Advertisement

Tumor Biology

, Volume 36, Issue 2, pp 1237–1244 | Cite as

Redox cycling of Cu(II) by 6-mercaptopurine leads to ROS generation and DNA breakage: possible mechanism of anticancer activity

  • Sayeed Ur Rehman
  • Haseeb Zubair
  • Tarique Sarwar
  • Mohammed Amir Husain
  • Hassan Mubarak Ishqi
  • Shamshun Nehar
  • Mohammad Tabish
Research Article

Abstract

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.

Keywords

6-mercaptopurine Copper Proxidant property DNA damage Comet assay 

Introduction

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 [5].

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 [11] Copper associated with DNA is believed to be involved in maintaining normal chromosome structure apart from assisting in gene regulatory processes [12]. 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 [10].

The interaction of copper with 6MP has been well documented [13]. The stability constant of 6MP-copper complex is known to exceed from those of several naturally occurring chelators of copper [14], 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 [15]. 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

Materials

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) [18]. 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) [19]. 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 [20] 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 [21]. 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 [22] 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 %.

TBARS assay

6MP-induced formation of TBA-reactive species (TBARS) as a measure of oxidative stress in lymphocytes was determined according to Ramanathan et al. [23]. 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.

Comet assay

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.

Statistical analysis

The statistical analysis of comet assay was performed as per Tice et al. [25] 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.

Results

Conversion of Cu(II) to Cu(I) in the presence of 6MP

UV-visible spectroscopy was employed to study the interaction of Cu(II) with 6MP. As seen in Fig. 1, with increasing concentration of Cu(II), there was a decrease in absorbance possibly due to the formation of 6MP-Cu(II) complex, indicating a direct interaction of 6MP with Cu(II). The interaction of 6MP and copper was extensively studied where strong interaction of 6MP and Cu(II) is described [13]. 6MP-induced conversion of Cu(II) to Cu(I) was studied by detecting the formation of bathocuproine-Cu(I) complex (Fig. 2a). Bathocuproine, a Cu(I)-specific chelator, gives a characteristic peak at 480 nm after chelating Cu(I) [18]. On addition of Cu(II) to a mixture containing fixed amount of 6MP and bathocuproine, there was an increase in the absorption at 480 nm. This confirmed the conversion of Cu(II) to Cu(I) in the presence of 6MP. In order to find the Cu(II) to Cu(I) conversion efficiency of 6MP, another experiment was performed (Fig. 2b). It was seen that 10 μM 6MP could convert 20 μM Cu(II) to Cu(I), and a plateau was obtained on further addition of Cu(II). Similarly, 20 μM 6MP could convert 40 μM of Cu(II) to Cu(I). This indicated that 6MP could convert twice the amount of Cu(II) to Cu(I). We further compared the copper redox cycling property of 6MP with other anticancer agents and found that 6MP was highly efficient in converting Cu(II) to Cu(I) as compared to other anticancer drugs tested (Fig. 3).
Fig. 1

Interaction of 6MP with Cu(II). Absorbance spectra of 6MP with increasing concentration of Cu(II). Concentration of 6MP was 50 μM in 10 mM Tris-HCl (pH 7.2) and increasing concentration of Cu(II) was added (0–50 μM)

Fig. 2

Reduction of Cu(II) to Cu(I) by 6MP. a 6MP-induced conversion of Cu(II) to Cu(I) was studied by detecting formation of bathocuproine-Cu(I) complex. Absorption spectra were taken between 400 and 600 nm with increasing concentration of Cu(II) while keeping the fixed concentrations of bathocuproine (1 mM) and 6MP (50 μM). (a bathocuproine only, b bathocuproine + 6MP). b Amount of Cu(II) converted to Cu(I) by 6MP. Increasing concentration of copper was added in the presence of bathocuproine and 6MP (10 and 20 μM). Formation of bathocuproine-Cu(I) complex was studied by recording absorbance at 480 nm and the result was plotted as function of Cu(II) concentration

Fig. 3

Reduction of Cu(II) to Cu(I) by anticancer drugs. Increasing concentration of copper was added in the presence of bathocuproine (1 mM) and different anticancer drugs (20 μM). Formation of bathocuproine-Cu(I) complex was studied by recording absorbance at 480 nm and the result was plotted as function of Cu(II) concentration. CHL chlorambucil, 6MP 6 mercaptopurine, ISF ifosfamide, ALP allopurinol, DAC dacarbazine

