Selective induction of apoptosis in various cancer cells irrespective of drug sensitivity through a copper chelate, copper N-(2 hydroxy acetophenone) glycinate: crucial involvement of glutathione Shilpak Chatterjee, Paramita Chakraborty, Kaushik Banerjee, Abhinaba Sinha, et al. BioMetals An International Journal on the Role of Metal Ions in Biology, Biochemistry and Medicine ISSN 0966-0844 Volume 26 Number 3 Biometals (2013) 26:517-534 DOI 10.1007/s10534-013-9637-z
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Author's personal copy Biometals (2013) 26:517–534 DOI 10.1007/s10534-013-9637-z
Selective induction of apoptosis in various cancer cells irrespective of drug sensitivity through a copper chelate, copper N-(2 hydroxy acetophenone) glycinate: crucial involvement of glutathione Shilpak Chatterjee • Paramita Chakraborty • Kaushik Banerjee Abhinaba Sinha • Arghya Adhikary • Tanya Das • Soumitra Kumar Choudhuri
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Received: 18 October 2012 / Accepted: 26 May 2013 / Published online: 4 June 2013 Ó Springer Science+Business Media New York 2013
Abstract Drug induced toxicity and drug resistance are the major impediments to successful application of cancer chemotherapy. Therefore, selective targeting of the key biochemical events of the malignant cells may have a great therapeutic potential in specifically kill the cancer cells. We have evaluated in vitro the cytotoxic efficacy of a previously reported copper complex viz. copper N-(2-hydroxy acetophenone) glycinate (CuNG) on different drug sensitive and resistant cancer cell lines by MTT, annexin V positivity and caspase 3 activation assays. We have also investigated the underlying signalling events in CuNG mediated apoptosis of cancer cells by Western blotting technique. We have found that CuNG preferentially induces apoptosis to malignant cells irrespective of drug sensitivity and spares the normal cells. Our studies disclose that CuNG causes cellular redox imbalance in cancer cells through depletion of intracellular GSH level. CuNG mediated depletion of intracellular GSH level induces mitochondrial superoxide generation, which detaches cyto C from mitochondrial membrane through lipid peroxidation.
The detached cyto C then release into the extra mitochondrial milieu in Bax mediated pathway where CuNG facilitates the binding of Bax through dissociation of hexokinase II from mitochondrial membrane. The present study opens the possibility of developing effective chemotherapeutic drugs by synthesizing numerous chemical compounds capable of targeting cellular redox environment and thus specifically kills cancer cells of broad spectrum. Keywords Glutathione Apoptosis Copper complex Mitochondrial superoxide Cytochrome C Bax Hexokinase II Abbreviations CuNG Copper N-(2-hydroxy acetophenone) glycinate GSH Glutathione Cytochrome C Cyto C HK II Hexokinase II
Introduction S. Chatterjee P. Chakraborty K. Banerjee A. Sinha S. K. Choudhuri (&) Department of In Vitro Carcinogenesis and Cellular Chemotherapy, Chittaranjan National Cancer Institute, S.P. Mukherjee Road, Kolkata 700026, India e-mail:
[email protected] A. Adhikary T. Das Department of Molecular Medicine, Bose Institute, Kolkata, India
Various attempts were made during past few decades to explore highly efficacious anticancer drugs with minimum cytotoxicity. The classical cancer chemotherapeutic drugs are highly cytotoxic and often cause collateral damage to host tissues (Drı´mal et al. 2006; Sargent et al. 2001). The development of multidrug resistance (MDR) in cancer patients is also another
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drawback of chemotherapeutic drugs (Fine and Chabner 1986). Therefore, to develop novel anticancer drugs, clear understanding of the biological differences between normal, drug sensitive and resistant cancer cells are of utmost importance. It will be wise to bank upon the biochemical difference amongst normal, drug resistant and sensitive cancer cells for the development of potential therapeutic agent. The most prevalent biochemical difference in cancer cells is the increase in intracellular glutathione (GSH) level compared to the normal cells (Huang et al. 2001). GSH, a tripeptide (L-g-glutamyl-L-cysteinylglycine) present virtually in all mammalian cells provides cellular protection from the damaging effect of reactive oxygen species (ROS), free radicals and other toxic compounds (Wang and Ballatori 1998). Cancer cells have higher rates of metabolic activity and suffer from persistent oxidative stress (Szatrowski and Nathan 1991) and to counteract the damaging effect of ROS, cancer cells require very high amount of intracellular GSH (Trachootham et al. 2006). GSH regulates various gene expressions involved in cell survival and apoptosis (Arrigo 1999) e.g., nuclear factor kappa B (NF-jB) as its activity gets reduced in the GSH depleted environment (Hutter and Greene 2000). Activation of NF-jB can turn on transcription of various anti-apoptotic and cell cycle regulator proteins crucial for cancer cell survival (Wang et al. 1998; Wu et al. 1998). Apart from regulation of gene expression, intracellular GSH level often determines the balance between cell death and cell survival. Depletion of intracellular GSH level causes mitochondrial dysfunction leading to apoptosis through mitochondrial pathway (Armstrong et al. 2002). In addition enhanced expression of anti-apoptotic proteins, viz., Bcl-2 or Bcl-XL is associated with high level of intracellular GSH (Wright et al. 1998; Xu et al. 1999). GSH plays important role in drug resistance in cancer (Ozols et al. 1990). The development of resistance to alkylating agents and cisplatin is associated with increased intracellular GSH level (Ozols et al. 1990; Gurtoo et al. 1981; Suzukake et al. 1983). GSH may reduce cytotoxicity by facilitating the metabolism of drugs to less active compounds or by detoxification of the free radicals (Arrick and Nathan 1984; Meister 1983). Additionally, GSH enhances the repair of drug-induced injury, primarily at the DNA level. Moreover, the sensitivity to alkylating agents
