Cellular uptake and distribution of cobalt complexes of ... - Springer Link

J Biol Inorg Chem (2008) 13:861–871 DOI 10.1007/s00775-008-0374-7

ORIGINAL PAPER

Cellular uptake and distribution of cobalt complexes of fluorescent ligands Natsuho Yamamoto Æ Sarah Danos Æ Paul D. Bonnitcha Æ Timothy W. Failes Æ Elizabeth J. New Æ Trevor W. Hambley

Received: 22 November 2007 / Accepted: 2 April 2008 / Published online: 17 April 2008 Ó SBIC 2008

Abstract The development of complexes that allow the monitoring of the release and distribution of fluorescent models of anticancer drugs initially bound to cobalt(III) moieties is reported. Strong quenching of fluorescence upon ligation to cobalt(III) was observed for both the carboxylate- and the hydroximate-bound fluorophores as was the partial return of fluorescence following addition of ascorbate and cysteine. The extent of the increase in the fluorescence intensity observed following addition of these potential reductants is indicative of the fluorophore being displaced from the complex by the action of ascorbate or cysteine, by ligand exchange. The cellular distribution of the fluorescence revealed that coordination to cobalt can dramatically alter the subcellular distribution of a bound fluorophore. This work shows that fluorescence can be an effective means of monitoring these agents in cells, and of determining their sites of activation. The results also reveal that the cytotoxicity of such agents correlates with their uptake and distribution patterns and that these are influenced by the types of ligands attached to the complex. Keywords

Anticancer drug Imaging

Electronic supplementary material The online version of this article (doi:10.1007/s00775-008-0374-7) contains supplementary material, which is available to authorized users. N. Yamamoto S. Danos P. D. Bonnitcha T. W. Failes E. J. New T. W. Hambley (&) Centre for Heavy Metals Research, School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia e-mail: [email protected]

Abbreviations ahaH2 Acetohydroxamic acid c343H Coumarin-343 c343haH2 Coumarin-343 hydroxamic acid cyclam 1,4,8,11-Tetraazacyclotetradecane DMF N,N0 -dimethylformamide DMSO Dimethyl sulfoxide ESI Electrospray ionisation HPLC High-performance liquid chromatography MTT 3-(4,5-Dimethylthiazole-2-yl)-2,5diphenyltetrazolium bromide tpa Tris(2-methylpyridine)amine

Introduction Many solid tumours contain regions of cells in hypoxic conditions [1], and this frequently leads to poorer prognosis due to the aggressive phenotypes [2] that develop resistance to chemotherapy and radiotherapy [3–5]. In addition, the treatment of solid tumours is hindered by the poor selectivity of chemotherapy. However, as extreme hypoxia is generally restricted to solid tumours [5], it can be used as a means of selectively activating prodrugs and the redox properties of metals can be exploited in this regard. Cobalt(III) complexes with cytotoxic ligands have been investigated as hypoxia-activated prodrugs and some have shown selective toxicity towards hypoxic cells [6–9]. While cobalt(III) complexes are kinetically inert, the cobalt(II) complexes are far more labile [10]. Thus, once a cobalt(III) complex is reduced, any cytotoxic ligands should be rapidly released and be able to effect their action [11]. The mechanism by which the hypoxia selectivity of such cobalt(III) complexes is achieved is currently unclear. It was thought

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that a redox cycling mechanism would operate, as many hypoxia-selective agents have been shown to achieve selectivity through this mechanism [12–14]. In this case, the cobalt(III) would be reduced by endogenous reducing agents to cobalt(II) and the cytotoxic agent released in hypoxic cells, whereas in normoxic cells it would be backoxidised to cobalt(III) by molecular oxygen. This requires that the back-oxidation of the cobalt(II) form is more rapid than the release of the ligands. A number of studies, such as investigations of the effect of denticity of the cytotoxic ligand [6] and pulse radiolysis studies [15], have led to the conclusion that the hypoxia selectivity of such cobalt complexes does not arise from redox cycling. Instead other mechanisms, such as competition with O2 for biological reductants, have been proposed [15]. While the exact mechanism of activity of these agents is unknown, ligand release, by reduction and/or ligand exchange, is essential for the activation of the cytotoxin. At the present time there is no means of establishing when and where ligand release occurs and determining this would greatly assist in the rational development of these agents. One way in which this could be done is by using cobalt(III) complexes containing fluorophores that are themselves cytotoxins or mimic the binding of cytotoxins to cobalt and whose fluorescence is quenched on ligation to cobalt(III). Following the reduction of the complex, the fluorophore should be released and the fluorescence of the molecules should return, allowing this key step to be monitored. To this end, two cobalt(III) complexes, [Co(c343)2 (cyclam)]ClO4 (1), where cyclam is 1,4,8,11-tetraazacyclotetradecane, and [Co(c343ha)(tpa)]ClO4 (2), where tpa is tris(2-methylpyridine)amine, containing the fluorescent ligands coumarin-343 (c343H) and its hydroxamic acid derivative coumarin-343 hydroxamic acid (c343haH2), respectively, were prepared and studied. A2780 ovarian cancer cells incubated with the complexes were examined using confocal fluorescence microscopy to evaluate the use of these complexes in determining the localisation of the fluorescence and the ligand release.

