Mixed chelate copper complex, Casiopeina IIgly®, binds and degrades nucleic acids: A mechanism of cytotoxicity

Chemico-Biological Interactions 165 (2007) 189–199

Mixed chelate copper complex, Casiopeina IIgly®, binds and degrades nucleic acids: A mechanism of cytotoxicity Adolfo Rivero-M¨uller a,∗ , Andrea De Vizcaya-Ruiz a,b , Nick Plant a , Lena Ruiz c , Miloslav Dobrota a a

b

School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK Secci´on Externa de Toxicolog´ıa, CINVESTAV-IPN, Col. San Pedro Zacatenco, CP 07360, Mexico D.F., Mexico c Department of Chemistry, University of Mexico, CP 04510, Mexico, D.F., Mexico Received 27 September 2006; received in revised form 7 December 2006; accepted 8 December 2006 Available online 15 December 2006

Abstract Metal-containing drugs that interact with DNA have been designed and studied for their anticancer activity. In this study, the mixed chelate copper-based anticancer drugs, the casiopeinas, were found to bind to DNA and to degrade DNA and RNA in the presence of reducing agents (e.g. ascorbic acid). Casiopeinas binding to DNA is high affinity, with harsh wash conditions failing to remove the interaction. The reaction requires oxygen, probably involved in the generation of • OH radicals, which would be responsible for the strand breakage. The reaction was diminished by catalase, and was completely abolished by copper chelators (e.g. trientine, EDTA); however, superoxide dismutase (SOD) had no significant effect on casiopeina-mediated DNA degradation. Casiopeina IIgly (casIIgly) in the presence of ascorbate was capable of degrading RNA, plasmid and genomic DNA, and chromatin and intranuclear genetic material. Moreover, catalase and/or SOD partially protected cells, ascorbic acid enhanced and trientine, a copper chelator, abolished the cytotoxicity of casIIgly. The generation of 8-oxodG in cells exposed to casIIgly suggests that the generation of ROS is the major cause of the cytotoxicity observed and underlies the high toxicity and anticancer activity of these compounds. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Mixed chelate; Copper complexes; Casiopeinas; Nucleic acid; Redox reaction; ROS; 8-oxodG

1. Introduction

Abbreviations: ROS, reactive oxygen species; AsA, ascorbic acid; ␤-ME, beta-mercaptoethanol; GSH, reduced glutathione; XOD, xanthine oxidase; OGS, oxygen generating system; BSA, bovine serum albumin; SDS, sodium dodecyl sulphate; 8-oxodG, 8-oxo-7,8-dihydro2 -deoxyguanosine ∗ Corresponding author at: Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. Tel.: +358 2 333 73 76; fax: +358 2 250 26 10. E-mail address: [email protected] (A. Rivero-M¨uller).

Several metal complexes have shown promising antineoplastic activity against cancer cells and tumors both in vitro and in vivo [1]. A group of such complexes is the casiopeinas, copper-based co-ordinated complexes with generic structure of [Cu(N–N)(O–N)]+ or [Cu(N–N)(O–O)]+ that have proven cytotoxic to cancer cells sensitive or resistant to cisplatin, and to xenograph tumors in mice [2,3]. casIIgly’s promising use as a chemotherapeutic agent in the treatment of cancer arises from various studies conducted in the in vitro and in vivo

0009-2797/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2006.12.002

190

A. Rivero-M¨uller et al. / Chemico-Biological Interactions 165 (2007) 189–199

systems, in which its capacity to reduce the size and volume of cultured and implanted tumors [4,5], have been shown. casIIgly might enter Phase I clinical trials as early as next year. The mode of action for casiopeinas is largely unknown but it has been suggested that mitochondrial permeability transition may be important [6]. Our own data (unpublished) confirms mitochondria as one intracellular site of cytotoxicty. However, as shown in this study, the mode of action may involve direct DNA damage. The hemotoxicity in rats points to more complex in vivo cytotoxicty of casiopeinas [7]. The binding and cleavage of nucleic acids has been reported for several metal complexes and this has been a rationale for the development of new chemotherapeutic drugs [8]. Of particular interest to this study is the ability of some redox-active coordination complexes (e.g. Cu-Chlorophyllin, [Cu(phenanthroline)2 ]+ , Cu(TAAB)2 , Cu(2,2 -bipyridyl)2 , Cu-nitrilotriacetate, [Fe-EDTA]2− ) to cleave DNA in the presence of a reducing agent and molecular oxygen (or H2 O2 ). Further to this, all-cis-2,4,6-triamino-1,3,5-trihydroxycyclohexane (TACl)-copper complex has been shown to induce degradation of DNA even in the absence of a reducing agent [9]. The cleavage of DNA by the [(Cu)(phenanthroline)2 ]+ seems to be site specific to regions on the genome where protein density is low; indeed, this activity has been used as a tool to study protein–DNA interactions (footprinting) [10]. Interestingly, complexes such as Fe, Mn or Cr-porphyrins, and Fe-phenanthroline also form oxo-complexes and generate ROS but do not damage DNA for steric reasons [11,12]. Such metal–DNA interactions, usually enhanced by a suitable pro-oxidant (reducing agent), can generate H2 O2 and further to • OH radicals, the latter being considered as the most reactive and damaging of all the ROS [13]. ROS produced in this manner are able to damage DNA [14], proteins [15], LDL and membranes in vitro [16]. Such damage may account for the cytotoxicity and mutagenicity observed for these interactions in bacteria [17], cytotoxicity in tumor cells [18], and the hemolytic effect of copper in sheep [19]. It is likely that the metal complexes damage DNA by binding to specific nucleotides or nucleotide sequences and thus generating the • OH radical in very close proximity to nucleic bases or the phosphate backbone resulting in the breakage of the DNA strand [20]. An example of such reaction is bleomycin, which interacts with DNA, but apparently does not damage DNA on its own. Instead, interactions with iron or copper, which in the presence of molecular oxygen (bleomycin-Fe(II)-