Copper-mediated formation of ROS by 6MP

A time-dependent increase in the generation of superoxide anion by 6MP was evidenced as a reduction of NBT (Fig. 4a). It is known that under in vitro conditions, superoxide anions undergo spontaneous dismutation to form H2O2 which further leads to the formation of hydroxyl radical in the presence of Cu+ via Fenton-like reaction. 6MP alone does not produce any detectable hydroxyl radical (result not shown). However, in the presence of Cu(II), a dose-dependent increase in hydroxyl radical production was obtained in the presence of 6MP (Fig. 4b). Intracellular ROS generation was determined using dichlorofluorescein assay. DCHF-DA (2, 7-dichlorodihydrofluorescein diacetate) is a nonfluorescent probe that easily diffuses inside the cell and is converted to DCHF by intracellular esterases. In the presence of ROS, DCHF is rapidly oxidized to fluorescent DCF. As seen in Fig. 5, fluorescent DCF formation was observed confirming intracellular ROS generation in 6MP concentration-dependent manner. In the presence of 25 μM exogenous copper, ROS generation was further enhanced by 6MP in a concentration-dependent manner. These results clearly indicated the enhanced pro-oxidant property of 6MP in the presence of copper.
Fig. 4

Generation of reactive oxygen species. a Time-dependent photogeneration of superoxide anion by 6MP. Concentration of 6MP was 100 μM. Samples were placed 10 cm from the light source for various time intervals as shown on x-axis, and absorbance was read at 560 nm. Values reported are ±SEM of three independent experiments. b Formation of hydroxyl radicals as a function of increasing concentration 6MP (0–150 μM) in the presence of 25 μM Cu(II). Incubation was done at 37 °C for 1 h. Absorbance was recorded at 532 nm. Values reported are ±SEM of three independent experiments

Fig. 5

6MP- and 6MP + Cu(II)-induced intracellular ROS production. Erythrocytes were preloaded with DCHF-DA and then exposed to 6MP in the absence and presence of 25 μM Cu(II) for 15 min at 37 °C. Fluorescence intensity was recorded at 530 nm using excitation wavelength of 485 nm. Results are mean ± SEM of three independent experiments

6MP-induced generation of TBARS in lymphocytes is enhanced in the presence of Cu(II)

6MP-induced DNA damage in lymphocyte is suggested to be caused by intracellular ROS generation. Damage to DNA by oxygen radicals gives rise to TBARS [20]. We have therefore determined the formation of TBARS as a measure of oxidative stress caused by 6MP and 6MP-Cu(II) in lymphocytes. Further role of neocuproine was also taken into account to ascertain the role of Cu(I) in the generation of intracellular ROS generation by 6MP. As seen in Fig. 6, concentration-dependent increase in the formation of TBARS was obtained in the presence of 6MP which further increased in the presence of exogenous copper. In the presence of neocuproine, formation of TBARS was significantly decreased as neocuproine removes the Cu(I) from the redox cycling and hence decreased ROS generation. These results further strengthen the role of copper ions in enhancing the pro-oxidant property of 6MP.
Fig. 6

Effect of 6MP on TBARS generation in lymphocytes, role of Cu(II) and neocuproine. Cells were preincubated for 30 min in the presence of 25 μM Cu(II) or 100 μM neocuproine where required and then treated with increasing concentration of 6MP for 2 h at 37 °C. Values reported are ±SEM of three independent experiments

Induced DNA breakage by 6MP in lymphocytes

Further, increasing concentrations of 6MP alone and in the presence of 25 μM Cu(II) were tested for DNA breakage in isolated human lymphocytes using alkaline single-cell gel electrophoresis. Increase in DNA damage was found with increasing concentration of 6MP as evident by the increase in tail length. However, in the presence of Cu(II), a significantly greater degree of DNA breakage was observed (Fig. 7a). The result clearly suggests that 6MP is capable of causing DNA damage which significantly increases in the presence of Cu(II).
Fig. 7