O

Materials and methods Diffuse reflectance IR Fourier transform spectra were collected using a Bio-Rad FTS-40 or FTS-70 spectrometer between 4,000 and 400 cm-1. KBr was used as a background and matrix for all samples. 1H and 13C NMR spectra were collected using a Bruker 300 MHz spectrometer at 27 °C. 3-(Trimethylsilyl)propionic acid was used as a reference for spectra measured in D2O. For all other solvents isotopic impurities were used as internal references. Mass spectra were collected with a Finnigan LCQ spectrometer using electrospray ionisation (ESI). Microanalysis (C, H, N) was performed by the Microanalytical Unit of the Australian National University. Synthesis All chemicals were reagent grade and were used without further purification. The starting materials tpa.nClO4 [16], [CoCl2(tpa)]ClO4 [17], [Co(aha)(tpa)]ClO4 [18], where ahaH2 is acetohydroxamic acid, and trans-[CoCl2(cyclam)]Cl [19] were prepared according to previously reported procedures. Synthesis of trans-[Co(OAc)2(cyclam)]ClO4 To a solution of trans-[CoCl2(cyclam)]Cl (0.30 g, 0.82 mmol) in water (10 mL), silver nitrate (0.30 g, 1.74 mmol) dissolved in a minimum volume of water was added and the solution was stirred for 2 h. The white silver chloride precipitate was removed by suction filtration and sodium acetate was added to the green solution with stirring until the solution turned a deep red colour (approximately 0.1 g). This was then stirred with heating for a further hour, and allowed to cool to room temperature. To this a saturated sodium perchlorate solution was added dropwise until a precipitate formed (approximately 1 mL). The pink precipitate was then removed by suction filtration, washed with ice-cold water and methanol and dried in a

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desiccator overnight (yield 0.28 g, 0.59 mmol, 72%). IR: KBr (4,000–400 cm-1), 3,231 m, 2,985–2,957 br; 1,616 s, 1,601 s m(carbonyl); 1,464 m, 1,419 m, 1,379 s, 1,364 s, 1,322 s, 1,098 s, 1,055 s, 1,023 s, 929 w, 885 w, 694 w, 625 w m(ClO), 501 w. 1H NMR (d4-MeOD): d 7.94 (s, NH), 2.97–2.94 (m, 2H, 1 9 CH2), 6.67–2.48 (m, 6H, 3 9 CH2), 2.30–2.16 (m, 2H, 1 9 CH2), 1.82 (s, 6H, 2 9 CH3). Owing to the sensitivity of c343H to light, all steps in the following procedures were carried out in the absence of light. Synthesis of trans-[Co(c343)2(cyclam)]ClO4 c343H (0.1 g, 0.35 mmol) was suspended in HPLC grade methanol dried with sodium sulfate (100 mL), and to it trans-[CoCl2(cyclam)]Cl (0.06 g, 0.16 mmol) in methanol (5 mL) was added. To the green solution silver oxide (approximately 0.2 g, 0.86 mmol) was added and the mixture was stirred over 2 days. The black silver oxide and silver chloride were then removed from the solution by suction filtration and the volume of the dark-orange filtrate was reduced to approximately 10 mL under a stream of nitrogen. Diethyl ether (100 mL) was added, leading to the formation of a light-orange/brown precipitate. The crude product was dissolved in acetone and the solution was filtered. Sodium perchlorate solution (0.1 M, 40 mL) was added and the volume of the solution was reduced under a stream of nitrogen until a precipitate formed. The solution was cooled on ice and the bright-orange precipitate collected by filtration, washed with ether and dried in a desiccator overnight (yield 30 mg, 32 lmol, 9.1%). IR: KBr (4,000–400 cm-1), 3,000–2,810 br, 1,718 m, 1,701 s, 1,623 s m(carbonyl); 1,597 s, 1,559 m, 1,435 w, 1,362 m, 1,310 s, 1,276 s, 1,212 m, 1,174 w, 1,157 w, 1,105 m, 1,050 w, 804 w, 624 m(ClO). 13C NMR (d6-DMSO, where DMSO is dimethyl sulfoxide): d 175.81, 158.73, 152.63, 147.72, 146.86, 126.99, 119.06, 111.72, 106.79, 104.88, 52.39, 49.80, 49.21, 27.12, 21.03, 20.09, 19.91. Anal. Required for CoN6ClC42H52O12: C, 54.4%, H, 5.65%, N, 9.06%. Found: C 54.4%, H 5.61%, N 8.68%. Mass spectrometry: m/z 827, for [Co(cyclam)(c343)2]+, 827. Synthesis of c343haH2 c343haH2 was synthesised using a modification of the procedure reported by Reddy et al. [20]. Ethyl chloroformate (0.1 mL, 1.0 mmol) and N-methylmorpholine (0.15 mL, 1.4 mmol) were added to a suspension of c343H (0.17 g, 0.9 mmol) in diethyl ether (2 mL) with the temperature kept below 5 °C using an ice bath. Dichloromethane (5 mL) was added to the stirred solution, and the