oxygen) becomes oxidised, are responsible for the DNA damage [21]. This study focuses on the copper-based casiopeina group of anticancer agents [22] and their effect on DNA degradation and on cytotoxicity using as example casiopeina IIgly (casIIgly), as part of a broader project to investigate its cytotoxic side effects and mode of action, particularly in vivo. 2. Materials and methods 2.1. Materials 4-(2-Hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), bovine serum albumin (BSA), catalase (from bovine liver), digested DNA marker lambda EcoR1/HindIII, ethidium bromide, EDTA, l-ascorbic acid, ␤-mercaptoethanol, proteinase K, ribonuclease-1, superoxide dismutase (Cu/Zn-SOD), tris(hydroxymethyl)aminomethane (Tris), Triton X-100 (t-Octylphenoxypolyethoxyethanol), Tween-20, Trientine (1.5 mg/L) and 4,7-dimethyl-1,10-phenanthroline were purchased from Sigma (Poole, UK). Agarose (electrophoresis grade) was purchased from Invitrogen (Paisley, UK). Acetic acid, boric acid, and glycine were obtained from Fisher Scientific (Loughborough, UK). Methanol, NaCl, and reduced glutathione (GSH) from BDH (Lutterworth, UK). RQ1: Bovine pancreas DNase (10 U/␮l), DNA marker lambda StyI was from Fermentas (York, UK). Loading dye (orange G), sodium dodecyl sulphate (SDS) and Chelex 100 resin from BioRad (Hemel Hempstead, UK). Casiopeina Igly [Cu(4,7biphenyl-1,10-phenanthroline) (glycinate)], IIgly [Cu (4,7-dimethyl-1,10-phenanthroline) (glycinate)] and IIIia [Cu(4,4 dimethyl) (2,2 -dipyridine) (acetylacetonate)] were synthesized as previously described [3,4]. In this paper the casiopeinas are referred to as casIgly, casIIgly and casIIIia, respectively. 2.2. DNA degradation 2.2.1. Plasmid DNA degradation Typically incubations in HEPES buffer 0.5 M pH 7.4 contained 250 ng of plasmid DNA (Stratagene, Amsterdam, Netherlands, pbluescript II SK+ , 3.5 kb), ascorbate (AsA), or beta-mercaptoethanol (␤-ME) at a final concentration of 1 mM, 4,7-dimethyl-1,10-phenanthroline, its complex with iron, casIgly, casIIgly or casIIIia at a final concentration of 10 ␮M. When required, 2 ␮l of SOD and/or catalase (40 mg/ml) was added; the final volume reaction was made up to 20 ␮l with HEPES buffer 0.5 M (pH 7.4) which was chelex-treated for 6 h before

A. Rivero-M¨uller et al. / Chemico-Biological Interactions 165 (2007) 189–199

191

use. MilliQ water was used for all solutions. All solutions, when possible, were metal-free (pre-treated with chelex 100 resin) and sterile. The mixture was incubated at 37 ◦ C for 20 min to 1 h, stopping the reaction by addition of EDTA (1 M, pH 8.0). Plasmid DNA damage was determined by horizontal electrophoresis (1% agarose). DNA was post-stained with ethidium bromide for 15 min and visualized under UV light. As a positive DNA degradation control, RQ1 DNase was used.

layered on the bottom of each tube. Tubes were centrifuged at 37,000 × g for 30 min. The upper layer and the sucrose cushion were removed by a suction aspirator. The resulting pellet of highly purified nuclei was resuspended in KPHM solution (125 mM KCl, 2 mM potassium phosphate, 25 mM HEPES, 4 mM MgCl2 , pH 7.0). Purity of nuclei was determined by lack of whole cells and mitochondria by phase contrast microscopy.

2.2.2. Genomic DNA degradation Human genomic DNA was extracted and purified from human liver (sourced from Keystone Bank as a kind gift from Roche) using the RecoverEase DNA isolation kit (Stratagene, UK) and resuspended in 1× TE at a concentration of 100 ng/ml. Human DNA at a final concentration of 0.5 ␮g, casIIgly (50 ␮M) and AsA (1 mM) were added in aliquots with HEPES buffer to a final volume of 20 ␮l. Samples were incubated at room temperature for 1 h in the presence or absence of albumin (4%) and the reaction was stopped by the addition of EDTA as previously described. Genomic DNA damage was assessed by electrophoresis.

2.3.2. Incubation of nuclei Rat-liver nuclei (7 × 105 ) or L1210 cells nuclei (6 × 105 ) were placed into 20 ␮l of HEPES buffer with or without the following reagents (see Fig. 7 legend for final concentrations): casIIgly, ascorbate (AsA) and BSA (5 ␮g) when specified. Samples were incubated at 37 ◦ C for 1 h and to stop the reaction 5 ␮l of EDTA (1 M, pH 8.0) was added to each sample. Nuclei were lysed through addition of 1 ␮l Triton X-100 and RNA degraded by addition of 6 ␮l of RNase (50 mg/ml). Nuclear DNA damage was determined by horizontal gel electrophoresis (1% agarose) using 0.5 V/cm for 16 h in TBE buffer. A strip of the running gel was cut about 1 cm from the comb to the upper end of the gel, and a digestion gel (containing 2% SDS, 0.5% Triton X-100 and Proteinase K (12.5 mg/10 ml) was poured (approximately 10 ml) into the gap in order to aid the disruption of the nuclear envelope and chromatin proteins allowing the DNA to run free. Finally the DNA was stained with ethidium bromide for 15 min and visualized with UV light.