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Biometals (2013) 26:517–534 Fig. 1 CuNG caused apoptosis of cancer cell lines irrespective c of their drug sensitivity while sparing the normal cells. Cytotoxic effect of CuNG on different drug sensitive, resistant cancer cell lines and normal cells were analysed by MTT assay. Briefly, 4 9 104 of cells per well were taken and next day treated with different dose of CuNG (e.g. 2, 4, 6, 8 and 10 lg/ml) for 24 and 48 h. Dose response curve of CuNG using a drug sensitive cancer cell lines (HCT-116, K562, CCRF-CEM, EAC and HeLa), b drug resistant cancer cell lines (CEM-ADR5000 and EAC-DR) and c normal cells (NIH3T3, human PBMC, Chang Liver, WI38 and human embryonic kidney cell line HEK293) was analyzed by MTT. CuNG induced apoptotic potential on different cell lines were measured by annexin V-FITC versus PI duel staining method. Cells were either treated with CuNG (8 lg/ml) or pretreated with NAC (5 mM) and then treated with CuNG (8 lg/ml) for different hours. End of treatment cells were labelled with annexin V-FITC and PI and percentage of annexin V positive population was determined in d drug sensitive cancer cell lines (HCT-116, K562, CCRF-CEM, EAC and Hela), e drug resistant cell lines (CEM-ADR5000 and EAC-DR) and f normal cells (NIH3T3, human PBMC, Chang liver, WI38 and HEK293) using flow cytometer. Representative data from three independent experiments is presented
can be restored by depletion of intracellular GSH (Hamilton et al. 1985; Canada et al. 1993). Previously we synthesized and characterized a novel copper chelate, viz., copper N-(2-hydroxy acetophenone) glycinate (CuNG) which proved to be a resistance modifying agent (Majumder et al. 2003; Majumder et al. 2006) and forms conjugate with GSH (Majumder et al. 2006; Basu et al. 2009) and is able to deplete GSH in EAC/DOX cells (Majumder et al. 2006). These observations incited us to speculate whether the ability of CuNG to deplete GSH can be harnessed to specifically kill the tumor cells irrespective of their drug sensitivity. Therefore, we studied the apoptotic potential of CuNG in various cancer cell lines including the drug resistant cells in vitro. Herein, we describe how depletion of intracellular GSH level of cancer cells by CuNG alters cellular redox status, which in turn induces apoptosis to cancer cells irrespective of their drug sensitivity. This study may open new avenue to synthesize a good number of chemical compounds targeting the cellular redox environment and thus specifically kill cancer cells of broad spectrum.
Materials and methods Synthesis and characterization of CuNG Synthesis of CuNG was synthesized by the reaction of potassium N-(2-hydroxy acetophenone) glycinate with hydrated copper sulfate according to the previously
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described method; characterization and structure determination were done by detailed spectroscopic studies (Majumder et al. 2003; Choudhuri 2005). The structure of CuNG (MW 308) is shown in Fig. 1. As CuNG is insoluble in water, DMSO stock solution was prepared following serial dilution with gradual addition of water or media.
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Cell culture Human T cell acute lymphoblastic leukemia cell lines CCRF-CEM and CEM-ADR5000 (doxorubicin resistant) were kindly provided by Prof T Efferth, University of Mainz, Germany, human erythromyeloblastoid leukemia cell line K562, human colon carcinoma cell line
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HCT-116, human cervical cancer cell line HeLa, normal mouse fibroblast cell line NIH3T3, normal human hepatocellular cell line Chang liver, normal human diploid lung fibroblast cell line WI38 and normal human embryonic kidney cell line HEK293 were purchased from ATCC, USA. Ehrlich ascites carcinoma EAC/ sensitive and EAC-DOX (doxorubicin resistant) cells are grown in Swiss albino mice obtained from National Institute of Nutrition (Hyderabad, India) and maintained in the institute animal facilities. Mice were used for experimental purpose with prior approval of the Institutional Animal Ethics committee. The experimental protocols described herein were approved by the IAEC (proposal no. IAEC-1.1/2008/AH-28, dated 02.12.2008; registration no.: 175/99/CPCSEA, dated 28.01.2000) in accordance with the ethical guidelines laid down by the Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA) by the Ministry of Social Justice and Empowerment, Government of India. CCRF-CEM, CEM-ADR5000, K562, EAC/sensitive and EAC-DOX cells were cultured in RPMI-1640 medium (Gibco, Invitrogen, USA); HCT-116 was cultured in McCoy’s 5A medium (Gibco, Invitrogen, USA), Hela, NIH3T3, Chang liver, WI38 and HEK293 were cultured in DMEM (Gibco, Invitrogen, USA). Culture medium was supplemented with 10 % foetal bovine serum (FBS, Gibco, Invitrogen, USA). Cells were grown in plastic tissue culture flasks (Nunc, USA) in a 5 % CO2 atmosphere at 37 °C. The doxorubicin resistant CEM-ADR5000 cell line was generated as reported previously and selectively expresses the MDR1/P-gp without concomitant overexpression of MRP1 or BCRP (Gillet et al. 2004; Efferth et al. 2003). Doxorubicin resistant EAC/DOX cell line expressing MRP1 was generated following the reported method and maintained in Swiss albino mice (Mookerjee et al. 2006). Cells from exponentially growing cultures were used for all experiments. All experiments were repeated three times. Isolation of lymphocytes Peripheral blood from healthy human volunteer was taken with prior approval from Institutional Ethics Committee (IEC), CNCI (project ref no. CNCI/IEC/ Aug-2010(5), dated 20.08.2010). IEC, CNCI was formed according to the guideline of Indian Council of Medical Research (ICMR), New Delhi and schedule
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Y-2005, CDSCO, New Delhi. Collected blood was heparinised and then diluted with equal volume of RPMI-1640. Lymphocyte-enriched mononuclear cells were isolated using Histopaque1077 (Sigma) by density gradient centrifugation. After completion of centrifugation lymphocyte-enriched layer was collected and washed twice with sterile phosphate buffer saline (PBS) and finally resuspended in cold RPMI1640 supplemented with 10 % heat inactivated foetal bovine serum (RPMI–FBS) (Chatterjee et al. 2009).