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yellow precipitate that formed was removed by filtration. In a second reaction vessel, a hydroxylamine solution was prepared by adding a solution of hydroxylammonium chloride (0.12 g, 1.7 mmol) in methanol (1.5 mL) dropwise to a solution of potassium hydroxide (0.10 g, 1.8 mmol) in methanol (1.5 mL) with the temperature kept below 5 °C using an ice bath. The white precipitate that formed was removed by filtration and the filtrate was stirred in an ice bath for a further 10 min. The filtrate from the first reaction vessel was added to the filtrate from the second reaction vessel and the mixture was stirred in the ice bath for a further 10 min before the volume of the solution was reduced to about half under a stream of nitrogen. A precipitate formed, and was collected by suction filtration and washed with generous amounts of methanol to give a bright-orange powder (yield 0.15 g, 0.49 mmol, 29%). Melting point 229 °C. 1H NMR (d6DMSO): d10.56 (1 s, 1H, NH), 9.19 (s, 1H, OH), 8.48 (s, 1H, pyranone), 7.26 (s, 1H, aromatic), 2.70 (m, 5H, quinolizine), 1.90 (m, 5H, quinolizine). 13C NMR (d6-DMSO): d148.32, 127.36, 119.77, 107.65, 105.06, 27.18, 20.91, 19.96. IR: KBr (4,000–400 cm-1), 2,951, 2,852, 1,684, 1,617, 1,582, 1,526, 1,313, 1,294, 1,201. Mass spectrometry (ESI positive ion): m/z 323 ([c343haH + Na]+; 301 ([c343haH]+). Anal. Required for C16H16N2O4: C, 63.99%, H, 5.37%, N, 9.33%. Found: C 64.40%, H 5.89%, N 8.82%. Synthesis of [Co(c343ha)(tpa)]ClO4 Silver oxide (0.30 g, 1.29 mmol) was added to a suspension of [CoCl2(tpa)]ClO4 (178 mg, 0.34 mmol) and c343aH2 (99 mg, 0.33 mmol) in methanol (50 mL), and the mixture was stirred for 10 h, after which it turned a dark-orange colour. The precipitate was removed by filtration and the filtrate was evaporated under vacuum to yield an orange–brown solid. To this, a few drops of saturated sodium perchlorate solution were added and the mixture was left in a fridge overnight. The resultant solid was collected by suction filtration, then dissolved in a solution of acetone/ethyl acetate (1:1) (approximately 100 mL) which had been dried over sodium sulfate. The solvent was evaporated slowly until the formation of a precipitate, which was collected by suction filtration and washed with generous amounts of diethyl ether to give an orange powder (yield 118 mg, 160 lmol, 48%). IR: KBr (4,000–400 cm-1), 1,120, 624 m(ClO); 3,080, 2,952, 1,735, 1,585, 1,524, 1,488, 1,310, 1,208, 1,155, 1,086, 771, 406. Mass spectrometry (ESI positive ion): m/z 648 ([Co(c343ha)(tpa)]+); 324 ([Co(c343haH)(tpa)]2+). Anal. Required for CoC34H34N6O13Cl2Na: C, 46.01%, H, 3.86%, N, 9.47%. Found: C 46.06%, H 3.79%, N 9.64%.

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Electrochemistry Cyclic voltammetry was conducted with a BAS 100B/W electrochemical analyser at a scan rate of 100 mV/s, using a glassy carbon working electrode, a platinum auxiliary electrode and a Ag|AgCl reference electrode at room temperature. Samples were prepared as 1 mM solutions in dimethylformamide (DMF) with 0.1 M tetrabutylammonium perchlorate. The samples were then degassed with argon for 10 min prior to measurement. The potentials were measured using ferrocene as an internal standard ([Fe(g5-C5H5)2]0/+ = +0.72 V versus the normal hydrogen electrode in DMF [21]). Fluorescence Fluorescence measurements were collected with an LS50B luminescence spectrometer using a 1 cm 9 1 cm silica cuvette, a scan rate of 100 nm/min, and excitation and emission slit widths of 2.5 nm. Emission scans were run between 400 and 600 nm using an excitation wavelength of 396 nm on 7 9 10-5 M solutions of c343H, c343haH2, [Co(c343)2(cyclam)]ClO4 and [Co(c343ha)(tpa)]ClO4 in acetone/water (1:1), prepared immediately prior to collection. Excitation scans were run on identical solutions with excitation wavelengths between 300 and 480 nm and detection at 493 nm. Emission spectra of [Co(c343)2 (cyclam)]ClO4 were measured at regular time intervals for up to 100 h following the addition of a tenfold excess of ascorbic acid or cysteine. Similar measurements were also made on solutions which had been deoxygenated by passing a stream of nitrogen through the solution for 10 min and sealing the cuvette.