2.3. Internucleosomal DNA degradation 2.3.1. Preparation of nuclei Nuclei from rat liver or L1210 cells (murine leukaemia; ECAC no. 87092804) were purified exactly as described by Jones et al. [23]. This highly purified nuclei suspension (1.8 × 106 /ml) was used immediately or stored in storage buffer (50% glycerol in TKM buffer) at −20 ◦ C until further usage. Briefly, livers were excised, diced in small pieces (1 g), washed with ice-cold sucrose–Tris–HCl buffer (pH 7.8) to remove blood, then homogenized with a glass–Teflon homogeniser (three strokes) in 40 ml of TKM solution (50 mM Tris–HCl, pH 7.5, 25 mM KCl, 5 mM MgCl2 ). The homogenate was filtered through at least three layers of cheesecloth. Nuclei were pelleted by centrifugation at 700 × g for 10 min. L1210 cells were cultured as described below and collected by centrifugation, washed twice with PBS, and homogenised in a glass–glass hand homogeniser (at least 20 strokes) on ice-cold lysis solution (TKM buffer containing 1% Triton X-100), 2 ml, on ice. Nuclei were pelleted as above. Nuclei pellets were resuspended in 24 ml of TKM solution. The solution (6 ml) was transferred to new tubes containing 12 ml of TKM solution containing 2.3 M sucrose. Tubes were gently mixed, and a 6 ml cushion (TKM containing 2.3 M sucrose) was carefully

2.4. Binding of casIIgly to DNA DNA, either plasmid or genomic (not shown), was co-incubated with casIIgly for 1 h in HEPES buffer. The DNA was then washed extensively with the same buffer by filtration through an Amicon ‘Centrifree’ system (cut off 10,000 Da) and recovered in 20 ␮l of HEPES buffer by inversion. DNA solution was incubated with 1 mM AsA for 1 h at room temperature and the DNA samples were separated by gel electrophoresis and visualized after ethidium bromide staining as previously described. For intranucleosomal binding, nuclei were incubated with casIIgly 50 ␮M 1 h in TKM buffer. BSA (4%) was added into some samples in order to elucidate if casIIgly has greater affinity for DNA or for albumin [24]. Nuclei were spun down at 1200 × g for 10 min, the supernatant discarded and pellet resuspended into 10 times the original volume; this procedure was repeated at least five times to ensure removal of any unbound complex from the nuclei. Nuclear DNA damage was then determined as previously described.

192

A. Rivero-M¨uller et al. / Chemico-Biological Interactions 165 (2007) 189–199

2.5. Cytotoxicity Cell viability was assayed using the CellTiter96 AQueous ® kit (Promega, UK). We choose green monkey kidney cells (COS1) for this study as they are “normal” cells and we were interested in the cytotoxicity of casIIgly in those conditions. Identical results however were obtained with L1210 cells (not shown). COS1 cells were maintained in 75 cm2 culture flasks with DMEM (w/glucose without sodium pyruvate) enriched with 10% (v/v) foetal calf serum (FCS) (both from ICN, UK), 1% gentamicin and 1% anti-PPLO (Sigma, Poole UK). COS1 cells were seeded in 96-well microplate at a concentration of 9 × 105 cells/well with 100 ␮l of medium and treated with the following solutions: casIIgly at concentration of 50 ␮M (in 5% glucose), catalase (50 ␮g/ml), SOD (50 or 200 U/ml), AsA (100 ␮M), GSH (1 mM), or trientine (100 ␮M). Control groups were treated with 5% glucose instead of the casIIgly. After 1 h of treatment cells were washed twice with PBS and fresh medium was added, plates were incubated at 37 ◦ C for 24 h. Viability was measured following manufacture’s instructions at 492 nm. Standards were performed with known number of cells per well (0–10 × 105 cells/well) and measured to determine the number of living cells. Time course of viability shows that cells treated with casIIgly (25, 50 or 100 ␮M) die in the period of 18–24 h mainly by apoptosis, thus, 24 h was chosen as the end-time point to measure the cytotoxicity. 2.6. Formation of 8-oxodG in cultured cells Cells were cultured and treated as described above, exact reagent concentrations are shown in Table 1. After 1 h incubation DNA was extracted by Pharmacia purification kit, dissolved in water and digested by P1 nuclease and alkaline phosphatase. Content of 8-oxodG was determined as described by Ito et al. and Tada-Oikawa et al., [25,26]. Briefly, cultured cells treated with casIIgly, AsA and/or antioxidants were collected by centrifugation after being scraped from the plates. Cells were lysed in 500 ␮l lysing buffer (100 mM NaCl, 50 mM Tris–HCl, pH 7.5, 10 mM EDTA, 0.5% SDS, 1 mg/ml proteinase K), and incubated for 3 h at 60 ◦ C. DNA was precipitated with isopropanol and centrifuged 3000 × g for 15 min. Pellets were washed three times with 70% ethanol and dissolved in 20 mM acetate buffer (pH 5.0), then digested to deoxynucleosides first by incubation with 5 U of nuclease P1 (Sigma) at 37 ◦ C for 30 min and then with 1 U of calf intestine alkaline phosphatase at 37 ◦ C for 1 h (in its commercial buffer, Promega). The resulting deoxynucleosides mixture was injected into a

Waters HPLC apparatus with a Beckman Ultrasphere ODS column (0.46 cm × 25 cm): eluent 10% aqueous methanol containing 12.5 mM citric acid, 25 mM sodium acetate, 30 mM NaOH, and 10 mM acetic acid: flow rate 1 ml/min. UV absorbance at 254 nm. The effluent was monitored by a UV detector (Waters, 254 nm), for quantification, peak areas of dG standards and pure 8-oxodG standards (Sigma) were analyzed each run. 2.7. Statistical analysis Statistical analyses of cell survival and generation of 8-oxodG were performed using ANOVA pair t–test using a StatView program for Windows (Version 5.0.1) (SAS Institute Inc., Cary, NC). All numerical data are presented as the mean ± S.E.M. and differences were considered as statistically significant at 95% confidence level (p < 0.05). 3. Results 3.1. casIIgly–ascorbate degradation of DNA Fig. 1 demonstrates that casIIgly was capable of degrading plasmid DNA in the presence of AsA or ␤ME, although AsA or ␤-ME (not shown) alone were ineffective. Both AsA and ␤-ME effectively activated casIIgly, with 20 min incubation completely degrading 250 ng of plasmid DNA (Fig. 1). Addition of copper chelators such as trientine or EDTA completely inhibited the casIIgly–AsA-mediated DNA degradation. This inhibition is indeed utilised in the methodology to stop the reaction (addition of 1 M EDTA). DNase treatment, used as a positive control for degradation, with RQ1 also degraded the plasmid DNA completely (Fig. 1, lane 2). 3.2. Effects of catalase and/or SOD on DNA degradation The activation of casiopeina-mediated nucleotide degradation by a reducing agent suggested the involvement of reactive oxygen species (ROS), generated between the metal complex bound to the nucleotides and a reducing agent; such ROS generation systems have been described for some chemical nucleases [18]. Thus, ROS scavengers (GSH, SOD and catalase) were added into the reaction mixture in order to elucidate the probable involvement of ROS, and the species responsible for the cleavage. SOD or GSH did not inhibit casIIgly–AsA induced DNA damage (Fig. 2) but catalase inhibited totally the