Treatment A stock solution of CuNG (20 mg/ml) was prepared just before the experiments by dissolving the lyophilized compounds in DMSO and finally suspended in culture medium to achieve final concentration of DMSO not more than at 0.1 % (v/v). For MTT assay, treatments were performed with concentration of 2, 4, 6, 8 and 10 lg/ml of CuNG. As control, equal volumes of medium containing DMSO (\0.1 %) were added to untreated cells. The antioxidant N-acetyl cystein (NAC) was used at a final concentration of 5 mM and incubated for 1 h. After incubation cells were washed twice with fresh media before addition of CuNG. 8 lg/ml concentration of CuNG (above IC50 value for all the cell line tested) was used for subsequent experiment unless otherwise mentioned. Cytotoxicity assay (MTT assay) Cell viability was determined by the MTT assay (Ganguly et al. 2010). Briefly, cells were seeded in 96-well plates at a density 4 9 104 cells/well. Cells were then allowed to settle for 24 h before treatment with increasing concentrations of CuNG and incubate it for further 24 and 48 h in 5 % CO2 at 37 °C. After completion of incubation cells were incubated with 5 mg/ml of MTT dye (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide; Sigma) for 4 h at 37 °C. The monolayer was suspended in 0.1 ml of DMSO and the absorbance at 540 nm was measured using ELISA reader (Tecan 200). The control value corresponding to untreated cells was taken as 100 % and the viability of treated samples were expressed as a percentage of the control. The IC50 values were determined as the concentration that reduced cell viability by 50 %.
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Apoptosis assay To distinguish between apoptosis and necrosis, annexin V FITC and propidium iodide (PI) binding assay was performed. Cells (5 9 105) from either untreated or treated groups were harvested and PI and annexin V FITC were added directly to the medium. The mixture was incubated for 15 min at 37 °C. Excess PI and annexin V-FITC were then washed off. Cells were then fixed and analyzed on flowcytometer using CellQuest software (Chatterjee et al. 2009; Ganguly et al. 2010). To confirm the CuNG induced apoptosis, caspase 3 activation assay was done following manufacturer’s protocol (Caspase3 Assay Kit, BD Bioscience, USA) (Chatterjee et al. 2009). Electron paramagnetic resonance (EPR) measurement EPR measurements were done at 90 K for both CuNG and different proportions of CuNG plus GSH using Bruker EMX equipped with a low temperature variable unit with sample tube mounted in a stream of cold nitrogen to minimize the disturbances in the cavity of the spectrometer. Determination of intracellular GSH contents Intracellular GSH content was measured using DTNB (5,50 -dithio-bis (2-nitrobenzoic acid) (Mookerjee et al. 2006). CuNG treated and untreated cells were washed twice with PBS and cell pellet (1 9 106 cells) was lysed with 100 ll cell lysis buffer. Thereafter 15 ll of 0.1 N HCl and 15 ll of 50 % sulphosalicylic acid were added. After centrifugation at 12,0009g for 15 min supernatants were collected. With 25 ll of cell lysate, 100 ll of DTNB (prepared in sodium phosphate buffer containing EDTA) was added and OD at 412 nm was taken immediately using spectrophotometer (Varian). RNA interference HCT-116 cells (1 9 106) were transiently transfected either with 40 pmol s/ml of control siRNA or siRNA specific for Bax (Santa Cruz Biotechnology) as per manufacturer’s instructions (Santa Cruz Biotechnology). After 24 h of transfection, cells were treated
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with CuNG (8 lg/ml) and analyzed for protein expression. Sub cellular fractionation To separate cytosolic and mitochondrial fraction, subcellular fractionation was done as reported previously (Latchoumycandane et al. 2006). Briefly, after completion of treatment, cells were washed twice with PBS. The cell pellet was resuspended in 200 ll of extraction buffer containing 200 mM mannitol, 70 mM sucrose, 20 mM HEPES–KOH, pH 7.4, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 0.1 mM PMSF and protease inhibitors (Complete Cocktail; Marck Bioscience). Following 20 min incubation on ice, cells were homogenized by 30–40 strokes with a glass Dounce homogenizer on ice. Homogenates were centrifuged at 6009g for 15 min at 4 °C, and resulting supernatant was further centrifuged at 12,0009g for 30 min at 4 °C. The resulting supernatant was the cytosolic fraction and the pellet was the mitochondrial fraction. Western blot analysis Protein concentration in either cytosolic or mitochondrial fraction was determined by Bradford method (Bradford 1976). For immunoblot analyses, 100 lg of protein lysate/sample were denatured in 29 SDSPAGE sample buffer and subjected to SDS-PAGE on 10 % Tris–glycine gel. The separated proteins were transferred onto PVDF membrane followed by blocking with 5 % BSA (w/v) in TBS (10 mM Tris, 100 mM NaCl, 0.1 % Tween 20) for 1 h at room temperature. Membrane was probed either with anti-cyto C (Santacruz Biotechnology, USA) or anti-Bax (Santacruz Biotechnology, USA) or anti-HK II (Sigma, USA) or anti-Tom-20 (Abcam) or anti-b actin (BD Pharmingen, USA) antibody overnight at 4 °C. After completion of incubation membrane was washed thrice with TBS containing 0.1 % Tween 20. After proper washing membrane was probed with HRP-conjugated secondary antibody (Sigma, USA) and incubated for 1 h at room temperature. After that membrane was vigorously washed and using a chemiluminescence kit (Lumi Glow, Cell signalling technology) chemiluminescence was identified which signified the presence of protein of interest on the membrane.
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Densitometric analysis Immunoreactive protein bands were scanned (Bio-Rad, model GS800) and then images were digitized and analyzed by using Bio-Rad QUANTITY 1 software. Immunoreactive bands were quantified and expressed as the ratio of each band density to corresponding loading control band density.