the growth medium, washed with phosphate-buffered saline solution and mounted onto microscope slides. Organelle staining Nuclear and lysosomal staining was performed using the green nucleic acid stain SYTO21 (Molecular Probes) and the green lysosomal stain Lyso Tracker Green DND-26 (Molecular Probes), respectively. For nuclear staining, cells prepared for confocal microscopy were treated with SYTO21 (1 mM in DMSO) 10 min prior to removing the cover slip, to give a final concentration of 2 lM in the medium. For lysosomal staining, the cells were treated with Lyso Tracker Green DND-26 (10 lM in DMSO) to give a final concentration of 0.075 lM in the medium, 1.5 h prior to removal of the cover slip. Confocal microscopy To ensure cells were still alive when viewed, all samples were examined within 30 min of preparation. Samples were observed by fluorescence microscopy using a Nikon Eclipse TE200 inverted fluorescence microscope and a Nikon Plan Fluor 9100/1.30 oil objective lens. Confocal images were collected using a Nikon DE-Eclipse confocal microscope C1 and Coherent RadiusTM 405-25 and SapphireTM 488.20 optically pumped semiconductor laser systems. For samples incubated without organelle stains confocal images were collected using an excitation wavelength of 405 nm. For those samples incubated with organelle stains sequential scans were collected using excitation wavelengths of 488 and 405 nm. A scan rate of 24 ms/pixel was used for all images and an average over five scans per image was collected.

Cell lines Cytotoxicity assays Parental A2780 human ovarian carcinoma cell lines were maintained as confluent monolayers in RPMI 1640 medium (TRACE Bioscience) or advanced Dulbecco’s modified Eagle’s medium (Gibco) containing 2.5 mM glutamine and 5% foetal calf serum at 37 °C in 5% CO2. Preparation of cells for confocal microscopy Cells were plated onto six-well tissue culture plates containing glass cover slips and grown to 40–60% confluence in the same medium at 37 °C in 5% CO2. Prior to dosing, the medium was removed and replaced with 1 mL of fresh medium. The cells were then treated with 24 lL of [Co(c343)2(cyclam)]ClO4, [Co(c343ha)(tpa)]ClO4, c343 or c343haH2 solutions prepared immediately prior to dosing, to give a final concentration of 12 lM in the medium. After 1- or 4–5-h incubations the cover slips were removed from

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Complexes to be tested were prepared as 1 mM aqueous solutions containing 5% DMF immediately prior to the assay. Cytotoxicity was determined using the 3-(4,5dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described [22]. Single-cell suspensions were obtained by trypsinisation of monolayer cultures, with cell counts performed using a heamocytometer counter (Weber). Approximately 1 9 105 cells in 100 lL culture medium were seeded onto each well of flatbottomed 96-well plates (Becton Dickinson) and allowed to attach overnight. Cobalt complex solutions were diluted in culture medium such that 5–40 lL of each drug solution was added to quadruplicate wells to produce the final desired concentrations spanning a 4-log range (final DMF concentrations were limited to 0.5%). Following incubation of the cells for 72 h, MTT (1.0 mM) was added to


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Results Synthesis

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Fluorescence studies The fluorescence spectra of the complexes and free coumarins were measured to examine the changes on coordination to cobalt(III). The free coumarins c343H and c343haH2 have similar emission and excitation spectra (Fig. S1), indicating that similar fluorescence intensities will result from equal concentrations. The emission spectra in Fig. 1 show that the fluorescence of c343H and that of c343haH2 are strongly quenched on coordination to cobalt(III), with the fluorescence intensity of the ligand decreased by approximately 100- and 50-fold, respectively (Fig. 1a, c). The fluorescence is not completely quenched in the complexes and an emission spectrum similar in shape and position to that of the free ligand is observed (Fig. 1b, d). The similarity of these spectra may indicate that the observed emission is due to trace amounts of free ligand rather than the complex. Further, as a control to show that the residual fluorescence arises from the coumarins, the emission spectra of [Co(OAc)2(cyclam)]ClO4 and [Co(aha)(tpa)]ClO4 were measured and showed no emission at the excitation wavelength (Fig. 1b, d).