A. Rivero-M¨uller et al. / Chemico-Biological Interactions 165 (2007) 189–199

193

Fig. 1. Gel elecrophoresis of plasmid DNA treated with casIIgly in the presence of AsA. Plasmid DNA was treated as described in each lane and was incubated for 20 min at 37 ◦ C. Lane 1, DNA lambda-StyI marker; lane 2, positive degradation control (DNase RQ1); lane 3, casIIgly (10 ␮M); lane 4, casIIgly + AsA (1 mM); lane 5, control DNA + AsA; lane 6, DNA; lane 7, casIIgly + mercaptoethanol (1 mM). Representative of at least three independent experiments.

cleavage at a concentration of 40 ␮g/ml when casIIgly was at a concentration of 10 ␮M. Higher casIIgly concentrations (25 and 50 ␮M) were able to partially overcome the inhibitory effect of catalase (data not shown). When boiled catalase was added no inhibition of DNA cleavage was observed. Addition of both catalase and SOD inhibited degradation, although it is not clear as to whether this was purely due to the catalase activity or co-operation between the two enzymes. The addition of catalase and/or SOD (40 ␮g/ml) used as indirect indicators of ROS generation, showed that DNA damage was caused not by the direct interaction of casIIgly with DNA, but by the production of ROS when casIIgly was reduced by AsA (Fig. 2).

3.3. DNA damage induced by other complexes and casIIgly ligand (4,7-dm-1,10-phenanthroline) DNA degradation profiles shown in Fig. 3 indicate that casIIIia and casIgly also exhibit nuclease activity when mixed with ascorbate. However, in the presence of AsA iron-4,7-dm-1,10-phenanthroline (made up by mixing equimolar concentrations of iron sulphate and 4,7-dimethyl-1,10-phenanthroline (10 ␮M) in 5% ethanol) did not elicit any effect on DNA degradation (Fig. 3). In addition, Cu(NO3 )2 (10 ␮M) and AsA elicited only minimal DNA degradation, suggesting that the Cu2+ alone is insufficient to catalyze nucleotide degradation, but requires to be complexed with a particular ligand. 4,7-dm-1,10-Phenanthroline and AsA also

Fig. 2. Effect of catalase/SOD and GSH on DNA degradation by casIIgly + AsA. Plasmid DNA was treated for 5 min with the following: lane 1, DNA lambda-StyI marker; lane 2, plasmid control; lane 3, casIIgly (10 ␮M); lane 4, casIIgly + AsA (1 mM); lane 5, casIIgly + AsA and catalase (40 ␮g/ml); lane 6, casIIgly + AsA and SOD (40 ␮g/ml); lane 7, casIIgly + AsA + catalase and SOD; lane 8, casIIgly + AsA + boiled catalase; lane 9, casIIgly + AsA + GSH. Samples were incubated at 37 ◦ C for 1 h.

194

A. Rivero-M¨uller et al. / Chemico-Biological Interactions 165 (2007) 189–199

Fig. 3. Plasmid DNA degradation by Cu2+ , ligand (4,7-dimethyl-1,10-phenanthroline) and other casiopeinas (all at a final concentration of 10 ␮M) under different conditions. Lane 1, DNA marker lambda-StyI; lane 2, plasmid DNA control; lane 3, Cu(NO3 )2 ; lane 4, Cu(NO3 )2 + AsA (1 mM); lane 5, 4,7-dm-1,10-phen; lane 6, 4,7-dm-1,10-phen + AsA; lane 7, Fe-4,7-dm-1,10-phen; lane 8, Fe-4,7-dm-1,10-phen + AsA; lane 9, casIIgly; lane 10, casIIgly + AsA; lane 11, casIgly; lane 12, casIgly + AsA; lane 13, casIIIia; lane 14, casIIIia + AsA. All samples were incubated at 37 ◦ C for 25 min.

generated small DNA damage which may be due to traces of copper in the reagents. 3.4. Involvement of oxygen in DNA degradation by casIIgly In order to elucidate the source of O2 radicals that initiate nucleotide degradation, the interaction of DNA and casIIgly was examined in an O2 •− producing system; xanthine oxidase (XOD) (50 ␮g) is oxidised by hypoxanthine (0.1 mM) to xanthine, which is then further oxidised by the same enzyme to uric acid, with both reactions generating O2 •− . This O2 •− generating system (OGS) alone did not degrade DNA, but when casIIgly was added to the same system (in presence or absence of AsA), DNA was rapidly degraded (Fig. 4). As previously demonstrated catalase was able to protect DNA from this degradative effect. Interestingly, under these conditions SOD was also effective, suggesting that this enzyme may function at a lower efficiency to catalase with respect to this scavenging reaction (Fig. 4). The reaction between casIIgly and AsA was also examined under anaerobic conditions, with all solutions being degassed with N2 for 45 min. Each tube contained:

HEPES buffer (50 mM, pH 7.4), DNA 480 ng, AsA (1 mM), casIIgly (10 ␮M), with a final volume of 100 ␮l, which was the minimum volume needed to allow N2 bubbling during the incubation time. Tubes were incubated for 25 min at 37 ◦ C under N2 atmosphere within sealed micro centrifuge tubes. Fig. 4 demonstrates that casIIgly alone, or with additional AsA, was not able to degrade DNA under anaerobic conditions (Fig. 4, lanes 11–13), proving the absolute requirement for oxygen in this reaction. These results suggest that in the presence of an OGS casIIgly may be reduced by O2 •− , or forms an oxocomplex, which in turn triggers the production of H2 O2 and later • OH by copper oxidation–reduction states [27]. SOD and catalase were effective in inhibiting these oxido-reduction reactions, consistent with ROS being the active species responsible for breaking DNA, and the absolute requirement for oxygen is confirmed by the lack of DNA degradation under anaerobic conditions. 3.5. Degradation of genomic DNA by casIIgly–AsA Human genomic DNA exposed to casIIgly–AsA showed similar results to those observed with plasmid

