J Biol Inorg Chem (2011) 16:695–713 DOI 10.1007/s00775-011-0771-1
ORIGINAL PAPER
Platinum(II) chloride indenyl complexes: electrochemical and biological evaluation Lisa Dalla Via • Saverio Santi • Vito Di Noto • Alfonso Venzo • Enzo Agostinelli • Annarica Calcabrini Maria Condello • Antonio Toninello
•
Received: 17 November 2010 / Accepted: 5 March 2011 / Published online: 29 March 2011 Ó SBIC 2011
Abstract Four platinum(II) complexes of general formula [PtCl(g1-C9H7)L2] [where L2 is 1,2-bis(diphenylphosphino)ethane (dppe) 1 or cycloocta-1,5-diene (cod) 3] and [PtCl2L2] (where L2 is dppe 2 or cod 4) were studied. Inhibition growth assays on human tumor cell lines evidenced for 1 and 3 an antiproliferative effect and, interestingly, the cytotoxic effect exerted by 1 is similar to that of cisplatin. Electrochemical and NMR measurements allowed us to determine the structural and redox properties. Investigation of the mechanism of action responsible for the cytotoxicity demonstrated a weak capacity of interacting with DNA. Some experiments performed on rat liver mitochondria indicate that 1 acts as an inducer of the mitochondrial permeability transition, thus leading to the release of proapoptotic factors, such as cytochrome c and apoptosis-inducing factor.
Electronic supplementary material The online version of this article (doi:10.1007/s00775-011-0771-1) contains supplementary material, which is available to authorized users.
Keywords Platinum(II) complexes Indenyl electrochemistry Antiproliferative activity DNA binding Mitochondrial permeability transition Abbreviations AIF Apoptosis-inducing factor cod Cycloocta-1,5-diene COSY Correlation spectroscopy CsA Cyclosporin A CV Cyclic voltammetry dach 1,2-Diaminocyclohexane DMSO Dimethyl sulfoxide dppe 1,2-Bis(diphenylphosphino)ethane EGTA Ethylene glycol bis(2-aminoethyl ether)N,N,N0 ,N0 -tetraacetic acid EXSY Exchange spectroscopy FITC Fluorescein isothiocyanate HEPES N-(2-Hydroxyethyl)piperazine-N0 ethanesulfonic acid
L. Dalla Via Department of Pharmaceutical Sciences, University of Padova, Via F. Marzolo, 5, 35131 Padua, Italy
E. Agostinelli Department of Biochemical Sciences, Institute of Molecular Biology and Pathology, University of Rome ‘‘La Sapienza’’ and CNR, Rome, Italy
S. Santi V. Di Noto (&) Department of Chemical Sciences, University of Padova, Via F. Marzolo, 1, 35131 Padua, Italy e-mail:
[email protected]
A. Calcabrini M. Condello Department of Technology and Health, Italian National Institute of Health, Viale Regina Elena 299, 00161 Rome, Italy
A. Venzo CNR-ISTM, Institute of Molecular Sciences and Technologies, Via F. Marzolo, 1, 35131 Padua, Italy
A. Toninello Department of Biological Chemistry, University of Padova, Via G. Colombo, 3, 35121 Padua, Italy
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ICP-AES MPT NEM NOESY PBS PI RLM SCE SDS TBS TE THF TMRM Tris
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Inductively coupled plasma atomic emission spectroscopy Mitochondrial permeability transition N-Ethylmaleimide Nuclear overhauser effect spectroscopy Phosphate-buffered saline Propidium iodide Rat liver mitochondria Standard calomel electrode Sodium dodecyl sulfate 2 mM tris(hydroxymethyl)aminomethane– HCl, 13.7 mM NaCl, pH 7.6 10 mM tris(hydroxymethyl)aminomethane, 1 mM EDTA, pH 7.4 Tetrahydrofuran Tetramethylrhodamine methyl ester Tris(hydroxymethyl)aminomethane
The design of new platinum derivatives often implies the use of active ligands such as DNA intercalators [13], estrogen analogues [14], and receptor binders [15, 16] in order to modulate the biological target and accumulation or to obtain synergistic activity. On the basis of the above-mentioned criteria, we synthesized and characterized a series of platinum compounds of general formula [PtCl(g1-C9H7)L2] (L2 is dppe [17] 1 or cycloocta-1,5-diene (cod) [18] 3) and [PtCl2L2] (L2 is dppe 2 or cod 4) (Structure 1) with the aim of studying the effect of the presence of bidentate ligands, dppe and cod, with different donor–acceptor ability. Moreover, the indenyl (C9H7) group is an aromatic olefin that, owing to its planarity, could allow an intercalative binding mode inside DNA base pairs.
