Redox cycling of Cu(II) by 6-mercaptopurine leads to ROS generation and DNA breakage: possible mechanism of anticancer activity
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6-Mercaptopurine (6MP) is a well-known purine antimetabolite used to treat childhood acute lymphoblastic leukemia and other diseases. Cancer cells as compared to normal cells are under increased oxidative stress and show high copper level. These differences between cancer cells and normal cells can be targeted to develop effective cancer therapy. Pro-oxidant property of 6MP in the presence of metal ions is not well documented. Redox cycling of Cu(II) to Cu(I) was found to be efficiently mediated by 6MP. We have performed a series of in vitro experiments to demonstrate the pro-oxidant property of 6MP in the presence of Cu(II). Studies on human lymphocytes confirmed the DNA damaging ability of 6MP in the presence of Cu(II). Since 6MP possesses DNA damaging ability by producing reactive oxygen species (ROS) in the presence of Cu(II), it may also possess apoptosis-inducing activity by involving endogenous copper ions. Essentially, this would be an alternative and copper-dependent pathway for anticancer activity of 6MP.
Keywords6-mercaptopurine Copper Proxidant property DNA damage Comet assay
6-Mercaptopurine (6MP) is an immunosuppressive drug that is also used as an anti-inflammatory and antineoplastic agent for treating childhood acute lymphoblastic leukemia, pediatric non-Hodgkin’s lymphoma, and psoriatic arthritis. Use of 6MP in the treatment of systemic autoimmune disease, ulcerative colitis, and other pathological conditions is also well documented [1, 2, 3, 4]. The known mechanism of action of 6MP is the activation of 6MP by hypoxanthine guanine phosphoribosyl transferase to form thioinosine monophosphate which is further metabolized to thioguanine monophosphate. The mononucleotides thus formed inhibit the first step of purine de novo synthesis. Moreover, it is also established that the metabolism of 6MP culminates in the formation of 6-thioguanine (6-TG) that further gets incorporated into DNA. The accumulated 6-TG in DNA gets methylated and the resulting DNA containing 6-meTG mispairs with thymine (T) in further replications. Consequently, the activation of mismatch repair system leads to cell death .
Several anticancer agents like arsenic trioxide, doxorubicin, bleomycin, and cisplatin are found to generate cellular reactive oxygen species (ROS) [6, 7, 8, 9]. Naturally occurring antioxidants such as polyphenols are known to interact with DNA and mediate its cleavage as a result of generation of ROS caused by redox cycling of copper [10, 11]. Redox cycling, in which a single electron may be accepted or donated by metal ions, catalyzes reactions that produce reactive radicals and thus produces ROS. It is likely that the formation of hydroxyl radical in close proximity of DNA may cause strand scission. Among the various metal ions found inside the cell, Fe3+ and Cu2+ are the most redox-active. Copper has important role in forming the essential redox-active centers in various metalloproteins. It is also found in nucleus closely associated with guanine bases of DNA  Copper associated with DNA is believed to be involved in maintaining normal chromosome structure apart from assisting in gene regulatory processes . It is known that chromatin-bound endogenous copper ions can be mobilized by metal chelating agents and generate ROS, leading to internucleosomal DNA fragmentation, a characteristic property of cells undergoing apoptosis .
The interaction of copper with 6MP has been well documented . The stability constant of 6MP-copper complex is known to exceed from those of several naturally occurring chelators of copper , making 6MP a suitable candidate for our study. It is well known that cancer cells contain elevated level of copper and is under oxidative stress . Thus, it is expected that metal chelating agents, like 6MP, can interact with endogenous copper ions and facilitate redox cycling that may further enhance ROS production in cancer cells. Any further increase in oxidative stress in cancer cells may exhaust their antioxidant capability, leading to apoptosis [16, 17]. In this paper, we have demonstrated that 6MP mediates the redox cycling of copper efficiently. Also, other in vitro experiments suggested for a possible role of copper in enhancing the ROS generation property of 6MP. Using a system of peripheral human lymphocytes and alkaline single-cell gel electrophoresis, we have documented for the first time that 6MP can cause oxidative DNA breakage in the absence and presence of added copper. Finally, we propose that the pro-oxidant property of 6MP in the presence of endogenous copper could play an important role in its anticancer property and can be utilized as a lead molecule for the synthesis of novel anticancer drugs with better copper chelating and pro-oxidant properties.
Materials and methods
6MP, calf thymus DNA (CT DNA), normal melting point agarose, low melting point agarose, RPMI (Roswell Park Memorial Institute) 1640, Triton X-100, trypan blue, Histopaque 1077, superoxide dismutase (SOD), neocuproine, catalase, and 2,7-dichlorodihydrofluorescein diacetate (DCHF-DA) were purchased from Sigma Chemical Co. (St. Loius, MO, USA). All other chemicals were of analytical grade. A 10 mM stock of 6MP was made in dimethyl sulfoxide (DMSO). All other reaction mixtures were prepared in 10 mM Tris-HCl (pH 7.2).
