European Journal of Pharmaceutical Sciences 47 (2012) 280–286

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European Journal of Pharmaceutical Sciences

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Apogossypolone, derivative of gossypol, mobilizes endogenous copperin human peripheral lymphocytes leading to oxidative DNA breakage

Haseeb Zubair a, Husain Y. Khan a, M.F. Ullah a,b, Aamir Ahmad a,c, Daocheng Wu d, S.M. Hadi a,⇑a Department of Biochemistry, Aligarh Muslim University, Aligarh 202002, Indiab Department of Pathobiology, University of Tennessee, Knoxville, TN 37996, USAc Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USAd Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, China

a r t i c l e i n f o

Article history:Received 13 December 2011Accepted 14 April 2012Available online 24 April 2012

Keywords:GossypolApogossypoloneEndogenous copperOxidative DNA breakageAnticancer mechanism

0928-0987/$ - see front matter � 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.ejps.2012.04.014

Abbreviation: ApoG2, apogossypolone.⇑ Corresponding author. Tel.: +91 614 293 3713, m

E-mail address: smhadi@vsnl.com (S.M. Hadi).

a b s t r a c t

Gossypol is a polyphenolic aldehyde that is produced in the cotton plant. Since long it has been reportedto possess antiproliferative activity against a variety of cancer cell lines as well as tumor regression inanimal models. However, the toxicity of gossypol does not permit it to be an effective antitumor agent.One of the derivatives of gossypol to show promising results is apogossypolone. For example, it has beenshown to specifically target tumor growth in hepatocellular carcinoma xenograft in nude mice withoutcausing any damage to normal tissue. Using human peripheral lymphocytes, in this paper we show thatboth gossypol and its semi-synthetic derivative apogossypolone cause oxidative DNA breakage in thesecells through the mobilization of endogenous copper ions. Such cellular DNA breakage is inhibited bycopper specific chelator but nor by iron or zinc chelating agents. Similar results are obtained with isolatednuclei indicating that chromatin bound copper is mobilized in this reaction. Further, apogossypoloneshowed enhanced DNA breakage and increased oxidative stress in whole lymphocytes as compared withgossypol indicating that this is possibly the result of greater permeability of apogossypolone. It is wellestablished that tissue, cellular and serum copper levels are considerably elevated in various malignan-cies. Therefore, cancer cells may be subject to greater electron transfer between copper ions and gossy-pol/apogossypolone to generate reactive oxygen species responsible for DNA cleavage. This may accountfor the preferential cytotoxicity of apogossypolone towards tumor cells.

� 2012 Elsevier B.V. All rights reserved.

1. Introduction

Gossypol [1,10,6,60,7,70-hexahydroxyl-5,50-diisopropyl-3,30-di-methyl-(2,20-binaphthalene)-8,80-dicarboxaldehyde], a polyphenolicaldehyde produced in the roots, leaves, stems and seeds of thecotton plant (Fig. 1). Gossypol has been reported to have antipro-liferative activity on a variety of cancer cell lines including breast(Ligueros et al., 1997), bladder (Macoska et al., 2008), pancreas(Mohammad et al., 2005), lung (Kilic et al., 2009), colon (Zhanget al., 2003), prostate (Huang et al., 2006), and head and neck (Bau-er et al., 2005; Wolter et al., 2006). It also inhibits proliferation ofDunning prostate cancer cells and reduces metastasis of these cellsin xenograft Copenhagen rat models (Jiang et al., 2000). It has beenshown that gossypol induces apoptosis and DNA fragmentation inDU-145 cells (Huang et al., 2006). However, the toxicity of gossypol

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does not permit it to be an effective antitumor agent. Nevertheless,gossypol represents a potentially interesting lead compound(Kitada et al., 2003; Zhang et al., 2009) for the synthesis of novelanticancer agents. One of the derivatives of gossypol to showpromising results is apogossypolone (ApoG2) (Arnold et al.,2008). It is assumed to have reduced non-specific reactivity, there-by decreasing toxicity of gossypol. The main modification is theremoval of the aldehyde groups of gossypol. Further, four quino-noid moieties have been added resulting in enhanced permeabilityof the compound.