6MP-induced human lymphocyte DNA breakage. a Comet tail length obtained after treatment with 6MP alone and in the presence of Cu(II). Lymphocytes were incubated with increasing concentration of 6MP alone or in the presence of 25 μM Cu(II) for 2 h at 37 °C and processed further for comet assay. b Role of membrane permeable Cu(I) chelator neocuproine and membrane impermeable Cu(I) chelator bathocuproine on 6MP-induced lymphocyte DNA breakage. Values reported are ±SEM of three independent experiments. *p value < 0.01 when compared to control. **p value < 0.01 when compared to *(6MP treated)

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

Scavengers of ROS such as superoxide dismutase (SOD), catalase, and thiourea (scavengers of superoxide, H2O2, and hydroxyl radical respectively) were found to inhibit the DNA damage caused by 6MP in human lymphocytes. All three caused significant inhibition of DNA breakage as evident by decreased tail lengths (Table 1). Thus, it can be inferred that superoxide anion, H2O2, and hydroxyl radical are essential components in the pathway that leads to DNA damage.
Table 1

Effect of active oxygen species scavengers on 6MP-induced lymphocyte DNA breakage. All values represent SEM of three independent experiments

Dose

Tail length (μM))

% Inhibition

Untreated

3.491 ± 0.194

6MP (150 μM)

22.601 ± 1.857*

6MP (150 μM) + catalase (100 μg/ml)

12.017 ± 0.598**

47

6MP (150 μM) + SOD (100 μg/ml)

10.213 ± 0.867**

55

6MP (150 μM) + thiourea (1 mM)

11.130 ± 0.613**

51

*p < 0.01 (when compared to control); **p < 0.01 (when compared to treated)

Discussion

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 [15]. 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 [15]. 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 [26], 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 [18], 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.

Role of ROS is well documented in inducing several responses in the cell [27]. It is reported that various anticancer drugs induce ROS generation which further activate cell death pathways. Anthracyclines, such as daunorubicin, generate superoxide, via series of reactions with cellular components that activate neutral sphingomyelinase enzyme and increase intracellular ceramide that activates the JNK/SAPK pathway leading to cell death [28]. Another member, doxorubicin, also generates superoxide and hydrogen peroxide which can mediate mitochondrial damage leading to apoptosis in a p53-independent pathway [29]. It has been shown that generation of ROS is an important mechanism of action of retinoid, N-4 hydroxyphenyl retinamide [30] which causes cardiolipin peroxidation and alteration in membrane permeability. This leads to cytochrome c release into the cytoplasm followed by caspase 3 activation causing apoptosis [31]. ROS are also reported as essential activators of apoptosis in cells having accumulated mutations [32]. It is expected that 6MP when administered as chemotherapeutic agent can possibly interact with endogenous copper associated with DNA and mobilize it. Subsequent generation of ROS and damage to DNA and protein may surpass the limit that can be tolerated by cancer cells that are already under increased oxidative stress. Formation of hypothesized ternary complex of DNA-6MP-Cu(II), where 6MP interacts with DNA and DNA bound copper is proposed to play a major role in causing strand scission. The summary of possible role of 6MP in ROS generation inside cancer cells is depicted in Fig. 8. Bioavailability of 6MP in the system can be of major issue. However, certain studies have reported that the activity of xanthine oxidase (XO), the enzyme responsible for metabolizing 6MP, is found to be reduced in malignancies [33]. It was also observed that Cu(II) behaves as potent inhibitor of XO [34]. Since copper level is elevated in various malignancies, this could be one of the possible reasons for the decrease in the activity of XO. Hence, there is possibility that 6MP remains for a longer time in cells when copper levels are elevated. It is concluded that 6MP, apart from acting as antimetabolite, may also possess anticancer/apoptosis-inducing activity by involving endogenous copper ions. If corroborated with subsequent studies, this would be an alternative, copper-dependent pathway for cytotoxic action of 6MP. The study can help us in identifying or synthesizing novel anticancer drugs with better copper chelating and pro-oxidant properties.
Fig. 8

Proposed role of 6MP in the generation of reactive oxygen species in cancer cells

