OP and Cu2+ has a redox potential (E◦for Cu2+/Cu+ = 0.17 V) that favors redox cycling, whereasthat for Fe3+/Fe2+ is 1.1 V, presumably because of stabiliza-tion in the ferrous state. Copper is also shown to be presentin chromosomes as Cu+ ions because of stabilization in thepresence of DNA. This overcomes the need for the reductionof Cu2+ to Cu+ and can directly generate the hydroxyl rad-icals. Finally, copper and zinc are major metal ions presentnaturally in chromosomes [15]. Because most polypheno-lics are also polycyclic compounds similar in size to OP,conceivably their metal binding properties are also similar.

5) Evidence suggests that the antioxidant properties ofpolyphenolics may not fully account for their chemopre-ventive effects. For example, it was shown that althoughellagic acid is an antioxidant ten times more potent thantannic acid, the latter was more effective in inhibiting skintumor promotion by 12-O-tetradecanoyl phorbol-13 acetatethan the former [18]. It was suggested that the antioxi-dant effects of these polyphenols might be essential but notsufficient for their antitumor promotion. In any case ROSscavenging properties of plant polyphenols may accountfor their chemopreventive effects but not for any thera-peutic action against cancer cells [22]. Expression of thebcl-2 proto-oncogene, which blocks apoptosis, decreasescellular production of ROS [37], whereas the coadministra-tion of antioxidant enzymes such as superoxide dismutase(SOD) and catalase prevents curcumin mediated apoptosisin human leukemia cells [5]. Further it has been shown thatthe programmed cell death induced by curcumin in humanleukemic T-lymphocytes is independent of the involvementof mitochondria and caspases suggesting the existence ofpathways other than the ‘classical’ ones [38]. Caspases areessential for both Fas and mitochondria mediated apop-tosis. However, inhibition of caspases or the use of cellswith defective apoptosis machinery has demonstrated thatalternative types of programmed cell death could occur aswell and such alternative death mechanisms are divided into“apoptosis-like” and “necrosis-like” [39].

6) Ascorbic acid is an essential micronutrient and is consideredto have an antioxidant function in living systems. However,it may also act as a prooxidant and site specific DNA cleav-

age by ascorbic acid in the presence of Cu(II) has beendescribed [40]. Relatively high concentrations of ascor-bic acid are able to induce apoptosis in various tumor celllines. Consequently, it has been shown to induce cell death,

RR

r Biology 17 (2007) 370–376 373

nuclear fragmentation and internucleosomal DNA cleavagein human myelogenous leukemia cell lines [41]. The apopto-sis inducing activity of ascorbic acid has been ascribed to itsprooxidant action and is inhibited by catalase, antioxidantslike N-acetylcysteine and GSH, Ca2+ and Fe3+ depletion butstimulated by H2O2, Cu2+, and iron chelators [42].

7) It has recently been shown that the polyphenol curcuminmediated apoptosis of HL60 cells is closely related to anincrease in the concentrations of ROS possibly generatedthrough the reduction of transition metals in cells [43].

. Oxidative DNA breakage by plant polyphenols inuman peripheral lymphocytes

Using the comet assay our laboratory has shown that plantolyphenols cause DNA breakage in isolated human peripheralymphocytes [44]. Photographs of comets seen on treatment withifferent concentrations of resveratrol are shown in Fig. 3. At0 and 100 �M concentrations resveratrol did not damage theymphocyte DNA to any significant extent whereas at 200 �Moncentration a comet with a tail indicative of DNA breakageas observed. The results demonstrate that resveratrol alone

s capable of DNA breakage in lymphocytes. We have furtherhown that several other polyphenols such as gallic acid, EGCGnd piceatannol are also able to catalyze DNA breakage in lym-hocytes. Table 1 gives the results of an experiment where threecavengers of ROS have been tested namely, SOD and catalase,hich remove superoxide and H2O2, respectively and thiourea,hich is a scavenger of several ROS. All three cause signif-

cant inhibition of DNA breakage as evidenced by decreasedail lengths. We concluded that superoxide anion and H2O2 aressential components in the pathway that leads to the forma-ion of hydroxyl radical and other species which would be theroximal DNA cleaving agents.

. Putative mobilization of endogenous copper by plantolyphenols

In a previous study [20] we have shown that theesveratrol–Cu(II) mediated DNA degradation of lymphocyteNA is inhibited by neocuproine which is a Cu(I) specific

helating agent and is membrane permeable [45]. Fig. 4 gives

esveratrol + catalase (100 �g/ml) 8.86 ± 0.29* 57esveratrol + thiourea (1 mM) 11.93 ± 1.01* 45

a All values represent S.E.M. of three independent experiments.* P <0.05 when compared to control.