ApoG2 has been shown to specifically target tumor growth inhepatocellular carcinoma xenograft in nude mice without causingdamage to normal tissue (Mi et al., 2008). Further, ApoG2 has alsobeen shown to induce apoptosis in follicular Small Cleaved CellLymphoma model, pre-B-acute lymphoblastic leukemia, marginalzone lymphoma and chronic lymphocytic leukemia (Arnold et al.,2008). It has also been shown to induce apoptosis and suppress tu-mor growth in nasopharyngeal carcinoma xenografts (Hu et al.,2008). The primary mechanism by which these compounds inhibitproliferation and induce apoptosis in cancer cells, sparing normal

Fig. 1. Chemical structure of gossypol and ApoG2.

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cells, is not known and has been the subject of considerable inter-est (Mehrpour et al., 2009).

Previous studies in our laboratory have centered on the mecha-nism of anticancer properties of plant derived polyphenoliccompounds. We have shown that various classes of plant polyphe-nols are able to cause oxidative breakage of cellular DNA eitheralone or in the presence of transition metal ions such as copper(Azmi et al., 2005; Bhat et al., 2007; Hanif et al., 2008; Khanet al., 2011). We have hypothesized that the cytotoxic action ofthese compounds against cancer cells involves mobilization ofendogenous copper and consequent prooxidant action (Hadiet al., 2000, 2007). Towards validation of our hypothesis consider-able evidence has been deduced over the years (Shamim et al.,2008; Nazeem et al., 2009; Ullah et al., 2010; Khan et al., 2011).Using human peripheral lymphocytes, in this paper also we showthat both gossypol and its derivative ApoG2 cause oxidative DNAbreakage in cells through mobilization of endogenous copper ions.Further, it is also suggested that in intact lymphocytes the en-hanced DNA breakage activity of ApoG2 is possibly the result ofgreater permeability of the compound.

2. Materials and methods

2.1. Materials

Gossypol, neocuproine, bathocuproine disulphonate, superox-ide dismutase (SOD), agarose, low melting point agarose, RPMI1640, Triton X-100, Trypan blue, Histopaque1077, and PBS(Ca2+ and Mg2+ free) were purchased from Sigma (St. Louis, MO).Laboratory synthesized ApoG2 was used (Zhan et al., 2009). Allother chemicals were of analytical grade. Fresh solutions of gossy-pol and ApoG2 were prepared as a stock of 3 mM in DMSO. In allstudies (except ferrous oxidation-xylenol orange (FOX) assay andTBARS estimation), the reaction mixture contained 62% DMSO(v/v). To test any effect of solvent on the studies, DMSO solutionwas added to the cells at the final concentration of 2% v/v, whichwas the highest concentration of DMSO used in the compound-treated reaction medium. No difference was observed with orwithout DMSO, indicating that the DMSO at the tested concentra-tions did not influence the results. For FOX assay and the

estimation of TBARS in lymphocytes, the solvent control concen-tration varied between 1% and 12% v/v and again no influence ofthe solvent was observed on the results. On addition to reactionmixtures, in the presence of buffers mentioned and at concentra-tions used, the polyphenols used remained in the solution. The vol-umes of stock solution added did not lead to any appreciablechange in the pH of reaction mixtures.

Lymphocytes were isolated from heparinized blood samplesusing Histopaque1077. Three milliliter blood was drawn from asingle healthy non-smoking donor for the entire study on severaloccasions by venepuncture and diluted suitably in Ca2+ and Mg2+

free PBS. The cells were finally suspended in RPMI 1640.

2.2. Detection of Cu(I) production

The selective sequestering agent neocuproine was employed todetect reduction of Cu(II) to Cu(I) by recording the formation ofneocuproine–Cu(I) complex which absorbs maximally at 450 nm.The reaction mixture (3 ml) contained 3 mM Tris–HCl (pH 7.5),fixed concentration (50 lM) of Cu(II), 300-lM neocuproine andfixed concentrations (50 lM) of gossypol/ApoG2. The reactionwas started by adding Cu(II) and the spectra were recorded imme-diately afterwards.