c343 [Co(c343) 2 (cyclam)]ClO4

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Fig. 1 a Emission spectra of 7 9 10-5 M acetone/water (1:1) solutions of [Co(c343)2(cyclam)]ClO4 and coumarin-343. b Emission spectra of 7 9 10-5 M acetone/ water (1:1) solutions of [Co(c343)2(cyclam)]ClO4 and [Co(OAc)2(cyclam)]ClO4. c Emission spectra of 7 9 10-5 M acetone/water (1:1) solutions of [Co(c343ha)(tpa)]ClO4 and coumarin-343-hydroxamic acid. d Emission spectra of 7 9 10-5 M acetone/water (1:1) solutions of [Co(c343ha)(tpa)]ClO4 and [Co(aha)(tpa)]ClO4. c343H is coumarin-343, cyclam is 4,8,11-tetraazacyclotetradecane, c343haH2 is coumarin-343 hydroxamic acid, tpa is tris(2-methylpyridine)amine and ahaH2 acetohydroxamic acid


Complexes of cobalt(III) with hydroxamic acids can exist in two different charged states, the singly deprotonated hydroxamate form and the doubly deprotonated hydroximate form, and both are known. Failes and Hambley [18] isolated cobalt–tpa complexes with ahaH2 and propionic hydroxamic acid in both the hydroxamate and the hydroximate forms, while only obtaining the hydroximate form with benzohydroxamic acid and marimastat complexes with cobalt–tpa. The cobalt complex of c343haH2 could therefore form in either the hydroxamate form, [Co(c343haH)(tpa)](ClO4)2, or the hydroximate form, [Co(c343ha)(tpa)]ClO4. The hydroximate form is assumed to have formed, as the elemental analysis suggests a mixture of (1:1:1) [Co(c343ha)(tpa)]ClO4/NaClO4/H2O. However, an analogous complex, [Co(aha)(tpa)]ClO4, was

found to have a pKa of 6.8, and assuming other tpa–hydroxamate complexes, including [Co(c343ha)(tpa)]ClO4, have a similar pKa leads to the expectation of a mixture of the hydroxamate and hydroximate forms at physiological pH.


each well and they were incubated for a further 4 h. The culture medium was then removed from each well and DMSO (150 lL) was added, the plate shaken for 5 s and the absorbance measured immediately at 600 nm with a Victor3V microplate reader (PerkinElmer). IC50 values were determined as the drug concentration that reduced the absorbance to 50% of that in untreated control wells.

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To investigate the effect of cellular reductants, the fluorescence of acetone/water (1:1) solutions of [Co(c343)2 (cyclam)]ClO4 was monitored over 100 h after the addition of a tenfold excess of ascorbic acid or cysteine [23] (Fig. 2). Figure 2a and b shows that following the addition of ascorbic acid or cysteine to a solution of [Co(c343)2(cyclam)]ClO4, the fluorescence intensity increases slowly. The increase in the fluorescence intensity of a solution of [Co(c343)2(cyclam)]ClO4 without added reductant over the same time was small in comparison, with a threefold as opposed to a 13-fold increase. This reveals that the addition of cysteine or ascorbate accelerates the return in fluorescence. The addition of a tenfold excess of the nonreducing acid HNO3 or of HCl led to a minimal return in fluorescence after 50 h. This suggests that the presence of acid or ligands such as the chloride anion are not responsible for the increase in fluorescence. Figure 2c shows a comparison between the rate of fluorescence return after the addition of ascorbic acid and cysteine. The addition of cysteine causes slower fluorescence return than does that of ascorbic acid, and to a lesser

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extent. Neither ascorbic acid nor cysteine caused the fluorescence to return to the level expected for an equivalent concentration of c343H. Addition of equivalent amounts of CoCl26H2O, cyclam and the reductants to 7 9 10-5 M solutions of c343H caused minimal quenching of the fluorescence intensity. Varying the pH between 3 and 12 caused a slight blueshift in the fluorescence spectra, but had little effect on the intensity of fluorescence. Therefore, the lower than expected return of fluorescence is most likely to be caused by incomplete ligand exchange or reduction rather than quenching of the fluorescence of the released ligand. Preliminary X-ray absorption near-edge spectroscopy studies on the oxidation state of the complexes over the course of the reaction suggest ligand exchange to be the likely mechanism, but further investigation will be required to determine the mechanism. Examination of the fluorescence of the complexes to which a tenfold excess of ascorbic acid had been added in the absence of oxygen and comparing this with the oxygenated solutions was used to provide insights into the mechanism of reduction. Figure 3 shows that the rates at which the fluorescence intensity returns following the addition of ascorbic acid to the oxygenated and deoxygenated solutions do not differ significantly. If the addition

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Fig. 2 Fluorescence emission spectra over 35 h of a 7 9 10-5 M acetone/water (1:1) solution of [Co(c343)2(cyclam)]ClO4 following the addition of a tenfold excess of a ascorbic acid and b cysteine using

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an excitation wavelength of 395 nm. c Maximum fluorescence intensity of emission spectra over 100 h after cysteine and ascorbic acid additions to [Co(c343)2(cyclam)]ClO4


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dependent on the other ligands. [CoCl2(tpa)]Cl has a positive reduction potential, whereas the two hydroximate complexes have substantially more negative reduction potentials, with the aromatic hydroxamate having the greatest stabilising effect. A similar stabilisation of the cobalt(III) state was observed for the equivalent naphthalene hydroxamate complex [25].