Measurement of cardiolipin peroxidation 10-N-Nonyl-acridin orange (NAO, Invitrogen, USA) (binds to mitochondria-specific cardiolipin) was used to measure cardiolipin peroxidation (Asumendi et al. 2002). Cells were treated with CuNG (8 lg/ml) for different hours, washed and labelled with 10 lg/ml NAO for 20 min. In some cultures 5 mM NAC was added 1 h before CuNG addition. After washing twice, fluorescence emitted by cardiolipin-bounded NAO was measured (excitation/emission: 485/530 nm) in a spectrofluorimeter (Varian). Measurement of mitochondrial superoxide Mitochondrial superoxide generation was assayed by using MitoSOXTM red reagent (Invitrogen, USA). Briefly, after completion of treatment cells (5 9 105) were taken, washed twice with PBS and resupended in HBSS. 5 lM MitoSOXTM red was then added and incubated for 10–15 min at 37 °C according to manufacturers’ protocol. After completion of incubation superoxide generation was measured using spectroflourimeter (Varian) excitation/emission: 510/580 nm. Hexokinase assay HK activity was determined spectrophotometrically (OD at 340 nm) by measuring the reduction of NAD? to NADH? in a coupling reaction using NAD-dependent glucose-6-phosphate dehydrogenase as reported previously (Canesi et al. 1998). Binding study of CuNG with hexokinase Binding study of CuNG with HK was done using spectrofluorimeter (Varian). Briefly, we incubated HK:CuNG at either 1:0.5 or 1:1 or 1:2 ratios for
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20 min at room temperature. Then the mixture was excited at 280 nm and emission was scanned. In some cases, 1 mM DTT was added to the protein and incubated for 15 min prior addition of CuNG.
Statistical analysis All data reported are the arithmetic mean from three independent experiments. The unpaired Student’s t test was used to evaluate the significance differences between groups, accepting P \ 0.05 as a level of significance. Data analyses were performed using the Prism software (GraphPad, San Diego, CA).
Result CuNG treatment caused apoptosis of different cancer cell lines irrespective of their drug sensitivity To determine antiproliferative effect of CuNG on different cancer cell lines, cells were treated with various concentrations of CuNG for different hours. MTT assay revealed that CuNG exert cytotoxic effect on different cancer cell lines, including the drug resistant cancer cell lines in a time and dose dependent manner (Fig. 1a, b). On the contrary, CuNG treatment was shown to be non-toxic to a series of normal cells like human PBMC, human hepatocellular Chang liver, human lung fibroblast WI38, human embryonic kidney HEK293 cells and towards mouse fibroblast cell NIH3T3 (Fig. 1c). We then enquired whether the antiproliferative effect of CuNG on different cancer cell lines occurred due to apoptosis. We treated different cancer cell lines including the drug resistant cancer cell lines with CuNG at a concentration of 8 lg/ml (above IC50 value of all cell lines tested, Table 1) for different hours and apoptosis was assayed by annexin V binding and also caspase 3 activity assay. CuNG at 8 lg/ml showed (Figs. 1d, e; 2a, b) significant level of apoptosis in both drug sensitive and resistant cell lines but failed to induce considerable level of apoptosis either in normal human PBMC, Chang liver, WI38, HEK293 cells or in normal mouse fibroblast cell line NIH3T3 (Figs. 1f; 2c).
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Table 1 IC50 values of CuNG for drug sensitive, drug resistant and normal cells Drug sensitive cells
IC50 values (lg/ml ± SD)
HCT-116
7.94 ± 0.54
K562
7.85 ± 0.72
Differences in intracellular GSH level between normal and cancer cells make the cancer cells vulnerable to CuNG mediated apoptosis
Anti-proliferative activity of CuNG was determined using drug sensitive cancer cells (HCT-116, K562, CCRF-CEM, EAC/ sensitive and Hela), drug resistant cancer cells (CEM-ADR5000 and EAC/DOX) and non-malignant cells (NIH3T3 and human PBMC) 48 h continuous incubation. All the data are representative of three similar experiments. Values represent mean ± SD
Since CuNG treatment caused apoptotic cell death in both drug sensitive and resistant cancer cell lines while sparing the normal cells, we tried to explore the underlying mechanism. Emerging evidences suggest that intracellular GSH level provide enormous support to the cancer cells’ survival (Trachootham et al. 2006) and depletion of intracellular GSH level causes imbalance in cellular redox homeostasis and thus lead to apoptotic cell death in various cancer cell lines (Trachootham et al. 2006; Armstrong et al. 2002). Previously, we showed that CuNG has the ability to form conjugate with GSH (Majumder et al. 2006; Basu et al. 2009) and therefore, we fervently desired to explore whether CuNG could deplete GSH in various cancer cell lines and thus make them apoptotic. To this end we first checked the intracellular GSH status of various malignant and normal cell lines and showed that
Fig. 2 CuNG caused activation of caspase 3 in various cancer cell lines. Cells were either kept untreated or treated with CuNG (8 lg/ml) or pretreated with NAC (5 mM) for 1 h following CuNG treatment (8 lg/ml) as indicated. Caspase 3 activity was measured in a drug sensitive cancer cell lines (HCT-116, K562, CCRF-CEM, EAC and HeLa), b drug resistant cancer cell lines
(CEM-ADR5000 and EAC-DR) and c normal cells (NIH3T3, human PBMC, Chang Liver, WI38 and HEK293) by measuring the release of fluorogenic substrate Ac-DEVD-AMC using spectrofluorimeter (Varian). Caspase activities are expressed as percent of untreated control and presented as mean ± SD of three independent experiments
CCRF-CEM
7.83 ± 0.41
EAC/sensitive
7.1 ± 0.83
Hela
7.33 ± 0.67
Drug resistant cells CEM-ADR5000
7.75 ± 0.44
EAC/DOX
7.55 ± 0.80
Normal cells NIH 3T3
Not determined
Human PBMC
Not determined
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all the cancer cell lines studied including the drug resistant cancer cells possess higher level of intracellular GSH as compare to normal human cells like PBMC, Chang liver, WI38, HEK293 and normal mouse fibroblast cell NIH3T3 (Fig. 3a). We further showed that CuNG treatment significantly depleted intracellular GSH level in all the cancer cell lines studied (Fig. 3b, c) whereas in normal cells no significant depletion of intracellular GSH was found (Fig. 3d). Nevertheless, in late hours of CuNG treatment the intracellular GSH level was found to be increased in normal human PBMC and normal mouse fibroblast. Next, we tried to determine whether the CuNG mediated apoptotic cell death of different cancer cell lines was due to depletion of intracellular GSH. Cells were pre-treated with NAC for 1 h prior CuNG treatment and apoptosis was assayed by both annexin V positivity and caspase 3 activity assays. We found that pre-treatment with NAC attenuated the CuNG mediated apoptotic cell death of different cancer cell lines (Figs. 1d, e; 2a, b). CuNG forms conjugate with GSH at higher GSH concentration We observed that CuNG caused significant depletion of intracellular GSH level in cancer cell lines where the GSH level remained considerably higher than that of normal cells. This simple observation led us to study whether the formation of CuNG–GSH conjugate depends on higher GSH:CuNG ratio because CuNG does not deplete GSH in normal cells having lower GSH level. To investigate the formation of GSH–CuNG conjugate, the mixture of CuNG and GSH was incubated in different proportions like 1:1 or 1:2 or 1:3 ratios and formation of CuNG–GSH conjugate was assayed by using low temperature (liquid nitrogen) EPR spectroscopy. Interestingly it was observed (Fig. 4a) that CuNG formed conjugate with GSH only when CuNG:GSH molar ratio lied above 1:1. When the ratio increased further in favour of GSH (e.g., CuNG:GSH = 1:3), the EPR signal completely disappeared, indicating the reduction of paramagnetic copper (Cu II) to diamagnetic copper (Cu I) species. This study reveals that when the level of GSH lied low, CuNG could not form conjugate with GSH and the formation of CuNG–GSH conjugate was optimal only when the GSH level remained high. We also studied the EPR spectrum of CuNG (Fig. 4b), which showed symmetrical singlet