2.3. Assessment of DNA breakage in isolated whole lymphocytes andlymphocyte nuclei

Treatment of whole lymphocytes with gossypol and ApoG2, andthe subsequent Comet assay was performed essentially as de-scribed earlier (Ullah et al., 2009; Khan et al., 2011). However, sincethe DNA breakage had to be compared with that in lymphocyte nu-clei, the treatment of cells with gossypol and ApoG2 was done onslides rather than in microcentrifuge tubes. Further, the lysis ofcells was carried out after the treatment. Briefly, approximately10,000 lymphocytes were mixed with 75 ll prewarmed 2% LMPAin PBS and immediately applied to a frosted microscope slide pre-viously layered with 75 ll of 1% standard agarose in PBS. The slideswere allowed to gel for 10 min at 4 �C. Each slide was then trans-ferred to a rectangular dish (8 cm � 3 cm � 5 mm) that containeda reaction mixture of gossypol/ApoG2 and other additions as men-tioned in various legends to figures and tables. The slides with thereaction mixture were incubated at 37 �C. The slides were thenwashed twice by placing in 0.4 M phosphate buffer pH 7.5 for5 min at room temperature. Lysis of cells was then performed bysubmerging the slides in a tank containing lysis solution in the ab-sence of light for 1 h at 4 �C. The use of a tank instead of a coplin jarallowed simultaneous processing of a number of slides. The lysissolution (pH 10) consisted of 2.5 M NaCl, 0.1 M EDTA, 10 mM Trisand 1% Triton X-100 added just prior to use. After lysis, slides weretransferred to another tank containing 0.4 M phosphate buffer (pH7.5) for 10 min. DNA unwinding and expression of alkali-labilesites were done by leaving the slides in the high-pH electrophore-sis buffer (1 mM EDTA, 300 mM NaOH, pH 13 prepared in PBS) at4 �C for 30 min. Subsequently, the electrophoresis, neutralizationand staining of the slides were carried out as described earlier(Azmi et al., 2005).

In the lysed version of comet assay, essentially all the stepswere carried out as above with the exception that the cells werelysed prior to treatment with reaction mixture. The lysed versionof the comet assay has been used to study the direct interactionof various agents with cell nuclei as it eliminates the effect of cellmembrane as a barrier and the cytoplasmic environment (Mazzan-ti, 1998). Comet images were observed at 100�magnification witha fluorescence microscope (Olympus CX41) and COHU 4910(equipped with a 510- to 560-nm excitation and 590-nm barrierfilters) integrated CC camera. Fifty images were randomly selected

Fig. 2. Detection of gossypol and ApoG2-induced Cu(I) production by neocuproine.Trace 1; neocuproine (300 lM) + Cu(II) (50 lM). Trace 2; neocuproine (300 lM) +gossypol (50 lM). Trace 3; neocuproine (300 lM) + ApoG2 (50 lM). Trace 4;neocuproine (300 lM) + gossypol (50 lM) + Cu(II) (50 lM). Trace 5; neocuproine(300 lM) + ApoG2 (50 lM) + Cu(II) (50 lM).

Fig. 3. Degradation of plasmid pBR322 DNA by gossypol and ApoG2 in absence andpresence of copper. Lane 1: DNA alone; Lane 2: DNA + Cu(II) (50 lM); Lanes 3,4:DNA + gossypol (100, 200 lM); Lane 5: DNA + gossypol (100 lM) + Cu(II) (50 lM);Lanes 6,7: DNA + ApoG2 (100, 200 lM); Lane 8: DNA + ApoG2 (100 lM) + Cu(II)(50 lM).

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from each sample, and their lengths (diameter of the nucleus plusmigrated DNA) were measured on the screen as automatically gen-erated by Komet 5.5 image analysis system of Kinetic Imaging, Liv-erpool, UK.

2.4. Viability assessment of lymphocytes

The lymphocytes were checked for their viability before thestart and after the end of the reaction using Trypan Blue ExclusionTest. The viability of lymphocytes was found to be more than 94%.

2.5. Determination of TBARS generation in lymphocytes and H2O2 inthe reaction medium

Thiobarbituric-acid-reactive substance (TBARS) was deter-mined according to the method of Ramanathan et al. (1994). A cellsuspension (1 � 105 cells/ml) was incubated with gossypol andApoG2 (0–400 lM) at 37 �C for 1 h and then centrifuged at1000 rpm. In some experiments, the cells were pre-incubated withfixed concentrations of neocuproine and thiourea. The cell pelletwas washed twice with PBS (Ca2+ and Mg2+ free) and suspendedin 0.1 N NaOH. The cell suspension (1.0 ml) was further treatedwith 10% TCA and 0.6 M TBA (thiobarbituric acid) in boiling waterbath for 10 min. The absorbance was read at 532 nm.