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Introduction During the last two decades great effort has been devoted to the design of metallodrugs having better pharmacological properties than cisplatin [cis-diamminedichloroplatinum(II)], such as reduced toxicity and wide spectrum of activity [1]. In this connection, a series of compounds of group 11 metal ion complexes of the chelating diphosphine 1,2-bis(diphenylphosphino)ethane (dppe) have been investigated. Among the various metal complexes based on the dppe ligand, those with AuI, AuIII, CuI, and CuII displayed a pronounced cytotoxic activity in several cell lines [2–6]. In particular, metal complexes of dppe cause DNA damage in vitro [2], inhibit the activity of DNA polymerase a [3], and determine mitochondrial dysfunction through an increased permeability of the inner mitochondrial membrane to cations [7]. Moreover, although the metal-free dppe showed by itself in vivo antitumor activity, it was found that gold–dppe and copper–dppe complexes were up to 1 order of magnitude more cytotoxic than dppe alone [3], thus suggesting that the nature of the metal ion plays a crucial role in the biological properties of the bidentate diphosphine. Conversely, no effect was observed for MgII, ZnII, FeII, CoII, and CdII complexes [3]. Despite the fact that currently the platinum-based drugs are among the most powerful antitumor agents and cisplatin is one of the most important anticancer drugs in clinical practice [8, 9], there are only few reports on the anticancer activity of PtII–dppe derivatives. In particular, the complex [Pt(cis-dach)(dppe)](NO3)2 (where dach is 1,2-diaminocyclohexane) displayed high antiproliferative activity on a number of tumor cell lines and a strong inhibitory effect on metastasis [10–12].
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4 4a 3
2 7a Cl 1 7 Ph Pt Ph PA PB Ph
a
Pt b
d c
Ph 3
1
Cl
Cl Ph Ph P
Pt P 2
Cl
Cl
Pt
Cl
Ph Ph 4
Structure 1
Compounds 1–4 were assayed on human tumor cell lines to evaluate their antiproliferative activity in comparison with that of cisplatin. The characterization and the molecular dynamics of complexes 1 and 3 were obtained by an in-depth NMR analysis. Moreover, electrochemical measurements on 1 and 3 were performed to assess their redox behavior and reactivity upon reduction, in an attempt to correlate the different biological activity found for the cod and dppe derivatives with the two distinct types of electrochemical characteristics. With the aim to elucidate the molecular mechanism accountable for the cytotoxicity, flow linear dichroism and circular dichroism spectroscopic analyses in the presence of DNA were performed. Moreover, the quantitative determination of drug bound to the macromolecule was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES).
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The effect of the most active compound 1 on isolated rat liver mitochondria (RLM) was also evaluated and the capacity to induce the mitochondrial permeability transition (MPT) is discussed. Furthermore, the release of the proapoptotic apoptosis-inducing factor (AIF) and cytochrome c by mitochondria was demonstrated. Finally, to correlate the induction of the MPT with the antiproliferative activity on whole cells, the effect of cyclosporin A (CsA) on HeLa cells was investigated.