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Fig. 3 Maximum fluorescence intensity of oxygenated and deoxygenated 7 9 10-5 M acetone/water (1:1) solutions of [Co(c343)2 (cyclam)]ClO4

of ascorbate was leading to reduction and subsequent ligand release, this result would indicate that back-oxidation by oxygen is occurring less rapidly than ligand displacement. It should be noted though that this is the expected result if ascorbate is displacing the coumarin ligands without reduction occurring. Electrochemistry Table 1 shows that [Co(OAc)2(cyclam)]ClO4 and [Co (c343)2(cyclam)]ClO4 have similar reduction potentials, which is to be expected given the similarity in their coordination spheres. They are significantly more negative than [CoCl2(cyclam)]Cl, indicating stabilisation of the cobalt(III) oxidation state by the carboxylate donor. The reduction potentials of the tpa complexes are highly

Table 1 Reduction potentials of cobalt(III) complexes in dimethylformamide Complex trans-[CoCl2(cyclam)]Cl

E°0 versus Fc/Fc+ (mV)

E°0 versus NHE (mV)

-808

-88

[Co(OAc)2(cyclam)]ClO4

-1,152

-432

[Co(c343)2(cyclam)]ClO4 [CoCl2(tpa)]Cl

-1,103 -389

-383 331

[Co(aha)(tpa)]ClO4

-1,288

-568

[Co(c343ha)(tpa)]ClO4

-2,054

-1,334

All values given versus the normal hydrogen electrode (NHE) were calculated using redox values of [Fe(g5-C5H5)2]0/+ = +0.72 V versus the NHE in dimethylformamide [24]. c343H is coumarin-343, c343haH2 is coumarin-343 hydroxamic acid and ahaH2 is acetohydroxamic acid Fc ferrocene, cyclam 4,8,11-tetraazacyclotetradecane, tpa tris(2methylpyridine)amine

The fluorescence of cells treated with c343H, c343haH2, [Co(c343)2(cyclam)]ClO4 and [Co(c343ha)(tpa)]ClO4 was examined using fluorescence confocal microscopy and cellular organelle stains. From these images (Fig. 4) it is clear that we can observe fluorescence within the cells, showing that there is cellular uptake of the complexes and/ or the free ligands, and that it is possible to use these coumarin compounds in cellular imaging. It also demonstrates that the strategy of using fluorophores coordinated to cobalt(III) is a viable means of monitoring the behaviour of these complexes within cells. Cells treated with c343H, c343haH2 and [Co(c343ha) (tpa)]ClO4 (Fig. 5a, c, d) show no colocalisation of the nucleic acid stain and coumarin. In contrast, Fig. 5b shows clear colocalisation following treatment with [Co(c343)2(cyclam)]ClO4 (evident as the aqua colour), showing that it or c343 dissociated from the complex is present in the nucleus of the cells. Fluorescence is also observed outside the nucleus, in the cytoplasm or other organelles. As c343H alone did not appear to enter the nucleus, we can conclude that [Co(c343)2(cyclam)]+ remained intact for long enough to enter the nucleus, whereupon the ligands were released, giving rise to the observed fluorescence. For all complexes and ligands, some lysosomal localisation is observed, indicated by the aqua colour of the superimposed images (Fig. S2). This is expected, as the lysosome is the organelle of the cell primarily responsible for enzymatic digestion of foreign molecules [26]. Cytotoxicity [Co(c343)2(cyclam)]ClO4 has a much higher cytotoxicity than either of the ligands, c343H and cyclam, or its nonfluorescent analogue, [CoCl2(cyclam)]Cl (Table 2). This may be because the complex, as a whole, is cytotoxic, while the components are not, or because the complex acts as a delivery system for the potentially cytotoxic components to a particular area of the cell where they would otherwise not reach, as suggested by the confocal microscopy studies. [Co(c343ha)(tpa)]ClO4 is more than tenfold less cytotoxic than [Co(c343)2(cyclam)]ClO4, and is only moderately more cytotoxic than CoCl26H2O or [CoCl2(tpa)]ClO4. For

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Fig. 4 Confocal images of A2780 cells incubated with a c343H, b [Co(c343)2(cyclam)]ClO4, c c343haH2 and d [Co(c343ha)(tpa)]ClO4 for 4–5 h using an excitation wavelength of 405 nm

this complex, the fluorescent ligand c343haH2 is not very toxic but the ancillary ligand tpa is. The fact that free tpa is so cytotoxic, while the complex is not, is most consistent with the tpa remaining bound to the cobalt.