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line due to exchange/dipole–dipole interactions between neighbouring copper ions. CuNG mediated apoptosis followed mitochondrial pathway CuNG treatment exhibited significant level of apoptosis in various cancer cell lines even in their resistant counterpart through depleting intracellular GSH level. Therefore, we investigated the signalling pathways underlying this phenomenon and focussed our study on a colon cancer cell line HCT-116 as it required highest dose of CuNG to induce apoptosis amongst all the cell lines we tested. Moreover, colon cancer is one of the most aggressive and common cancer worldwide. Since, apoptosis follows either extrinsic (involvement of transmembrane death receptor) or intrinsic (involvement of mitochondrial pathway) pathway, we investigated which pathway was involved in CuNG mediated apoptosis. We treated HCT-116 cells with CuNG (8 lg/ml) for different hours and assayed the expression of FasR (to determine extrinsic pathway) or release of cyto C from mitochondria to cytosol (to determine intrinsic pathway). We observed that CuNG treatment did not induce the expression of FasR on cell surface (data not shown) but caused the release of cyto C from mitochondria to cytosol (Fig. 5a) in a time dependent fashion. Since we had shown that the restoration of intracellular GSH level by pre-treatment with NAC prevent CuNG mediated apoptosis, we enquired whether NAC pre-treatment could prevent CuNG induced release of cyto C in cytosol. NAC pre-treatment almost completely suppressed the release of cyto C from mitochondria to cytosol (Fig. 5b). CuNG induced generation of mitochondrial superoxide leads to cardiolipin peroxidation and thereby detach cyto C from mitochondrial membrane Release of cyto C from mitochondria is considered a key initial step in apoptosis. Evidences suggest that release of cyto C from mitochondria is initiated by the detachment of cyto C from mitochondrial membrane that requires peroxidation of cardiolipin (Ott et al. 2002). So we investigated whether CuNG treatment caused cardiolipin peroxidation in HCT-116. We
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Fig. 3 CuNG caused significant depletion of intracellular GSH level of various drug sensitive and resistant cancer cell lines. a Intracellular GSH of various drug sensitive, drug resistant and normal cells were analyzed by spectrophotometric method as described in ‘‘Materials and methods’’ section. Cells were either kept untreated or treated with CuNG (8 lg/ ml) for 1–7 h. Intracellular GSH level in b drug sensitive cancer cell lines (HCT-116, K562, CCRFCEM, EAC and HeLa), c drug resistant cell lines (CEM-ADR5000 and EAC/ Dox) and d normal cells (NIH3T3, human PBMC, Chang liver, WI38 and HEK293) was measured as described in ‘‘Materials and methods’’ section. Results are presented as mean ± SD of three independent experiments. Differences between untreated control and CuNG treated cells are significant *P \ 0.05, **P \ 0.01, ***P \ 0.001 by unpaired Student’s t test
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Fig. 4 CuNG forms conjugate with GSH at particular molecular ratio. a Formation of conjugate between CuNG and GSH was measured by EPR spectroscopy. In the ratio CuNG:GSH (1:1) there are slight, but visible changes in the EPR spectra suggesting an interaction between CuNG complex and GSH. When the ratio CuNG:GSH increases in favour of GSH (e.g. CuNG:GSH between 1:1 and 1:3) the changes in the EPR spectra became prominent but when the CuNG:GSH is 1:3, the EPR signal completely disappears. This undoubtedly indicates
that the paramagnetic copper (II) ion has been reduced by GSH to diamagnetic, d10 (EPR silent) copper (I) species. This EPR spectrum clearly suggested that formation of CuNG–GSH conjugate optimally occur at a particular molecular ratio of CuNG and GSH. b EPR spectrum of CuNG (polycrystalline powder form). The spectrum shows symmetrical singlet line due to the exchange/dipol–dipol interactions between neighboring copper ions
observed (Fig. 6a) that CuNG treatment caused extensive peroxidation of cardiolipin as determined by the decrease in fluorescence of NAO. We also observed that pre-treatment with NAC diminished mitochondrial cardiolipin peroxidation in CuNG treated HCT-116 cells. Evidences suggest that cardiolipin peroxidation is increased by the generation of mitochondrial ROS (Asumendi et al. 2002). Since, generation of
superoxide is the major source of mitochondrial ROS, so we hypothesized that CuNG treatment could induce the generation of mitochondrial superoxide that ultimately caused peroxidation of cardiolipin, the initial step in the release of cyto C from mitochondria. So, we treated HCT-116 cells with CuNG for different hours and measured the generation of mitochondrial superoxide. We observed that after 12 h of CuNG treatment mitochondrial superoxide level started to
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Fig. 5 CuNG induced apoptosis follows mitochondrial pathway. a Involvement of mitochondrial pathway of apoptosis following CuNG treatment was confirmed by the release of cyto C. HCT-116 cells were either kept untreated or treated with CuNG for indicated hours. Cytosolic fraction was prepared as described in ‘‘Materials and methods’’ section and release of cyto C was analyzed by western blotting technique. b Actin was taken as loading control. Immunoreactive bands were quantified and expressed as the ratio of each band density to corresponding loading control (b actin) band density and values were represented after normalization to untreated control. b HCT-
116 cells were either treated with CuNG (8 lg/ml) for 24 h or pre-treated with NAC (5 mM) followed by CuNG (8 lg/ml) treatment as indicated and release of cyto C was analyzed in the cytosolic fraction by western blotting technique. Immunoreactive bands were quantified and expressed as the ratio of each band density to corresponding loading control (b actin) band density and values were represented after normalization to CuNG treated control. Difference between untreated and CuNG treatments are significant *P \ 0.05, **P \ 0.01, ***P \ 0.001 by unpaired Student’s t test
increase, peaked at 16 h and came to the basal level at 18 h in HCT-116 cells (Fig. 6b). We also observed that pre-treatment of NAC almost completely abolished CuNG induced generation of mitochondrial superoxide in HCT-116 cells. However, it is not yet understood how CuNG-mediated depletion of GSH induces the generation of mitochondrial superoxide.