H2O2 was determined in the incubation medium (RPMI 1640)by FOX (Ferrous ion Oxidation-Xylenol orange) assay as describedby Long et al. (2000). The simplified reaction sequence involves theoxidation of ferrous (Fe2+) to ferric (Fe3+) ions by H2O2 with thesubsequent binding of the Fe3+ ion to the ferric-sensitive dye xyle-nol orange, yielding an orange to purple complex, which is mea-sured at 560 nm. The reaction mixture contained gossypol orApoG2 along with the incubation medium used in the treatmentof lymphocytes. After incubation for 2 h at 37 �C, an aliquot of200 lL was analyzed for H2O2 formation (Shamim et al., 2008).

2.6. Statistics

The statistical analysis was performed as described by Tice et al.(2000) and is expressed as ±SEM of three independent experi-ments. A student’s t-test was used to examine statistically signifi-cant differences. Analysis of variance was performed using ANOVA.p-values 60.05 were considered statistically significant.

3. Results

3.1. Reduction of Cu(II) to Cu(I) by gossypol and apogossypolone

To analyze the copper reducing capacity of these two com-pounds, in the experiment shown in Fig. 2 we used neocuproinethat sequesters Cu(I) selectively forming a complex that absorbsmaximally at 450 nm. Under the conditions employed in theexperiment neither Cu(II) nor the compounds alone interfere withthis maxima whereas, gossypol as well as ApoG2 react with Cu(II)to generate Cu(I) as evidenced by the peak appearing at 450 nm.The result shows that both the compounds are capable of reducingCu(II) to Cu(I) to a significant degree.

3.2. Cleavage of supercoiled pBR322 plasmid DNA by gossypol andApoG2

To assess the DNA cleavage efficiency of gossypol and its syn-thetic derivative ApoG2, we analyzed the cleavage of supercoiledplasmid DNA to other topological forms. Fig. 3 shows that evenat 200 lM concentration, gossypol and ApoG2 alone do not causeany change in the structure of the plasmid. Further, at the tested

concentration (50 lM) copper alone does not cause any DNAbreakage. However, in the presence of copper and at relatively low-er concentration (100 lM) of the compounds, both the compoundsshow conversion of supercoiled DNA to open circular form. How-ever, despite the DNA cleavage similarity, the relative cytotoxicityof ApoG2 has been found to be considerably greater in prostatecancer cells (Zhan et al., 2009). Presumably the cytoxic mechanismof ApoG2 involves additional mechanisms for its cytotoxic actionsuch as enhanced cell permeability.

3.3. Comparison of lymphocyte DNA breakage by gossypol and ApoG2

Alkaline version of comet assay makes it possible to detect DNAsingle-strand breaks and alkali-labile sites in cellular DNA. In thepresent experiment, we tested increasing concentrations of gossy-pol and ApoG2 (10, 20 and 50 lM) for DNA breakage in peripheralhuman lymphocytes using alkaline comet assay with whole lym-phocytes and lymphocyte nuclei. Table 1 shows that there is a dosedependent increase in DNA breakage in whole lymphocytes as well

Table 1Comparison of DNA breakage in intact lymphocytes and lymphocyte nuclei by gossypol and ApoG2 as a function of comet tail length.

Concentration (lM) Whole lymphocytes Lymphocyte nuclei

Gossypol ApoG2 Gossypol alone ApoG2 alone

Alone With 20 lM copper Alone With 20 lM copper

0 3.04 ± 0.2 3.09 ± 0.12 3.03 ± 0.3 3.10 ± 0.19 4 ± 0.17 4.1 ± 0.0910 8.60 ± 0.6⁄ 16.41 ± 1.02# 12.58 ± 0.97⁄⁄ 19.77 ± 0.88## 10.9 ± 0.42 12.7 ± 0.6620 18.11 ± 1.09⁄ 23.77 ± 1.61# 22.23 ± 1.7⁄⁄ 28.84 ± 1.92## 22.26 ± 0.71 23.8 ± 0.9250 25.89 ± 1.77⁄ 37.05 ± 2.03# 30.17 ± 2.1⁄⁄ 42.69 ± 2.23## 36.6 ± 0.95 38.4 ± 0.98

#Values were found to be significant when compared to gossypol alone at the same concentration ⁄at P < 0.05. ##values were found to be significant when compared to ApoG2alone at the same concentration ⁄⁄At P < 0.05.

Table 2Effect of various scavengers of ROS on DNA breakage induced by Gossypol and ApoG2in whole cells.