Discussion Our goal in this study was to develop complexes that would allow the monitoring of the release and distribution of fluorescent models of anticancer drugs initially bound to cobalt(III) moieties. The fluorescence studies have established that this is possible and have also shown that coordination to cobalt can change the subcellular distribution of a bound fluorophores. Strong quenching of fluorescence upon ligation to cobalt(III) was observed for both the carboxylate- and the hydroximate-bound fluorophores as was the partial return of fluorescence following addition of ascorbate and cysteine. The extent of the increase in the fluorescence intensity observed following addition of these potential reductants is indicative of the fluorophore being displaced from the complex by the

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action of ascorbate or cysteine by ligand exchange. Ascorbate [27, 28] and cysteine [29, 30] have been shown to reduce cobalt complexes, as well as to coordinate to cobalt(III) [31–33], but their reduction potentials are 58 and -340 mV, respectively [34], suggesting that reduction is not expected for either of the complexes investigated here. Ascorbate caused a more complete return of fluorescence than did cysteine, which is more consistent with ligand exchange rather than with reduction being responsible for the release of the coumarin. The similarity of the return of the fluorescence of c343H in oxygenated and deoxygenated environments provides further insight into the mechanism of ligand release. While many tumour-activated prodrugs work via a redox cycling mechanism, it is currently unknown if this is how cobalt prodrugs act. For a redox cycling mechanism to operate, the rate of back-oxidation by molecular oxygen would have to be quicker than the rate of ligand release, which seems unlikely as the rate of aquation of cobalt(II) complexes is on the order of 106 s-1 [35]. If a redox cycling mechanism is operative, where the complexes are reduced to the cobalt(II) complex and are then reoxidised by molecular


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Fig. 5 A2780 cells incubated with SYTO21 and a c343H, b [Co(c343)2(cyclam)]ClO4, c c343haH2 and d [Co(c343ha)(tpa)]ClO4 for 4–5 h. For each incubation, 1, 2 and 3 correspond to the

fluorescence from the complex or ligand (405 nm excitation), SYTO21 (488 nm excitation) and the superposition of these images, respectively. Scale bars represent 20 lm

oxygen to the cobalt(III) complex before the ligands are released, we would expect the fluorescence of the oxygenated solution to increase more slowly than that of the deoxygenated solution, as no back-oxidation would be possible. The fact that this was not observed suggests that the level of oxygen in the environment does not affect the

rate of ligand release in the complex, consistent with the mechanism of ligand release being ligand exchange. Finally, the low cytotoxicity of the tpa complex suggests this cytotoxic ligand is not released to any significant extent, consistent with retention of the cobalt(III) oxidation state.

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Table 2 Cytotoxicity (IC50) of cobalt complexes and ligands in A2780 ovarian cancer cells following 72-h incubation Complex

IC50 (lM) ± SD

CoCl26H2O

138 ± 7

c343H

[200

Cyclam

[200

[CoCl2(cyclam)]Cl

[200

[Co(c343)2(cyclam)]ClO4

4.8 ± 0.3

c343haH2

117 ± 14

tpa.nClO4 [CoCl2(tpa)]ClO4 [36]

5.3 ± 0.5 77 ± 6

[Co(c343ha)(tpa)]ClO4

55 ± 3

Values are the means of data from at least three independent experiments with quadruplicate readings in each experiment. SD standard deviation

The fluorescence seen in the confocal microscopy images is most likely due to free ligands since the fluorescence of the complexes is 50–100-fold lower, but a contribution from the complexes cannot be ruled out. When the complexes are taken up into cells it is to be expected that the ligands will be displaced from the cobalt(II) or cobalt(III) complex rapidly because of the high concentrations of species such as cysteine, ascorbate and glutathione that are capable of reduction as well as displacement of ligands. High levels of fluorescence in cells were observed in a short time following treatment with both complexes, suggesting that the reduction potential does not greatly affect the rate of release of fluorophores from the complexes. The subcellular localisation of the ligands seen in the confocal studies is in accord with the cytotoxicity assays. In particular, the cytotoxicity of [Co(c343)2(cyclam)]ClO4 being so much higher than that of c343H (Table 2) corresponds with the observation of fluorescence in the nucleus of cells treated with the complex, but not in those treated with the free fluorophore. This may indicate that the delivery of the c343H ligand to the nucleus by the complex potentiates its cell killing abilities. Neither the cyclam ligand nor any fragment of Co(cyclam) is considered to cause the cytotoxicity of [Co(c343)2(cyclam)]ClO4 since [CoCl2(cyclam)]Cl is noncytotoxic. The lack of nuclear localisation of the coumarin-derived fluorescence following treatment with [Co(c343ha)(tpa)]ClO4 suggests that this complex does not deliver the ligand to the nucleus, and this too is in accord with its low cytotoxicity. These results demonstrate that variation of the co-ligand and/or the ligating groups of the cytotoxic ligand can be used to control the disposition of the latter. This work shows that the strategy of using fluorescent molecules coordinated to cobalt(III) complexes is an effective means of monitoring ligand displacement. It also shows that it may be an effective means of monitoring

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these agents in cells, and to determine their sites of activation. The results of some of the experiments using the fluorescent molecules also reveal that the cytotoxicity of such agents correlates with their uptake and distribution patterns and that these are influenced by the types of ligands attached to the complex.