membrane forms selective channel and thus release the soluble cyto C from mitochondria (Nechushtan et al. 1999; Kuwana et al. 2002). So we investigated whether CuNG treatment allowed the translocation of Bax on to the mitochondrial membrane so that cyto C could release. To investigate, we performed subcellular fractionation of HCT-116 following CuNG treatment for different hours. Western blot analysis (Fig. 7a) revealed that CuNG treatment significantly increased the level of Bax associated with the mitochondrial fraction. Our study also disclosed that NAC pre-treatment had no effect on CuNG mediated translocation of Bax to mitochondria. So, we conclude that the translocation of Bax to mitochondria in CuNG treated HCT-116 cells was independent of GSH depletion. To further ascertain the involvement of mitochondrial Bax localization with the CuNG
CuNG mediated release of cyto C is Bax dependent Release of cyto C from mitochondria is a two step process, first is the detachment of cyto C from inner mitochondrial membrane, and then permeabilization of outer membrane to release cyto C into the extra mitochondrial milieu (Ott et al. 2002). Accumulating evidences suggest that translocation followed by oligomerization of Bax on the mitochondrial
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CuNG mediated dissociation of HK II from mitochondrial membrane allows Bax to bind with the mitochondrial membrane
Fig. 6 CuNG caused cardiolipin peroxidation through generation of mitochondrial superoxide. a HCT-116 cells were either kept untreated or treated with CuNG (8 lg/ml) or pre-treated with NAC (5 mM) followed by CuNG (8 lg/ml) treatment for indicated hours and mitochondrial membrane cardiolipin peroxidation was measured by analyzing the decrease in fluorescence of NAO by using spectrofluorometer (Varion) as described in ‘‘Materials and methods’’ section. Data are expressed as the percent of untreated control and are presented as mean ± S.D of three independent experiments. Difference between untreated and CuNG treated cells are significant *P \ 0.05, **P \ 0.01, ***P \ 0.001 by unpaired Student’s t test. b Generation of mitochondrial superoxide by measuring the fluorescence of MitoSox were performed in HCT-116 cells that either kept untreated or CuNG (8 lg/ml) treated or pretreated with NAC (5 mM) and then treated with CuNG (8 lg/ ml) for different hours. Representative data of three independent experiments are presented here
mediated release of cyto C, RNA interference of Bax was employed to HCT-116 cells and release of cyto C was analysed following CuNG treatment. We observed that transfection with Bax siRNA caused a sharp reduction in CuNG mediated release of cyto C from mitochondria (Fig. 7b) whereas transfection with control siRNA produced no effect on cyto C release (data not shown).
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In various cancer cells around 80 % of HK II has been found to be attached to the mitochondria (Arora and Pedersen 1988; Nakashima et al. 1986) and interferes with the binding of Bax to mitochondrial membrane and thus makes the cancer cells resistant to apoptosis (Pastorino et al. 2002). Therefore, binding of Bax to mitochondria requires detachment of HK II from mitochondrial membrane. So, we enquired whether CuNG can dissociate HK II from mitochondria so that Bax can bind and subsequently release cyto C in the cytosol. We observed that following CuNG treatment (12 h) of HCT-116 cells, level of HK II in the cytosolic fraction (Fig. 8a) started to increase whereas in the corresponding mitochondrial fraction, HK II level significantly decreased as compare to untreated control (Fig. 8b). We then tried to investigate how CuNG treatment caused dissociation of HK II from mitochondrial membrane. Recent study shows that application of 3BrPA causes dissociation of HKII from mitochondrial membrane through interaction with the thiol (–SH) groups of the HK II protein (Chen et al. 2009). Moreover, many copper chelates react with thiol group and form copper sulphur bond (Elo 1987). We also earlier reported the reaction of CuNG with thiol group of cysteine (Basu et al. 2009). Since CuNG has the ability to react with the –SH groups of cysteine, we presume that CuNG can also react with –SH groups of HK II protein and cause the dissociation of HK II from mitochondria. To validate, purified HK II protein was incubated with different concentration of CuNG and we observed a decrease in fluorescence of HK II when excited at 280 nm (Fig. 8c). To further substantiate, we added 1 mM DTT before addition of CuNG and found (Fig. 8d) that preincubation of DTT prevented the reaction between CuNG and HK II. Our findings indicate that CuNG react with the HK II through the – SH groups of the protein. We also investigated whether interaction of CuNG with HK II affect the activity of the protein. To this end, we treated HCT116 cells with CuNG for different hours and HK II activity was assayed in the cell lysate. We observed that CuNG treatment caused no significant change on the HK II activity as compare to untreated control (Fig. 8e).