374 S.M. Hadi et al. / Seminars in Cancer Biology 17 (2007) 370–376

F al lymphocytes showing comets (100×) after treatment with different concentrationso

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Fig. 5. Effect of preincubation of lymphocytes with neocuproine and thiourea onTBARS generated by increasing concentrations of resveratrol. Resveratrol alone(�); resveratrol + neocuproine (1 mM) (©); resveratrol + thiourea (1 mM) (�).The isolated cells (1 × 105) suspended in RPMI 1640 were preincubated with the

ig. 3. Alkaline single cell gel electrophoresis (comet assay) of human peripherf resveratrol: (A) untreated, (B) 50 �M, (C) 100 �M and (D) 200 �M.

entration was seen. From the results we concluded that theNA breakage by the polyphenol involves endogenous copper

ons and that Cu(I) is an essential intermediate that leads toNA breakage. We presume that the lymphocyte DNA break-

ge is the result of the generation of hydroxyl radicals andther ROS in situ. Oxygen radical damage to deoxyribose orNA is considered to give rise to thiobarbituric acid reactive

ubstance (TBARS) [46,47]. We therefore determined the for-ation of TBARS in lymphocytes as a measure of oxidative

tress with increasing concentrations of resveratrol. The effectf preincubating the cells with neocuproine and thiourea waslso studied. Results given in Fig. 5 show that there is a doseependent increase in the formation of TBARS in lymphocytes.owever, when the cells were preincubated with neocuproine

nd thiourea there was a considerable decrease in the rate oformation of TBARS by resveratrol. These results indicate thatoth DNA breakage and oxidative stress in cells is inhibited byu(I) chelation and scavenging of ROS. Similar results werebtained with caffeic acid [48], a major polyphenolic compo-ent of coffee. Thus, it can be concluded that the formation ofOS by polyphenols in lymphocytes involves their interaction

ith intracellular copper as well as its reduction to Cu(I).It is well known that polyphenols autooxidize in cell cul-

ure media to generate H2O2 and quinones that can enter cellsausing damage to various molecules [49–51]. This may lead to

ig. 4. Effect of increasing concentrations of neocuproine on resveratrol inducedNA breakage in human lymphocytes. Values reported are ±S.E.M. of three

ndependent experiments.

indicated concentrations of neocuproine and thiourea for 30 min at 37 ◦C. Afterpelleting the cells were washed twice with PBS (Ca2+ and Mg2+ free) beforeresuspension in RPMI and further incubation for 1 h in the presence of increasingrnt

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esveratrol concentrations. Viability of lymphocytes after preincubation witheocuproine and thiourea was more than 90%. Values reported are ±S.E.M. ofhree independent experiments.

xtracellular production of ROS that could account for lympho-yte DNA breakage. However, this does not appear to be the casen our system since we have previously shown that no lympho-yte DNA breakage is observed on preincubating the cells withesveratrol alone up to a concentration of 50 �M. DNA breakageould only be seen after incubating the pre-treated cells furthern the presence of Cu(II) [20]. Further, we could not detect any

2O2 formation on incubating resveratrol (up to a concentrationf 300 �M) in the suspension medium (RPMI) [44].

. Conclusions

Most studies on anticancer mechanisms of plant polyphenolsnvoke the induction of cell cycle arrest at S/G2 phase transition

rought about by an increase in cyclins A and E and inactivationf cdc 2. Other mechanisms have also been proposed [52]. Iniew of the above findings in our laboratory and those of oth-rs in literature we suggest that the conclusion of our studies is

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hat the plant polyphenols possessing anticancer and apoptosisnducing activities are able to mobilize endogenous copper ionsossibly the copper bound to chromatin. Essentially this woulde an alternative, non-enzymatic and copper-dependent path-ay for the cytotoxic action of certain anticancer agents that are

apable of mobilizing and reducing endogenous copper. As suchhis would be independent of Fas and mitochondria mediatedrogrammed cell death. It is conceivable that such a mechanismay also lead to internucleosomal DNA breakage (a hallmark of

poptosis) as internucleosomal spacer DNA would be relativelyore susceptible to cleavage by ROS. Indeed such a commonechanism better explains the anticancer effects of polyphenolsith diverse chemical structures as also the preferential cytotox-

city towards cancer cells. The generation of hydroxyl radicalsn the proximity of DNA is well established as a cause of strandcission. It is generally recognized that such reaction with DNAs preceded by the association of a ligand with DNA followedy the formation of hydroxyl radicals at that site. Among oxy-en radicals the hydroxyl radical is most electrophilic with higheactivity and therefore possesses a small diffusion radius. Thus,n order to cleave DNA it must be produced in the vicinity ofNA [53]. The location of the redox-active metal is of utmost

mportance because the hydroxyl radical, due to its extreme reac-ivity, interacts exclusively in the vicinity of the bound metal54]. As already mentioned cancer cells are known to containlevated levels of copper [32–35] and therefore may be moreubject to electron transfer with antioxidants to generate ROS13]. Thus, because of higher intracellular copper levels in can-er cells it may be predicted that the cytotoxic concentrations ofolyphenols required would be lower in these cells as comparedo normal cells. Studies by Chen et al. [55] have earlier shownhat EGCG inhibited the growth of SV40 virally transformed

138 cells but not their normal counterparts. The IC50 value ofGCG was estimated to be 120 and 10 �M for normal versus

he transformed cells respectively. EGCG also showed a simi-ar differential growth inhibitory effect with two other humanancer cell lines, Caco-2 colorectal adenocarcinoma cells ands578T breast ductal carcinoma cells. These authors have also

hown that the black tea polyphenol theaflavin-3′-monogallatelso exhibits a similar differential inhibitory effect for these can-er cell lines [56]. We have recently shown that ascorbate whichlso acts as a prooxidant in the presence of copper ions is cyto-oxic to normal lymphocytes [57]. Indeed it had been earlierhown that ascorbate is cytotoxic to a leukemic cell line at aower concentration than normal lymphocytes [58].

cknowledgment

The authors acknowledge the financial assistance providedy University Grants Commission, New Delhi, under the DRSrograme.

eferences

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