Notes

Acknowledgments

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

None

References

  1. 1.
    Lawrance IC. What is left when anti-tumour necrosis factor therapy in inflammatory bowel diseases fails? World J Gastroenterol. 2014;20:1248–58.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Scott FI, Osterman MT. Medical management of Crohn disease. Clin Colon Rectal Surg. 2013;26:67–74.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Frei P, Biedermann L, Nielsen OH, Rogler G. Use of thiopurines in inflammatory bowel disease. World J Gastroenterol. 2013;19:1040–8.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Elion GB. The purine path to chemotherapy. Science. 1989;244:41–7.CrossRefPubMedGoogle Scholar
  5. 5.
    Swann PF, Waters TR, Moulton DC, Xu YZ, Zheng Q, Edwards M, et al. Role of post replicative DNA mismatch repair in the cytotoxic action of thioguanine. Science. 1996;273:1109–11.CrossRefPubMedGoogle Scholar
  6. 6.
    Jing Y, Dai J, Chalmers-Redman RM, Tatton WG, Waxman S. Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a hydrogen peroxide-dependent pathway. Blood. 1999;94:2102–11.PubMedGoogle Scholar
  7. 7.
    Serrano J, Palmeira CM, Kuehl DW, Wallace KB. Cardioselective and cumulative oxidation of mitochondrial DNA following subchronic doxorubicin administration. Biochim Biophys Acta. 1999;1411:201–5.CrossRefPubMedGoogle Scholar
  8. 8.
    Hug H, Strand S, Grambihler A, Galle J, Hack V, Stremmel W, et al. Reactive oxygen intermediates are involved in the induction of CD95 ligand mRNA expression by cytostatic drugs in hepatoma cells. J Biol Chem. 1997;272:28191–3.CrossRefPubMedGoogle Scholar
  9. 9.
    Miyajima A, Nakashima J, Yoshioka K, Tachibana M, Tazaki H, Murai M. Role of reactive oxygen species in cis-dichlorodiammineplatinum-induced cytotoxicity on bladder cancer cells. Br J Cancer. 1997;76:206–10.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hadi SM, Bhat SH, Azmi AS, Hanif S, Uzma S, Ullah MF. Oxidative breakage of cellular DNA by plant polyphenols: a putative mechanism for anticancer properties. Semin Cancer Biol. 2007;17:370–6.CrossRefPubMedGoogle Scholar
  11. 11.
    Zubair H, Khan HY, Sohail A, Azim S, Ullah MF, Ahmad A, et al. Redox cycling of endogenous copper by thymoquinone leads to ROS-mediated DNA breakage and consequent cell death: putative anticancer mechanism of antioxidants. Cell Death Dis. 2013;4:e660.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Nazeem S, Azmi AS, Hanif S, Kumar KS. Reactive oxygen-dependent dna damage resulting from the oxidation of plumbagin by a copper-redox cycle mechanism: implications for its anticancer properties. Aust-Asian J Cancer. 2008;7:72.Google Scholar
  13. 13.
    Kela U, Vijayvargiya R. Studies on the mechanism of action of 6-mercaptopurine. Interaction with copper and xanthine oxidase. Biochem J. 1981;193:799–803.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Foye WO. Role of metal-binding in the biological activities of drugs. J Pharm Sci. 1961;50:93–108.CrossRefPubMedGoogle Scholar
  15. 15.
    Gupte A, Mumper RJ. Elevated copper and oxidative stress in cancer cells as a target for cancer treatment. Cancer Treat Rev. 2008;35:32–46.CrossRefPubMedGoogle Scholar
  16. 16.
    Pelicano H, Feng L, Zhou Y, Carew JS, Hileman EO, Plunkett W, et al. Inhibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated mechanism. J Biol Chem. 2003;278:37832–9.CrossRefPubMedGoogle Scholar
  17. 17.
    Sastre J, Pallardo FV, Vina J. Mitochondrial oxidative stress plays a key role in aging and apoptosis. IUBMB Life. 2000;49:427–35.CrossRefPubMedGoogle Scholar
  18. 18.
    Li Z, Yang X, Dong S, Li X. DNA breakage induced by piceatannol and copper(II): mechanism and anticancer properties. Oncol Lett. 2012;3:1087–94.PubMedPubMedCentralGoogle Scholar