Fig. 4. Effect of casIIgly under an oxygen generating system (OGS), also casIIgly–AsA effect under anaerobic conditions on plasmid DNA degradation. Lane 1, DNA marker lambda-StyI; lane 2, DNA + OGS; lane 3, casIIgly (10 ␮M); lane 4, casIIgly + OGS; lane 5, casIIgly + XOD; lane 6, casIIgly + hypoxanthine; lane 7, casIIgly + OGS + catalase (40 ␮g/ml); lane 8, casIIgly + OGS + boiled catalase; lane 9, casIIgly + OGS + SOD (40 ␮g/ml); lane 10, casIIgly + OGS + catalase + SOD; lane 11* , casIIgly + AsA (1 mM); lane 12* , casIIgly; lane 13* , plasmid DNA. All samples were incubated at 37 ◦ C for 25 min. OGS refers to XOD (50 ␮g) + hypoxanthine (0.1 mM). * Samples were assayed under anaerobic conditions.

A. Rivero-M¨uller et al. / Chemico-Biological Interactions 165 (2007) 189–199

Fig. 5. Degradation of genomic DNA by casIIgly after 1 h incubation. Lane 1, genomic DNA control; lane 2, DNA + casIIgly (50 ␮M); lane 3, DNA + AsA (1 mM); lane 4, DNA + casIIgly (50 ␮M) + AsA (1 mM); lane 5, lambda-StyI marker.

DNA. The genetic material was rapidly degraded by casIIgly in the presence of AsA (Fig. 5). 3.6. DNA binding by casIIgly Some metal complexes, such as [Fe-EDTA]n− , do not bind to DNA but act as a source of • OH radicals, which when generated in solution close to DNA may result in DNA damage [28]. In order to determine if casIIgly acts in a similar fashion, or is physically associated with

195

Fig. 7. Degradation of whole nucleus DNA by casIIgly: Lanes 2–6 correspond to L1210 nuclei while lanes 7–10 correspond to rat liver nuclei. Lane 1, DNA marker lambda-EcoRI/HindIII; lanes 2–4 were exposed to AsA (50 ␮M) in combination with: in lane 2, no casIIgly; in lane 3, casIIgly (50 ␮M); in lane 4, casIIgly (25 ␮M); lanes 5–6 were exposed to AsA (1 mM) in combination with: in lane 5, casIIgly (25 ␮M); in lane 6, casIIgly (50 ␮M); lane 7, control AsA (1 mM); lane 8, casIIgly (50 ␮M) + AsA (1 mM); lane 9, casIIgly(50 ␮M); lane 10, control nuclei; lane 11, DNA marker lambda-StyI. Results from two separate gels were apposed for display.

DNA, extensive washing was undertaken to remove any unbound complex. As expected, DNA incubated with casIIgly only did not show any degradation, but addition of AsA resulted in severe degradation (Fig. 6). casIIgly was not removed by extensive washing or by competition of binding proteins (BSA) (Fig. 6).

Fig. 6. casIIgly binding to DNA: nuclear DNA was incubated with or without casIIgly (25–50 ␮M) for 1 h, then spun down and washed as stated in the text. AsA (1 mM) was added and incubated with DNA for 15 min at room temperature. Lane 1, DNA Lambda-EcoRI/HindIII marker; lane 2, nuclear DNA control; lane 3, DNA + later addition of AsA; lane 4, DNA + casIIgly (50 ␮M); lane 5, DNA + casIIgly (50 ␮M) + later addition of AsA; lane 6, DNA + casIIgly (25 ␮M); lane 7, DNA + casIIgly (25 ␮M) + later addition of AsA; lanes 8–13 exactly same order as 2–7 but in the presence of 4% BSA; lane 14, DNA Lambda-EcoRI/HindIII marker.

196

A. Rivero-M¨uller et al. / Chemico-Biological Interactions 165 (2007) 189–199

Fig. 8. Viability of COS1 cells treated with casIIgly in the absence and presence of antioxidant agents: casIIgly was added at a final concentration of 50 ␮M, AsA 100 ␮M, catalase (Cat) 50 ␮g, SOD 50 U, GSH 1 mM, and trientine 100 ␮M (before or after addition of casIIgly into the media). COS1 cells were incubated with the corresponding reagents for 1 h then washed twice with PBS and incubated for 24 h at 37 ◦ C with 100 ␮l of Eagle’s modified medium. Cytotoxicity was measured with CellTiter96 kit. Control cells were untreated with casIIgly. * Statistically significant (p < 0.05) against control samples; ** statistically significant (p < 0.05) against casIIgly treated samples.

3.7. Intranucleosomal DNA degradation by casIIgly Isolated nuclei exposed to casIIgly–AsA showed significant DNA damage as judged by DNA degradation products visualized after electrophoresis (Fig. 7), suggesting that the presence of a nuclear envelope or other intra-nuclear elements failed to provide any type of protection from the degradative effects of casIIgly–AsA. Addition of BSA (4%) to this system also did not provide protection, indicating that casIIgly has a much higher affinity for DNA binding than for BSA (data not shown). 3.8. Effect of radical scavengers on casIIgly’s-mediated nucleotide functioning CasIIgly alone (50 ␮M) killed more than 70% of cells. This effect was enhanced in wells where AsA (100 ␮M) was also added, with viability was significantly lower (<40%). The viability of cells with AsA alone was not significantly different compared with controls (Fig. 8). Presence of GSH (1 mM) did not show any enhancement or protection of casIIgly’s toxicity, the viability

Fig. 9. Enhancement of cytotoxicity by high concentration SOD: casIIgly was added as in Fig. 8 but SOD was added at 50 and 200 U in the presence or absence of catalase (Cat, 50 ␮g). Cells were incubated with the corresponding reagents for 1 h then washed twice with PBS and incubated for 24 h at 37 ◦ C with 100 ␮l of Eagle’s modified medium. * Statistically significant (p < 0.05) against control samples; ** statistically significant (p < 0.05) against casIIgly treated samples.