Treatment Tail length % Inhibition

None 2.03 ± 0.07§ –Gossypol (50 lM) 26.53 ± 1.74⁄ –

+SOD (100 lg/ml) 11.93 ± 0.98# 55+Catalase (100 lg/ml) 12.42 ± 0.74# 53+Thiourea (1 mM) 15.46 ± 1.35# 41+Mannitol (1 mM) 14.23 ± 1.23# 46

Apogossypolone (50 lM) 29.92 ± 2.43⁄⁄ –+SOD (100 lg/ml) 15.21 ± 1.22## 47+Catalase (100 lg/ml) 14.13 ± 1.04## 51+Thiourea (1 mM) 12.64 ± 0.62## 57+Mannitol (1 mM) 13.84 ± 0.97## 52

Cells were preincubated with scavengers of ROS and then treated with the com-pounds for 1 h in dark at 4 �C. ⁄Value was found to be significant when compared to§control (no treatment) value and #value at P 6 0.01. Similarly, ⁄⁄value was found tobe significant when compared to §control (no treatment) value and ##value atP 6 0.01.

Fig. 4. Gossypol (50 lM, h) and ApoG2 (50 lM, 4)-induced DNA degradation inwhole lymphocyte cells (A) and in lymphocyte nuclei (B) in presence of Cu(I)specific chelating agents, neocuproine (—) or bathocuproine disulphonic acid (----).Values reported are mean ± SEM of three independent experiments. ⁄⁄value werefound to be significant when compared to ⁄value (in absence of chelators) atP < 0.01.

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as lymphocyte nuclei by both the compounds. However, ApoG2causes a significantly greater degree of DNA breakage in wholelymphocytes. Further, the result also shows that such DNA break-age is expressively enhanced in presence of exogenously addedcopper in whole lymphocytes. We further tested the effect of gos-sypol and ApoG2 on lymphocyte nuclei at the concentrations givenabove. Since the treatment of cell nuclei eliminates the cell mem-brane and cytoplasmic barrier, the compounds are able to directlyinteract with the nucleus. Thus the degree of permeability is not afactor in compound-induced DNA breakage. It is known that thenuclear pore complex is permeable to small molecules (Mazzanti,1998). The DNA damage induced in cell nuclei is a result of directinteraction of the compounds with the nucleus. Therefore, thistreatment should result in a higher degree of DNA breakage ascompared to whole cells. Thus as expected, an enhanced level ofDNA breakage is seen in cell nuclei. Both the compounds induceapproximately similar degree of DNA breakage, in the case of nu-clei. Thus the enhanced DNA breakage seen in whole lymphocyteswith ApoG2 is possibly the result of greater permeability of thegossypol derivative.

Table 2 gives the results of the experiment where the effect ofvarious scavengers of ROS; i.e. SOD, catalase, thiourea and manni-tol, was tested on compound-induced DNA breakage in whole lym-phocytes using comet assay. SOD and catalase remove superoxideand H2O2, respectively whereas; thiourea and mannitol scavengehydroxyl radicals. All four scavengers caused significant inhibitionin gossypol and ApoG2-induced DNA breakage as evidenced by thedecreased tail lengths. From the data it is suggested that cellularDNA breakage by both the compounds is mediated by ROS. Furtherit is indicated that the hydroxyl radical is the proximal DNAcleaving agent. It may be mentioned that due to its high reactivity

and short diffusion radius, it is difficult for any trapping moleculeto intercept the hydroxyl radical completely (Czene et al., 1997).

We also used copper chelators (neocuproine and bathocuproinedisuphonic acid), to study their effect on DNA breakage by both thecompounds in whole lymphocytes as well as in lymphocyte nuclei.In whole lymphocytes, a clear inhibition was seen in presence ofneocuproine (a cell membrane permeable Cu(I) specific chelator)on gossypol as well as ApoG2-induced DNA breakage (Fig. 4A).Further, no such DNA breakage inhibition was observed in wholelymphocytes with bathocuproine disulphonic acid (a water solublecell membrane impermeable analog of neocuproine). However, in

Fig. 5. Effect of preincubation of lymphocytes with neocuproine and thiourea onTBARS generated by increasing concentration (lM) of gossypol (A) and ApoG2 (B).Compound alone (}); compound + neocuproine (1 mM) (h); compound + thiourea(1 mM) (4). The isolated lymphocyte cells (1 � 105) suspended in RPMI 1640 werepreincubated with the indicated concentrations of neocuproine and thiourea for30 min at 37 �C. Values reported are mean ± SEM of three independent experiments.P < 0.05, when ⁄⁄values were compared to ⁄values.