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Fig. 7 CuNG caused translocation of Bax to mitochondria. a Mitochondrial enriched fraction was isolated from HCT-116 cells from different treatment group and translocation of Bax was analyzed by immunoblotting technique. TOM-20 expression was taken as loading correction for mitochondrial protein. Densitometric analysis of immunoreactive bands were performed and expressed here as the ratio of each band density to corresponding loading control (TOM-20) band density and presented as percent of untreated control. b CuNG induced
release of cyto C was determined in the HCT-116 cells transfected with Bax SiRNA. HCT-116 cells were either treated with CuNG (8 lg/ml) or transfected with Bax SiRNA (40 pmol s/ml) and then treated with CuNG (8 lg/ml) for indicated hours. Cytosolic fraction was isolated and membrane was probed with anti cyto C antibody followed by incubation with HRP conjugated secondary antibody. b actin expression was taken as loading control
Discussion
through regulation of proper cellular redox status (Hammond et al. 2001). Number of studies discloses that cancer cells bear elevated basal ROS level due to extensive metabolic activity and hence their survival depends mainly on their antioxidant system (Szatrowski and Nathan 1991; Trachootham et al. 2006). Our studies reveal that all the cancer cell lines, including the drug resistant cells possess higher intracellular GSH level as compared to normal cells (Fig. 3). Our study reveals that drug resistant cancer cells possess higher level of intracellular GSH as compared to their sensitive counterpart. A number of factors are responsible for the accumulation of higher amount of GSH in drug resistant cancer cells. Higher intracellular GSH level in drug resistant cancer cells may cause either effective efflux of anticancer drugs through MRP1 mediated ABC transporter (Mu¨ller et al. 1994) or simply confer protection trough neutralizing cytotoxic effect of anticancer drugs (Meister 1983). The dependence of cancer cells to their antioxidant system had previously been exploited e.g., DL-buthionine (S,R) sulfoximine (BSO), ethacrynic acid (EA) were applied to deplete the level of intracellular GSH
Drug mediated toxicity and cancer drug resistance limit the use of different chemotherapeutic agents (Drı´mal et al. 2006; Sargent et al. 2001). Therefore, selective killing of malignant cells may be the ideal criteria for anti-cancer drug development. The present study tries to establish a paradigm where malignant cells can selectively be killed irrespective of their drug sensitivity by using a non-toxic copper chelate (CuNG). Reports show that free copper causes severe cytotoxcity (Rae et al. 1999). In our study we always used nontoxic dose of CuNG. Previously, we showed the toxicity of CuNG at different doses and also observed that 50 % of the applied dose entered in the cell (Majumder et al. 2003; Mookerjee et al. 2006; Ganguly et al. 2011). Herein, we disclose that CuNG mediated depletion of intracellular GSH level in cancer cells trigger a sequence of biochemical events that ultimately induce apoptosis to cancer cells irrespective of their drug sensitivity. GSH, the important cellular redox buffer, maintains a delicate balance between cell survival and cell death
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Fig. 8 CuNG caused dissociation of HK II from mitochondria through physically interact with it. HCT-116 cells were either kept untreated or treated with CuNG (8 lg/ml) for different hours and after completion of incubation cytosolic and mitochondrial fraction was separated as described in ‘‘Materials and methods’’ section. HK II level was analyzed in both a cytosolic fraction and b mitochondrial fraction using immunoblotting. b Actin expression was taken as loading control for cytosolic protein and TOM-20 expression was taken as loading control for mitochondrial protein and immunoreactive bands are expressed as percent of untreated control after
normalization. Difference between untreated and CuNG treatments are significant *P \ 0.05, **P \ 0.01, ***P \ 0.001 by unpaired Student’s t test. c Purified hexokinase was allowed to incubated with CuNG at different ratio and emission spectra was scanned after excitation at 280 nm. d Purified hexokinase was incubated with 1 mM DTT for 15 min before addition of CuNG at different ratio. Thereafter, emission spectra were scanned after excitation at 280 nm. e HCT-116 cells were either kept untreated or treated with CuNG (8 lg/ml) for different hours and hexokinase activity was measured as described in ‘‘Materials and methods’’ section
and induce apoptosis to drug resistant cancer cells (Schneider et al. 1995; Rhodes and Twentyman 1992). However, most of the GSH-depleting agents have been found to be toxic in vivo (Wu and Kang 1998; Yamamoto et al. 2002). Earlier a number of workers also had studied the role of GSH in the cytotoxicity of copper chelates in detail (Elo 1987). Previously we showed that CuNG has the ability to deplete GSH without causing any severe toxicity at selected doses in animal model (Mookerjee et al. 2006). In the present study we determined the killing effect of CuNG on different cancer cell lines. Initially we determined the
IC50 value of CuNG on different cancer cell lines and found that IC50 values of CuNG remains within 7–8 lg/ml (Table 1). The effect of CuNG is dose dependent and we have selected 8 lg/ml dose of CuNG. Moreover, the selected dose (8 lg/ml) of CuNG is absolutely non-toxic to a series of normal cells and bears preferential cytotoxicity to cancer cells (Fig. 1). The apoptotic potential of CuNG on different cancer cells including the drug resistant cancer cells was confirmed by annexin V positivity and caspase 3 activity assays (Figs. 1, 2). In both the assays, CuNG exhibited extensive apoptotic cell death to different