being similar to the ones treated with casIIgly alone (Fig. 8). GSH added alone had no effect on cell viability. When catalase (50 ␮g) and/or SOD (50 U) were added into the medium of cells co-treated with casIIgly viability was significantly (p < 0.05) increased (Fig. 8), with both enzymes being equally as effective. This protection occurs only when catalase/SOD are added at the same time as casIIgly, with addition 1 h after casIIgly being ineffective at protecting cells. Interestingly, increasing the SOD concentration to 200 U showed a reverse effect, significantly (p < 0.05) increasing the cytotoxicity of casIIgly by 20%. The presence of catalase (50 ␮g) in the mixture diminished this effect to some degree, but little net protection was observed by the mixture (Fig. 9). However, when AsA and casIIgly were added together, neither catalase SOD nor the mixture of the two could protect the cells from casIIgly’s toxicity. Trientine completely protected cells from casIIgly’s redox insult but only when added prior to casIIgly; 10 min after casIIgly’s treatment, trientine showed only a modest cytoprotective effect, which indicates the cytotoxic effect occurs inside the cell (Fig. 8). Cells treated with high doses of casIIgly died rapidly, while low doses (used in this study) elicited cell death mainly via apoptosis [2]. This seems to be as a result of the oxidative insult, with high doses causing the cell membrane to be perforated, cells to swell and conse-

A. Rivero-M¨uller et al. / Chemico-Biological Interactions 165 (2007) 189–199 Table 1 DNA damage in living cells (COS1), treated with casIIgly, CuNO3 (in the presence or absence of AsA, catalase, or SOD), expressed as the formation of 8-oxodG (results are means and S.D. of values obtained from four independent experiments) casIIgly (␮M)

8-oxodG/104 dG ± S.D.

0 10 50 100 50 + AsA (100 ␮M) 50 + catalase (50 ␮g/ml) 50 + SOD (50 ␮g/ml) 50 + SOD (200 ␮g/ml) CuNO3 50 ␮M CuNO3 100 ␮M

0.21 0.35 0.56 0.83 2.35 0.31 0.35 0.72 0.28 0.34

*

± ± ± ± ± ± ± ± ± ±

0.01 0.08 0.04* 0.16* 0.32* 0.06 0.12 0.23* 0.06 0.13

Statistically significant (p < 0.05) compared to control values.

quently burst. However, at low doses the toxicity is not so overt, allowing signals to undergo apoptosis to prevail. Ascorbic acid strongly exacerbates the activity of casIIgly, which suggests that ROS are involved in the cytotoxic effect, and moreover that OH and O2− are the active species, since SOD at high concentration increased the cytotoxic effect. Determination of 8-oxdG is in close concordance with these findings proving that casIIgly damages DNA in living cells by ROS generation (Table 1). 4. Discussion The interaction between DNA and metal-containing drugs has become an important area of research since platinum drugs were discovered to be active against a number of tumors [29,30]. The activity of metal complexes in the inhibition of DNA replication and tumor cell cytotoxicity [31] is now recognized as a component of their anticancer activity. In addition, it is now considered that the ‘nuclease’ activity of copper complexes makes a significant contribution towards their chemotherapeutic activity [32]. Our data show that casIIgly degrades DNA in the presence of AsA (or ␤-ME), and we suggest a binding of this complex with DNA since even after extensive washing to remove unbound casIIgly DNA degradation was observed. Degradation of the DNA from within whole nuclei exposed to casIIgly was also consistent with this hypothesis, with BSA being ineffective in preventing the binding of casIIgly to DNA. As described by Knorre et al. [33] DNA degradation by metal-containing drugs may involve the formation of a metal oxo-complex which needs a reducing reagent to generate • OH radicals, the ultimate toxic agent. Interestingly, only strong reducing

197

agents (AsA and ␤-ME) result in degradation of DNA by casiopeinas, whereas reduced glutathione (GSH) was not active (data not shown). We hypothesize that, in the presence of a suitable reducing agent, casIIgly could bind to DNA in such a way that the spacing corresponds with the copper moiety generating • OH radicals in close proximity to the relatively labile phosphodiester bonds of the nucleotide chain. Reports indicate that reactive • OH ˚ of their site of production species react within ∼60 A [34], which could explain why protection mechanisms such as catalase did not totally prevent DNA degradation by casIIgly complex; the enzyme can not access the sites of • OH production. A further possibility could be that casIIgly links an O2 −• ion instead of transforming it into H2 O2 (mimicking SOD-like activity) when binding to DNA. This may lead to oxidation of casIIgly’s copper to a Cu3+ state (oxo-complex) which is unstable and when a reducing agent is added the O bound transforms into • OH. Paradoxically, some Cu (II) complexes including casiopeina compounds [27] have been reported to exhibit SOD-like activity [35,36], and thus might be expected to protect biomolecules against formation of ROS. However, casIIgly in the presence of a reducing agent (ascorbate or ␤-ME), degraded DNA (and RNA) by a mechanism involving redox reactions and ROS generation. Subsequently, the requirement for oxygen was demonstrated with casIIgly–AsA under anaerobic conditions leading to no cleavage of DNA, while in the presence of an oxygen generating system (OGS) the DNA was totally degraded. The inhibition, or reduction, of casIIgly-induced DNA damage by catalase but not by SOD in the presence of AsA, suggests that • OH could be involved in the DNA breakage. This is consistent with data presented herein suggesting that O2 −• is not the primary reactive species, as demonstrated by the lack of SOD-mediated protection. The most likely mechanism for the production of • OH is the reduction of copper, from Cu3+ O to Cu+ in a Fenton-like reaction. Von Sonntag [37] reported that • OH produces strand breakage in DNA via chain reactions and chemical purine and pyrimidine base modification. The oxidation of DNA commonly involves the nucleobases because the • OH radical attacks preferentially heterocycles. While base damage involving apurinic or apyrimidinic sites [38] may not result in DNA strand scission the oxidation of the phosphodiester backbone (e.g. C1 position in deoxyribose) is a common site for DNA degradation by metal complexes with affinity for DNA [33,39]. The observation that trientine and EDTA inhibit DNA degradation by casIIgly–AsA sup-