Interaction of 6MP with Cu(II)
In order to detect the interaction of Cu(II) with 6MP, absorption spectra of 6MP (50 μM) in the absence and presence of increasing concentration of Cu(II) (0–50 μM)) in 10 mM Tris-HCl (pH 7.2) was recorded using Shimadzu spectrophotometer (Japan). Further, detection of 6MP-induced redox cycling of Cu(II) to Cu(I) was done utilizing bathocuproine. Bathocuproine is a Cu(I)-specific chelator that gives a characteristic peak at 480 nm when complexed with Cu(I) . Increasing concentration of Cu(II) was added to the reaction mixture that also contained 6MP (50 μM), 1 mM bathocuproine, and 10 mM Tris-HCl (pH 7.2). Absorption spectra were recorded from 400 to 600 nm. To check the amount of Cu(II) converted to Cu(I) by 6MP, fixed concentration of 6MP (10 or 20 μM) was taken along with 1 mM bathocuproine and increasing concentration of Cu(II) was added (0–80 μM). Absorbance was recorded at 480 nm. Similarly, different anticancer drugs where compared for redox cycling of copper. Details are mentioned in the legend to the figure.
Assay of reactive oxygen species
The time-dependent generation of superoxide anion by 6MP was detected by the reduction of nitroblue tetrazolium (NBT) . Assay mixture containing 10 mM sodium phosphate buffer (pH 7.8), 0.5 mM NBT, 0.1 mM EDTA, and 0.06 % Triton X-100 along with 6MP was placed in front of white fluorescent lamp at a distance of 10 cm and absorbance was recorded at 560 nm. No change in the temperature of the solution was observed at the end of the experiment. Hydroxyl radical formation was assayed essentially by the method described by Quinlan and Gutteridge  with some changes. CT DNA (300 μg) was treated with increasing concentration of 6MP (0–150 μM) in the presence of 25 μM Cu(II) in 10 mM Tris-HCl (pH 7.2). Incubation was done for 60 min at 37 °C. The generation of malonaldehyde was detected by the reaction with thiobarbituric acid (TBA), and the colored adduct formed was recorded by taking absorbance at 532 nm.
Intracellular ROS generation—dichlorofluorescein assay
6MP- and 6MP-Cu(II)-induced intracellular ROS production was assayed using DCHF-DA . Erythrocytes at 5 % hematocrit were incubated with 10 μM DCHF-DA for 1 h at 37 °C. Cells were washed twice with phosphate-buffered saline (PBS). The erythrocytes were then resuspended in PBS and exposed to varying concentration of 6MP alone and in the presence of 25 μM Cu(II) for 15 min at 37 °C. The emission fluorescence was recorded at 530 nm after excitation at 485 nm on spectrofluorometer (Shimadzu, Japan). The amount of ROS generated is directly proportional to DCF formation that was plotted as percent change from control.
Isolation and viability assessment of lymphocytes
Fresh blood samples (3 ml) were obtained from healthy volunteers by vein puncture and stored in the presence of heparin. Lymphocytes were isolated from diluted blood using Histopaque 1077 (HiMedia) and suspended in RPMI 1640. Trypan blue exclusion test  was performed before start and after the end of the experiment to check the viability of lymphocytes. Viability of cells was found to be more than 95 %.
6MP-induced formation of TBA-reactive species (TBARS) as a measure of oxidative stress in lymphocytes was determined according to Ramanathan et al. . Fixed number of cells (1 × 105 cells/ml) were incubated with 6MP or 6MP + Cu(II) or 6MP + Cu(I) chelator (neocuproine) in different sets of experiments. Cells were preincubated with 25 μM Cu(II) and 100 μM neocuproine for 30 min prior to 6MP addition where required. After incubation for 2 h in the presence of 6MP at 37 °C, cells were centrifuged and washed twice with PBS (Ca2+ and Mg2+ free) and suspended in 0.1 N NaOH. Cell suspension was further treated with 10 % TCA and 0.6 M TBA and incubated in boiling water for 10 min. Absorbance was read at 532 nm.
A fixed number of lymphocyte cells were treated with different concentrations of 6MP in a total reaction volume of 500 μl composed of Ca2+ and Mg2+-free PBS and RPMI 1640. Lymphocytes were incubated with Cu(II) or ROS scavengers or neocuproine/bathocuproine for 1 h prior to 6MP treatment. Incubation in the presence of 6MP was performed for 2 h at 37 °C followed by centrifugation at 5000 rpm to collect the lymphocyte. The cell pellet was further suspended in 100 μl Ca2+ and Mg2+-free PBS and further processed for comet assay and performed as described earlier [24, 25]. Analysis of the slides was done the same day and scored using image analysis system (Komet 5.5; Kinetic Imaging, Liverpool, UK) attached to an Olympus (CX41) fluorescent microscope (Olympus Optical Co, Tokyo, Japan) and a COHU 4910 integrated CC camera equipped with 510–560 nm excitation and 590 nm barrier filters (COHU, San Diego, CA, USA). Images from 50 cells were analyzed. Migration of DNA from the nucleus, i.e., tail length, was measured as the main parameter to assess lymphocyte DNA damage.