References 1. West JB (2005) Respiratory physiology: the essentials, 7th edn. Lippincott Williams & Wilkins, Philadelphia 2. Graeber TG, Osmanian C, Jacks T, Housman DE, Koch CJ, Lowe SW (1996) Nature 379:88–91 3. Grey LH, Conger AD, Ebert M, Hornsey S, Scott OC (1954) Br J Radiol 26:638–648 4. Brown MJ (1999) Cancer Res 59:5863–5870 5. Denny WA (2000) Lancet Oncol 1:25–28 6. Ware DC, Brothers PJ, Clark GR, Denny WA, Palmer BD, Wilson WR (2000) J Chem Soc Dalton 925–932 7. Teicher BA, Abrams MJ, Rosbe KW, Herman TS (1990) Cancer Res 50:6971–6975 8. Ware DC, Wilson WR, Denny WA, and Richard CEF (1991) J Chem Soc Chem Commun 1171–1173 9. Ware DC, Palmer BD, Wilson WR, Denny WA (1993) J Med Chem 36:1839–1846 10. Zhang CX, Lippard SJ (2003) Curr Opin Chem Biol 7:481–489 11. Simic M, Lilie J (1974) J Am Chem Soc 96:291–292 12. Belcourt MF, Hodnick WF, Rockwell S, Sartorelli AC (1996) Proc Natl Acad Sci USA 93:456–460 13. Lee AE, Wilson WR (2000) Toxicol Appl Pharmacol 163:50–59 14. Breider MA, Pilcher GD, Graziano MJ, Gough AW (1998) Toxicol Pathol 26:234–239 15. Anderson RF, Denny WA, Ware DC, Wilson WR (1996) Br J Cancer 27:S48–S51 16. Anderegg G, Wenk F (1967) Helv Chim Acta 50:2330–2332 17. Mandel JB, Maricondi C, Douglas BE (1988) Inorg Chem 27:2990–2996 18. Failes TW, Hambley TW (2006) J Chem Soc Dalton 1895–1901 19. Bosnich B, Tobe ML, Webb GA (1965) Inorg Chem 4:1109– 1112 20. Reddy AS, Kumar MS, Reddy GR (2000) Tetrahedron Lett 41:6285–6288 21. Barrette WC, Johnson HW, Sawyer DT (1984) Anal Chem 56:1890–1920 22. Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB (1987) Cancer Res 47:943–946 23. Cieslak-Golonka M, Raczko M, Staszak Z. (1992) Polyhedron 11:2549–2555 24. Barrette WC Jr, Johnson HW Jr, Sawyer DT (1984) Anal Chem 56:1890–1898 25. Bonnitcha P (2006) An investigation into cobalt prodrugs for hypoxic tumours. MSc thesis, University of Sydney, Sydney 26. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002) Molecular biology of the cell, 4th edn. Garland, Oxford 27. Saha SK, Ghosh S, Gould ES (1992) Inorg Chem 31:5439–5443 28. Abdur-Rashid K, Dasgupta TP, Bergess J (1996) J Chem Soc Dalton Trans 1393–1398 29. Jackson WG, Sargeson AM, Tucker PA (1977) J Chem Soc Chem Commun 199–200 30. Connet PH, Wetterhahn KE (1985) J Am Chem Soc 107:4282– 4288 31. Tajmir-Riahi HA (1986) Biophys Chem 25:37–41

J Biol Inorg Chem (2008) 13:861–871 32. Freeman HC, Moore CJ, Jackson WG, Sargeson AM (1978) Inorg Chem 17:3513–3521 33. Yonemura T, Bai Z-P, Okamoto KI, Ama T, Kawaguchi H, Yasui T, Hidaka J (1999) J Chem Soc Dalton Trans 2151–2158 34. Fasman GD (ed) (1976) Handbook of biochemistry and molecular biology, 3rd edn, vol I. CRC, Cleveland

871 35. Cotton FA, Wilkinson G, Gaus PL (1967) Basic inorganic chemistry. Wiley, New York 36. Failes TW, Cullinane C, Diakos CI, Yamamoto N, Lyons JG, Hambley TW (2007) Chem Eur J 17:2974–2982

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