198

A. Rivero-M¨uller et al. / Chemico-Biological Interactions 165 (2007) 189–199

ports the idea that the phenanthroline family of copper complexes exhibits a unique ability of binding to DNA with a specific stoichiometry that leads to the degradation. We have no data on the exact binding site on the DNA or the size of the products but these appear to be very small (<100 bp) since they could not be observed on the gels after degradation of plasmid DNA. Assuming that casIIgly binds stoichiometrically along the DNA and that it produces one break per molecule the relative molar ratios suggest fragments of approximately 370 bp for plasmid DNA, and in the case of genomic DNA > 30 kbp. However, since the cleavage fragments are likely to be smaller than 100 bp this suggests that each casIIgly molecule is able to cleave several DNA bonds. Our results suggest that casIIgly cleaves DNA in vivo as well as in vitro by generation of ROS (mainly • OH radicals) and that this potential to react within biological systems directly correlates with casIIgly cytotoxicity. The lack of specificity to damage cancer cells DNA only by this highly reactive complex hampers its use in vivo [40] but indeed correlates with its acute toxicity [41]. Nevertheless, our data illustrates the potential of casIIgly to react with biological molecules and indeed degrade them by generation of ROS, and that such reactivity is a main mechanism for its cytotoxicity.

[6]

[7]

[8]

[9]

[10] [11] [12]

[13]

[14]

Acknowledgements We would like to thank Dr. L.R. Kelland and Dr. M.R. Orr at the Institute of Cancer Research, Sutton, UK, for providing us with the L1210 cell line. This work was funded by CONACyT, Mexico, Grants 93006 and 93044. References [1] U.Z. Guo, P.I. Sadler, Metals in medicine, Angew. Chem. Int. Ed. 38 (1999) 1512–1531. [2] A. De Vizcaya-Ruiz, A. Rivero-Muller, L. Ruiz-Ramirez, G.E. Kass, L.R. Kelland, R.M. Orr, M. Dobrota, Induction of apoptosis by a novel copper-based anticancer compound, casiopeina II, in L1210 murine leukaemia and CH1 human ovarian carcinoma cells, Toxicol. In Vitro 14 (2000) 1–5. [3] L. Ruiz-Ramirez, I. Gracia-Mora, R. Moreno-Esparza, D. Diaz, L. Gasque, L. Huerta, L. Mayet, V. Ortiz, C. Lomeli, The antitumor activity of several transition metal complexes, J. Inorg. Biochem. 43 (1991) 615. [4] I. Gracia-Mora, L. Ruiz-Azuara, C. G´omez, M. Tinoco, A. M´arquez, L. Romero, A. Mar´ın, L. Mac´ıas, M.E. Bravo, Knight’s move in the periodic table, from copper to platinum, novel antitumour mixed chelate copper compounds, Casiope´ınas, evaluated by an in vitro human and murine cancer cell line panel, Metal Based Drugs 8 (2001) 19–28. [5] C. Trejo-Sol´ıs, G. Palencia, S. Z´un˜ iga, A. Rodr´ıquez-Ropon, L. Osorio-Rico, T.L. S´anchez, I. Gracia-Mora, L. M´arquez-Rosado, A. S´anchez, M.E. Moreno-Garc´ıa, A. Cruz, M.E. Bravo-G´omez,

[15]

[16]

[17]

[18]

[19]

[20] [21] [22]

L. Ru´ız-Ram´ırez, S. Rodr´ıguez-Enriquez, J. Sotelo, Cas IIgly induces apoptosis in glioma C6 cells in vitro and in vivo through caspase-dependent and caspase-independent mechanisms, Neoplasia 7 (2005) 563–574. A. Marin-Hernandez, I. Gracia-Mora, L. Ruiz-Ramirez, R. Moreno-Sanchez, Toxic effects of copper-based antineoplastic drugs (Casiopeinas) on mitochondrial functions, Biochem. Pharmacol. 65 (2003) 1979–1989. A. De Vizcaya-Ruiz, A. Rivero-Muller, L. Ruiz-Ramirez, J.A. Howarth, M. Dobrota, Hematotoxicity response in rats by the novel copper-based anticancer agent: casiopeina II, Toxicology 194 (2003) 103–113. S.J. Lippard, Platinum complexes: probes of polynucleotide structure and antitumor drugs, Acc. Chem. Res. 11 (1978) 211– 217. C. Sissi, F. Mancin, M. Gatos, M. Palumbo, P. Tecilla, U. Tonellato, Efficient plasmid DNA cleavage by a mononuclear copper(II) complex, Inorg. Chem. 44 (2005) 2310–2317. D.S. Sigman, C.H. Chen, Chemical nucleases: new reagents in molecular biology, Annu. Rev. Biochem. 59 (1990) 207–236. J.H. Lee, J.W. Park, A manganese porphyrin complex is a novel radiation protector, Free Radic. Biol. Med. 37 (2004) 272–283. I.G. de Avellar, M.M. Magalhaes, A.B. Silva, L.L. Souza, A.C. Leitao, M. Hermes-Lima, Reevaluating the role of 1,10phenanthroline in oxidative reactions involving ferrous ions and DNA damage, Biochim. Biophys. Acta 1675 (2004) 46–53. B. Halliwell, J.M. Gutteridge, Role of free radicals and catalytic metal ions in human disease: an overview, Meth. Enzymol. 186 (1990) 1–86. J. Ueda, M. Takai, Y. Shimazu, T. Ozawa, Reactive oxygen species generated from the reduction of copper(II) complexes with biological reductants cause DNA strand scission, Arch. Biochem. Biophys. 357 (1998) 231–239. M. Kito, Y. Takenaka, R. Urade, The 1,10-phenanthroline micelles–copper(I) complex catalyses protein degradation, FEBS Lett. 362 (1995) 39–42. S.V. Avery, N.G. Howlett, S. Radice, Copper toxicity towards Saccharomyces cerevisiae: dependence on plasma membrane fatty acid composition, Appl. Environ. Microbiol. 62 (1996) 3960–3966. F.C. Furtado, N.R. Asad, A.O. Leitao, Effects of 1,10phenanthroline; hydrogen peroxide in Escherichia coli: lethal interaction, Mutat. Res. DNA Repair 385 (1997) 251–258. Y. Ma, L. Cao, T. Kawabata, T. Yoshino, B.B. Yang, S. Okada, Cupric nitrilotriacetate induces oxidative DNA damage and apoptosis in human leukemia HL-60 cells, Free Radic. Biol. Med. 25 (1998) 568–575. A.S. Sansinanea, S.I. Cerone, A. Elperding, N. Auza, Glucose6-phosphate dehydrogenase activity in erythrocytes from chronically copper-poisoned sheep, Comp. Biochem. Physiol. 114 (1996) 197–200. A.G. Papavassiliou, Chemical nucleases as probes for studying DNA–protein interactions, Biochem. J. 305 (1995) 345–357. R.M. Burger, Cleavage of nucleic acids by bleomycin, Chem. Rev. 98 (1998) 1153–1169. Process to obtain new mixed copper aminoacidate complexes from phenylatephenanthroline to be used as anticancerigenic agents, April 21 (1992) Number 5, 107, 005. U.S Patent Re35, 458, February 18 (1997) Process to obtain new mixed copper aminoacidate from methylate phenanthroline complexes to be used as anticancerigenic agents.U.S. Patent Patent no. 5,576,326 November 19 (1996).