  19. 19.
    Nakayama T, Kimura T, Kodama M, Nagata C. Generation of hydrogen peroxide and superoxide anion from active metabolites of naphthylamine and amino azo dyes: its possible role in carcinogenesis. Carcinogenesis. 1983;4:765–9.CrossRefPubMedGoogle Scholar
  20. 20.
    Quinlan GJ, Gutteridge JMC. Oxygen radical damage to DNA by rifamycin SV and copper ions. Biochem Pharmacol. 1987;36:3629–33.CrossRefPubMedGoogle Scholar
  21. 21.
    Ahmad MK, Amani S, Mahmood R. Potassium romated causes cell lysis and induces oxidative stress in human erythrocytes. Environ Toxicol. 2014;29:138–45.CrossRefPubMedGoogle Scholar
  22. 22.
    Pool-Zobel BL, Guigas C, Klein RG, Neudecker CH, Renner HW, Schmezer P. Assessment of genotoxic effects by lindane. Food Chem Toxicol. 1993;31:271–83.CrossRefPubMedGoogle Scholar
  23. 23.
    Ramanathan A, Das NP, Tan CH. Effects of Ƴ-linolenic acid, flavonoids and vitamins on cytotoxicity and lipid peroxidation. Free Radic Biol Med. 1994;16:43–8.CrossRefPubMedGoogle Scholar
  24. 24.
    Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1998;175:184–91.CrossRefGoogle Scholar
  25. 25.
    Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 2000;35:206–21.CrossRefPubMedGoogle Scholar
  26. 26.
    Sarkar B, Roberts EA. The puzzle posed by COMMD1, a newly discovered protein binding Cu(II). Metallomics. 2011;3:20–7.CrossRefPubMedGoogle Scholar
  27. 27.
    Hensley K, Robinson KA, Gabbita SP, Salsman S, Floyd RA. Reactive oxygen species, cell signaling, and cell injury. Free Radic Biol Med. 2000;28:1456–62.CrossRefPubMedGoogle Scholar
  28. 28.
    Mansat-de Mas V, Bezombes C, Quillet-Mary A, Bettaieb A, D’orgeix AD, Laurent G, et al. Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin. Mol Pharmacol. 1999;56:867–74.PubMedGoogle Scholar
  29. 29.
    Tsang WP, Chau SP, Kong SK, Fung KP, Kwok TT. Reactive oxygen species mediate doxorubicin induced p53-independent apoptosis. Life Sci. 2003;73:2047–58.CrossRefPubMedGoogle Scholar
  30. 30.
    Suzuki S, Higuchi M, Proske RJ, Oridate N, Hong WK, Lotan R. Implication of mitochondria-derived reactive oxygen species, cytochrome c and caspase-3 in N-(4-hydroxyphenyl)retinamide-induced apoptosis in cervical carcinoma cells. Oncogene. 1999;18:6380–7.CrossRefPubMedGoogle Scholar
  31. 31.
    Asumendi A, Morales MC, Alvarez A, Arechaga J, Perez-Yarza G. Implication of mitochondria-derived ROS and cardiolipin peroxidation in N-(4 hydroxyphenyl) retinamide-induced apoptosis. Br J Cancer. 2002;86:1951–6.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Mishra A, Awate R, Namrata S, Mishra N, Soni R, Sharma P. Synthesis and characterization of transition metal (Cu, Co, Fe) complexes of 6-methyl-5-arylhydrazono-2 thio-4-oxo-pyrimidine ligand. Phosphorus Sulfur Silicon Relat Elem. 2009;184:2624–35.CrossRefGoogle Scholar
  33. 33.
    Prajda N, Morris HP, Weber G. Imbalance of purine metabolism in hepatomas of different growth rates as expressed in behavior of xanthine oxidase (EC 1.2.3.2). Cancer Res. 1976;36:4639–46.PubMedGoogle Scholar
  34. 34.
    Sau AK, Mondal MS, Mitra S. Interaction of Cu2+ ion with milk xanthine oxidase. Biochim Biophys Acta. 2001;544:89–95.CrossRefGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  • Sayeed Ur Rehman
    • 1
  • Haseeb Zubair
    • 1
  • Tarique Sarwar
    • 1
  • Mohammed Amir Husain
    • 1
  • Hassan Mubarak Ishqi
    • 1
  • Shamshun Nehar
    • 2
  • Mohammad Tabish
    • 1
  1. 1.Department of Biochemistry, Faculty of Life SciencesA. M. UniversityAligarhIndia
  2. 2.PG Section, Department of ZoologyRanchi UniversityRanchiIndia

Personalised recommendations