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cancer cells including the drug resistant cancer cells and spare the normal cells. We then tried to envisage the underlying mechanism of CuNG mediated preferential killing of cancer cells. Since, intracellular GSH level plays decisive role in dictating balance between cell death and survival and CuNG has the ability to deplete GSH, we assume the involvement of GSH in CuNG mediated apoptosis of cancer cells. To substantiate our assumption, we designed relevant experiments and found that pre-treatment of NAC, a precursor for GSH synthesis (Schreck et al. 1991), almost completely abrogated the apoptotic potential of CuNG on cancer cells (Figs. 1, 2). Since GSH is present in almost all the cell types, we raised the question why CuNG caused depletion of GSH only in malignant cell types. To address the relevant point we studied the mechanism of GSH depletion by CuNG. Previous studies disclose that CuNG forms conjugate with GSH both in vitro and in vivo (Majumder et al. 2006; Basu et al. 2009). Herein, we show that depletion of GSH proceeds through the formation of conjugate between CuNG and GSH and the formation of conjugate occurs at high molecular ratio of GSH over CuNG (CuNG:GSH [ 1:1) (Fig. 4). Since, cancer cells possess higher intracellular GSH level compared to normal cells, and higher GSH concentration favours CuNG–GSH conjugation, we propose that in cancer cells CuNG forms conjugate with GSH more efficiently than in normal cells. Due to the formation of watersoluble CuNG–GSH conjugate in cancer cell, the intracellular GSH level is significantly depleted and the resulting oxidative cellular milieu leads to apoptosis. To justify our proposition we worked with GSH precursor, e.g., NAC assuming NAC pre-treatment would cause elevation of intracellular GSH level (Schreck et al. 1991) high enough so that applied dose of CuNG (8 lg/ml) may not be sufficient to impart redox imbalance and consequent apoptosis. Apoptosis follows either extrinsic or intrinsic pathways. Extrinsic pathway is determined through the expression of various cell surface trans-membrane death receptors (Ashkenazi and Dixit 1998) whereas cytosolic release of cyto C is considered to be the hallmark for intrinsic pathway (Liu et al. 1996). Our studies demonstrate that CuNG treatment causes increase in cyto C level in the cytosolic fraction in a time dependent fashion and hence we conclude that CuNG mediated apoptosis of cancer cells follow the intrinsic pathway (Fig. 5). However, pre-treatment of
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NAC prevents the release of cyto C in CuNG treated HCT-116 cells; this observation thus suggests the involvement of CuNG mediated depletion of GSH with the concomitant release of cyto C. We further studied the underlying mechanism of CuNG mediated cyto C release in the cytosol. Evidences disclose that cyto C remains bound to the inner mitochondrial membrane through the interaction with membrane cardiolipin and peroxidation of cardiolipin causes detachment of cyto C from mitochondrial membrane (Ott et al. 2002; Nechushtan et al. 1999; Nomura et al. 2000). Herein, we noted that CuNG treatment led to the generation of mitochondrial superoxide which is possibly the crucial mediator involved in extensive cardiolipin peroxidation in HCT-116 cells (Fig. 6a, b). We presume that CuNG mediated generation of mitochondrial superoxide may lead to the accumulation of H2O2 in mitochondria that actually cause the peroxidation of cardiolipin. We argue that CuNG mediated depletion of intracellular GSH level critically regulates the generation of mitochondrial superoxide in HCT-116 cells because our study reveal that pre-treatment of NAC abrogated the effect. However, the precise mechanism(s) underlying the aforesaid observation remains elusive. Release of cyto C into the cytosol requires mitochondrial outer membrane permeabilization (Ott et al. 2002). A number of studies pointed towards the fact that mitochondrial docking followed by oligomerization of Bax induces the formation of pores in the mitochondrial outer membrane and thus releases cyto C into the cytosol (Nechushtan et al. 1999; Kuwana et al. 2002). Herein, we observed that CuNG treatment increases the Bax localization onto the mitochondrial fraction (Fig. 7a). Furthermore, we also found that RNA interference of Bax in HCT-116 cells prevents the release of cyto C following CuNG treatment (Fig. 7b). However, we conclude that that CuNG mediated translocation of Bax to mitochondria is independent of intracellular GSH depletion and such translocation is unable to induce apoptosis; such conclusion is based on the fact that NAC pre-treatment caused no effect on CuNG mediated Bax translocation rather NAC-pretreatment prevented apoptosis of HCT-116 cells. To summarise CuNG mediated translocation of Bax to mitochondrial membrane is only associated with the release cyto C from mitochondria. Recent studies suggest that elevated expression of mitochondria associated HK II in cancer cells prevents
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the binding of Bax to the mitochondria. Evidences show that the association of HK II with VDAC inhibits the binding of Bax with VDAC and thus prevent subsequent pore formation and detachment of HK II from mitochondria potentiates Bax dependent apoptosis (Pastorino et al. 2002). Herein, we showed that CuNG reacts with HKII and cause its dissociation from mitochondria as evidenced by the increased level of HK II in cytosolic fraction following CuNG treatment of HCT-116 cells (Fig. 8a, b). Our further investigations disclose that chemical reaction of CuNG with HK II possibly occurs through the –SH groups of the protein as pre-incubation of DTT prevent the reaction (Fig. 8c, d). –SH group and copper reaction However, the exact number of CuNG molecules bound to each HK II protein and which cysteine residues in HK II protein are targets of CuNG remain unclear. The result of our study also disclose that binding of CuNG with HK II, a key enzyme in glycolytic pathway does not hamper the activity of this protein (Fig. 8e) and arrive at the conclusion that its binding perhaps occurred at non active cites of the enzyme. In summary, CuNG has the potentiality to selectively kill cancer cells through depleting intracellular GSH level that in turn induce mitochondrial superoxide generation and ultimately disrupt the interaction of cyto C with cardiolipin. CuNG treatment also detaches the HK II from mitochondrial membrane and thus facilitates the binding of Bax to the mitochondrial membrane. These two phenomena lead to release of cyto C from mitochondria and eventually induce apoptosis. Our study indicates the possibility of targeting cellular redox milieu to trigger apoptosis in cancer cells, irrespective of drug sensitivity or drug resistance. Acknowledgments Authors acknowledge the help of Prof. Marian Valko, Slovak Technical University, Bratislava, Slovakia for doing EPR work. This investigation received financial support from Indian Council of Medical Research (ICMR), New Delhi, No. 5/13/18/2007/NCD-III, 3/2/2/177/ 2009-NCDIII and 5/13/53/2008/NCDIII. Conflict of interest The authors declare that they have no conflict of interest.
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