A. Rivero-M¨uller et al. / Chemico-Biological Interactions 165 (2007) 189–199 [23] D.P. Jones, D.J. McConkey, P. Nicotera, S. Orrenius, Calciumactivated DNA fragmentation in rat liver nuclei, J. Biol. Chem. 264 (1989) 6398–6403. [24] I. Fuentes-Noriega, L. Ruiz-Ram´ırez, A. Tovar Tovar, H. RicoMorales, I. Gracia-Mora, Development and validation of a liquid chromatographic method for casiopeina IIIi in rat plasma, J. Chromatogr. B 772 (2002) 115–121. [25] K. Ito, S. Inoue, K. Yamamoto, S. Kawanishi, 8-Hydroxydeoxyguanosine formation at the 5 site of 5 -GG-3 sequences in double-stranded DNA by UV radiation with riboflavin, J. Biol. Chem. 268 (1993) 13221–13227. [26] S. Tada-Oikawa, S. Oikawa, S. Kawanishi, Determination of DNA damage, peroxide generation, mitochondrial membrane potential, and caspase-3 activity during ultraviolet A-induced apoptosis, Meth. Enzymol. 319 (2000) 331–342. [27] G. Ferrer-Sueta, L. Ruiz-Ramirez, R. Radi, Ternary copper complexes and manganese(III) tetrakis(4-benzoic acid)porphyrin catalyze peroxynitrite-dependent nitration of aromatics, Chem. Res. Toxicol. 10 (1997) 1338–1344. [28] M. Fojta, T. Kubicarova, E. Palecek, Electrode potentialmodulated cleavage of surface-confined DNA by hydroxyl radicals detected by an electrochemical biosensor, Biosens. Bioelectron. 3–4 (2000) 107–115. [29] D.B. Zamble, S.J. Lippard, Cisplatin and DNA repair in cancer chemotherapy, Trends Biochem. Sci. 20 (1995) 435–439. [30] D.B. Lovejoy, D.R. Richardson, Novel “hybrid” iron chelators derived from aroylhydrazones and thiosemicarbazones demonstrate selective antiproliferative activity against tumor cells, Blood 100 (2002) 666–676. [31] Y. Hiraku, S. Inoue, S. Oikawa, K. Yamamoto, S. Tada, K. Nishino, S. Kawanishi, Metal-mediated oxidative damage to

[32]

[33] [34]

[35]

[36]

[37] [38]

[39] [40]

[41]

199

cellular and isolated DNA by certain tryptophan metabolites, Carcinogenesis 16 (1995) 349–356. C.A. Detmer, F.V. Pamatong, J.R. Bocarsly, Non-random double strand cleavage of DNA by a monofunctional metal complex: mechanistic studies, Inorg. Chem. 35 (1996) 6292–6298. D.G. Knorre, O.S. Fedorova, E.I. Frolova, Oxidative degradation of nucleic acids, Russ. Chem. Rev. 62 (1993) 65–86. R. Roots, S. Okada, Estimation of life times and diffusion distances of radicals involved in X-ray-induced DNA strand breaks or killing of mammalian cells, Radiat. Res. 64 (1975) 306–320. K.R. Huber, R. Sridhar, E.H. Griffith, E.L. Amma, J. Roberts, Superoxide dismutase-like activities of Cu(II) complexes tested in serum, Biochim. Biophys. Acta 915 (1987) 267–276. J. Casanova, G. Alzuet, J. Borras, J. LaTorre, M. Sanau, S. Garcia-Granada, Coordination behaviour of sulfatiazole. Crystal structure of (Cu(sulfatiazole)(py)3Cl) superoxide dismutase activity, J. Inorg. Biochem. 60 (1995) 219–230. C. Von Sonntag, The Chemical Basis of Radiation Biology, Taylor and Francis, London, 1987. M. Casadevall, A. Kortenkamp, The generation of apurinic/ apyrimidinic sites in isolated DNA during the reduction on chromate by glutathione, Carcinogenesis 15 (1994) 407–409. A.V. Peskin, Interaction of reactive oxygen species with DNA. A review, Biochem. Moscow 62 (1997) 1341–1347. M. Valko, M. Izakovic, M. Mazur, C.J. Rhodes, J. Telser, Role of oxygen radicals in DNA damage and cancer incidence, Mol. Cell. Biochem. 266 (2004) 37–56. E. Garcia, M. Medina, Y. Rojas, L. Ruiz, P. Ostrosky, R. Rodriguez, Acute toxicity of casiopeine, a new type of cytotoxic agent, Proc. West. Pharmacol. Soc. 34 (1991) 65–67.