The statistical analysis of comet assay was performed as per Tice et al.  and is expressed as ±standard error of the mean (SEM) of three experiments. All other experiments were also statistically analyzed by one-way ANOVA. p values <0.01 were considered statistically significant.
Conversion of Cu(II) to Cu(I) in the presence of 6MP
Copper-mediated formation of ROS by 6MP
6MP-induced generation of TBARS in lymphocytes is enhanced in the presence of Cu(II)
Induced DNA breakage by 6MP in lymphocytes
Neocuproine ameliorates 6MP-induced DNA damage in human lymphocyte
Redox cycling of Cu(II) and the formation of Cu(I) in 6MP-Cu(II) complex is essential for the generation of ROS, which act as the mediators of DNA breakage. In Fig. 7b, Cu(I)-specific chelators were used to assess their efficacy in inhibiting 6MP-mediated DNA breakage in comet assay. With the increasing concentration of neocuproine (a Cu(I)-specific, membrane permeable chelator), there was a gradual decrease in the tail length. However, bathocuproine (a membrane impermeable Cu(I) chelator) did not have any significant effect on the DNA breakage efficacy of 6MP. This suggests that the 6MP-mediated redox cycling of intracellular copper leads to DNA breakage in human lymphocytes.
ROS scavengers protect 6MP-induced human lymphocyte DNA breakage
Effect of active oxygen species scavengers on 6MP-induced lymphocyte DNA breakage. All values represent SEM of three independent experiments
Tail length (μM))
3.491 ± 0.194
6MP (150 μM)
22.601 ± 1.857*
6MP (150 μM) + catalase (100 μg/ml)
12.017 ± 0.598**
6MP (150 μM) + SOD (100 μg/ml)
10.213 ± 0.867**
6MP (150 μM) + thiourea (1 mM)
11.130 ± 0.613**
Living organisms are constantly under attack of free radicals, including superoxide anion (·O2 −) and hydroxyl radical (·OH). Evidences suggest that cancer cells are under increased oxidative stress as compared to normal cells . Moreover, several anticancer drugs currently used for cancer treatment have been shown to increase cellular ROS levels, leading to cancer cell death [6, 7, 8, 9]. Thus, in chemotherapy involving these agents, cancer cells are expected to face further ROS insult that may surpass the survival limit ultimately leading to apoptosis.
Superoxide anion, formed in almost all aerobic cells, promotes the formation of hydrogen peroxide (H2O2), which in the presence of suitable transition metal ions such as Fe3+ or Cu2+ is converted to highly reactive ·OH radicals. In our experiments, we have used Cu(II) concentration close to the concentrations reported in most of the cancer cells . Our results indicate that 6MP in the presence of Cu(II) is able to cause DNA breakage by the redox cycling of copper and subsequent generation of ROS. Moreover, since most of the intracellular copper ions are present in Cu(I) form , its reoxidation to Cu(II) by H2O2 in a Fenton-like reaction is very much feasible. 6MP further participates to complete the cycle by converting Cu(II) to Cu(I). We have shown that 6MP mediates the reduction of Cu(II) to Cu(I). The Fenton-like reaction culminates in the formation of hydroxyl radical (·OH) in the presence of Cu(II). It is highly expected that the hydroxyl radicals formed in these reactions can cause strand scission in DNA. However, due to strong electrophilic nature and a small diffusion radius, formation of these highly reactive hydroxyl radicals should occur in close proximity of DNA in order to cleave it. Since the copper ions are also closely associated with DNA , 6MP can interact with bound copper ions and produce hydroxyl radicals close enough to DNA and cause damage. Increase in 6MP-induced intracellular ROS was observed in the presence of Cu(II) by dichlorofluorescein assay. Similarly, 6MP-induced generation of TBARS was enhanced in the presence of Cu(II), while reduction in TBARS generation occurred in the presence of neocuproine, suggesting a significant role of copper in 6MP-induced DNA damage.
Subsequent studies conducted on cellular system of isolated human peripheral lymphocytes using alkaline single-cell gel electrophoresis further confirmed that 6MP is capable of causing DNA degradation. Moreover, such DNA degradation is significantly enhanced in the presence of exogenous copper. With the increasing concentration of neocuproine, a Cu(I)-specific membrane permeable chelator, decrease in tail length was recorded by comet assay confirming the participation of copper in 6MP-mediated DNA degradation. Lowering of the DNA damage due to presence of various ROS scavengers further suggested the generation of ·O2 −,, H2O2, and ·OH in the presence of 6MP.
We thank the Council of Scientific and Industrial Research (C.S.I.R.), New Delhi, India, for the award of Senior Research Fellowship to Sayeed Ur Rehman (File no. 09/112(0470)/2011-EMR1). We are also thankful to the Department of Biochemistry, A.M.U. for providing the necessary facilities.
Conflicts of interest
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