Fig. 6. A comparison of the rate of H2O2 formation by tannic acid (grey bars), gossypol anmean ± SEM of three independent experiments. P < 0.01, when ⁄⁄values were compared

284 H. Zubair et al. / European Journal of Pharmaceutical Sciences 47 (2012) 280–286

lymphocyte nuclei treated with gossypol and ApoG2, both neo-cuproine and bathocuproine disulphonic acid were found to inhibitDNA breakage (Fig. 4B). In this case both neocuproine and batho-cuproine disulphonic acid are directly able to interact with nuclei.We also tested the effect of desferoxamine (iron specific chelator)and histidine (zinc specific chelator) on compound induced DNAbreakage using whole lymphocytes as well as nuclei. However,both the chelators did not cause any inhibition of DNA breakageby gossypol or ApoG2. We take these results to suggest that gossy-pol and its derivative ApoG2 mobilize chromatin bound copperleading to oxidative DNA breakage Fig. 4.

3.4. Comparison of TBARS generation in lymphocytes by gossypol andApoG2

Damage by hydroxyl radicals and other reactive oxygen speciesto deoxyribose gives rise to thiobarbituric acid (TBA) reactive sub-stances (Smith et al., 1992; Quinlan and Gutteridge, 1987). There-fore, we have determined the formation of TBA reactive substances(TBARS) in lymphocytes as a measure of oxidative stress withincreasing concentration of gossypol and ApoG2. The effect ofpreincubating cells with neocuproine and thiourea on the level ofTBARS, was also tested for both the compounds. Fig. 5A and Bshows that both the compounds give a dose-dependent increasein TBARS generation. As seen from the results, ApoG2 generatessignificantly higher levels of TBARS than gossypol. This may beattributed to greater cell permeability of ApoG2 and subsequentlya higher degree of hydroxyl radical generation by ApoG2 in cells.Moreover, when the cells were preincubated with neocuproineand thiourea there was a considerable decrease in TBARS level byboth the compounds. Thus, it can be suggested that both DNAbreakage and oxidative stress in cells involves the interaction ofgossypol and ApoG2 with intracellular copper and its reductionto Cu(I).

3.5. Generation of hydrogen peroxide in the incubation medium

Polyphenols have been shown to auto-oxidize in cell culturemedia and lead to the release of H2O2 and quinone that can enterthe cells/nuclei causing damage to various biomacromolecules(Long et al., 2000). This extraneous production of ROS couldalso account for DNA breakage observed above. Therefore, wedetermined the production of H2O2 by gossypol and ApoG2 in

d ApoG2 in the incubation medium as determined by FOX assay. Values reported areto ⁄values.

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the incubation medium RPMI 1640 and compared it to a knownproducer, tannic acid. As can be seen in Fig. 6, where the generationof H2O2 is notably significant by tannic acid, gossypol and ApoG2do not produce H2O2 in any significant amount. This indicates thatthe DNA breakage by the compounds observed above is not a resultof extraneous generation of H2O2 in the reaction medium. More-over, we have earlier shown that there exists no correlation be-tween the extraneous generation of H2O2 and the ability ofpolyphenols to cause oxidative DNA breakage (Ullah et al., 2009).

4. Discussion

The above results lead to the following conclusions: (1) the cel-lular DNA breakage in normal lymphocytes by gossypol and ApoG2involves redox-cycling of endogenous copper, possibly chromatinbound copper; (2) the relatively higher DNA breakage efficiencyof ApoG2 is possibly accounted for by the presence of quinonoidmoieties which impart a greater degree of membrane permeability.Copper mediated further oxidation of ortho-hydroxyl groups inApoG2 may lead to an even greater degree of DNA breakage capac-ity. As already mentioned both gossypol and ApoG2 have beenshown to possess anticancer and apoptosis inducing properties invarious cancer cell lines (Huang et al., 2006; Zhang et al., 2009;Mi et al., 2008; Arnold et al., 2008). Thus gossypol and ApoG2 fallunder the category of plant polyphenols such as flavonoids andcatechins (considered to possess anticancer properties) that areable to mobilize endogenous copper leading to the formation ofreactive oxygen species and consequent cellular DNA breakage(Hadi et al., 2000, 2007; Hanif et al., 2008; Shamim et al., 2008; Ul-lah et al., 2009; Khan et al., 2011). We believe that this could be animportant mechanism for the anticancer properties of gossypoland ApoG2. We give below several lines of evidence in support ofthe above mechanism:

1. Copper is an important metal ion present in the chromatinand is closely associated with DNA bases particularly guanine(Bryan, 1979; Kagawa et al., 1994). Copper transporters areover expressed in malignant cells, which aid the uptake andaccumulation of copper (Peng et al., 2006). It is well estab-lished that tissue, cellular and serum copper levels are con-siderably elevated in various malignancies (Gupte andMumper, 2008). Therefore, cancer cells may be subject togreater electron transfer between copper ions and gossypol/ApoG2 to generate ROS. There are a number of studies whichhave focused on determining the concentrations of fourimportant elements; copper, zinc, iron and selenium inpatients with cancer (Kuo et al., 2002; Zuo et al., 2006). Thesestudies showed that while zinc, iron and selenium concentra-tions were significantly lower in patients with cancer, thecopper concentrations were almost always found to be signif-icantly elevated (up to two- to threefold) compared to age-matched samples from normal tissue.

2. Fe3+ and Cu2+ are the most redox active of the metal ions inliving cells. Wolfe et al. (1994) have proposed that a coppermediated Fenton reaction, generating site-specific hydroxylradicals, is capable of inducing apoptosis in thymocytes. Ina study with thiol-containing compounds, apoptosis wasinduced in different cell lines when either free copper or ceru-loplasmin (a copper binding protein) was added; such activitywas not observed, however, when either free iron or the iron-containing serum protein transferrin was added (Held et al.,1996).

3. Among oxygen radicals, the hydroxyl radical is most electro-philic with high reactivity and therefore possesses a smalldiffusion radius. Thus, in order to cleave DNA, it must be

produced in the vicinity of cellular DNA (Pryor, 1988). Thelocation of the redox-active metal is of utmost importancebecause the hydroxyl radical, due to its extreme reactivity,interacts exclusively in the vicinity of the bound metal. Thisis also in concurrence with the observation that gossypolinduces apoptosis-like DNA fragmentation in HL-60 cells (Jar-vis et al., 1994). Such internucleosomal DNA laddering oftenused as an indicator of apoptosis may reflect DNA fragmenta-tion by non-enzymatic processes by metal-chelating agentsthat promote the redox activity of endogenous copper ionsand the resulting production of hydroxyl radicals (Burkittet al., 1996).

4. In normal cells, there exists a balance between the free radicalgeneration and the antioxidant defense (Devi et al., 2000).However, it has been clearly documented that tumor cellsare under persistent oxidative stress and have an altered anti-oxidant system (Schumacker, 2006) and thus further ROSstress in these malignant cells reaching a threshold levelcould result in apoptosis (Gupte and Mumper, 2008). Theseobservations further suggest that neoplastic cells may bemore vulnerable to oxidative stress because they functionwith a heightened basal level of ROS due to increased rateof growth and metabolism (Kong et al., 2000). Thus, in cancercells, an enhanced exposure to ROS, generated through theredox activity of endogenous copper, can overwhelm the cellsantioxidant capacity, leading to irreversible damage and celldeath.

5. Conclusion

The significance of the results presented above lies not so muchin the use of gossypol or its derivatives as antitumor agents but asin further confirmation of our hypothesis which envisages themobilization of endogenous copper and the consequent generationof ROS. Since tumor cells are already under considerable oxidativestress any further increase in ROS levels is likely to be cytotoxic(Schumacker, 2006). Thus, the mechanism proposed by us wouldbe an alternative, non-enzymatic and copper dependent pathwayfor the cytotoxic action of certain anticancer agents that arecapable of mobilizing and reducing endogenous copper. Such acommon mechanism better explains the anticancer effects of poly-phenols with diverse chemical structures as also the preferentialcytotoxicity towards cancer cells. Further, this also leads to theprospect of synthesizing novel anticancer compounds with greaterpermeability and half-life in cells and more efficient copper-chela-tion and -reducing capabilities.

Acknowledgments

The authors acknowledge the financial assistance Provided bythe University Grants Commission, New Delhi, Under the BSR Pro-gramme. The authors also acknowledge the financial support ofCSIR, New Delhi.

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