Materials and methods General methods Inert-atmosphere techniques were used for all preparations. Tetrahydrofuran (THF) was distilled from sodium/benzophenone just before use. Complexes 2 [17] and 3 [18] were prepared according to previously reported procedures; complex 4 was purchased from Sigma-Aldrich. [Pt(g5-C9H7)(dppe)]? (I?) and [Pt(g5-C9H7)(cod)]? (III?) were prepared in situ following previously reported procedures [18]. 1 13 H, C{1H} and 31P{1H} NMR spectra were obtained at 298 K as acetone-d6 solutions with a Bruker Avance DXR400 spectrometer operating at 400.13, 100.61, and 161.96 MHz, respectively, and equipped with a Bruker BVT-100 temperature controller (dT ± 0.1 K). The d values are referenced to internal Me4Si (for 1H and 13C) and to external 85% aqueous H3PO4 (for 31P). The assignments of the proton resonances were performed by standard chemical shift correlations, correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and nuclear Overhauser effect spectroscopy (NOESY) experiments. The 13C resonances were attributed through 2D-heterocorrelated COSY experiments (heteronuclear multiple quantum correlation with bird sequence [19] and quadrature along F1 achieved using the TPPI method [20, 21] for the hydrogen-bonded carbon atoms and for the phosphorous nuclei, heteronuclear multiple bond correlation [22] for the quaternary carbons). For atom labeling, see Structure 1.
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with toluene and then concentrated under reduced pressure. The solution was cooled to 253 K and then to 195 K overnight, giving greenish white powder of 1 (0.30 g, 0.40 mmol, 61% yield). Anal. Calcd. for C35H31ClP2Pt: C, 56.50; H 4.20. Found C: 56.39; H 4.27. 1H NMR (298 K, acetone-d6): d 7.96, 7.65, and 7.21 (Ph2PA protons), 7.55, 7.52, and 7.42 (Ph2PB protons), 7.68 (m, H7), 6.79 (m, H6), 6.73 (m, H5) 6.68 (m, H4), 6.33 [m, J(1H,31P) 12 Hz, H2], 5.81 [m, J(1H,31P) 17 Hz, H3], 4.99 [dm, 3J(1H,31P) 17.4 Hz, 2J(1H,195Pt) 118.6 Hz, H1], 2.31 and 2.18 (CH2B), 2.08 and 1.99 (CH2-A). 13C{1H} NMR (298 K, acetone-d6): d 151.64 (C7a), 143.5 (C3a), 141.60 [2J(Pt,C) 12 Hz, C2], 133.93, 134.20, and 128.86 [Ph(A)], 131.70, 131.14, and 128.73 (Ph2PB carbons), 123.28 [3J(195Pt,13C) 10 Hz, C3], 124.22 (C7), 121.93 (C6), 121.09 (C5), 120.20 (Ph2PA carbons), 119.97 (C4), 52.21 [1J(195Pt,13C) 392 Hz, C1], 34.97 [2J(1H,31P) 42.9 Hz, 3J(1H,31P) 16.9 Hz, CH2PA], 25.47 [2J(1H,31P) 34.89 Hz, 3J(1H,31P) 6.9 Hz, CH2PB]. 31P (298 K, acetone-d6, ppm): d 42.52 [d, 3J(31P,31P) 5.2 Hz; 1J(31P,195Pt) 4,223 Hz, PA], 44.51 [d, 3J(31P,31P) 5.2 Hz; 1J(31P,195Pt) 2,077 Hz, PB]. Synthesis of 3 Complex 3 was synthesized following the procedure previously reported [18]. Anal. Calcd. for C17H19ClPt: C, 43.99; H 4.22. Found C: 43.65; H 4.26. 1H NMR (298 K, ppm): d 7.56 (m, H7), 7.42 (m, H4), 7.17 (m, H5), 7.14 (m, H6), 6.76 [m, J(195Pt,1H 15.2 Hz, H3], 6.70 [m, J(195Pt,1H approximately 9 Hz, H2], 5.30 [s, J(195Pt,1H) 157 Hz, H1], 5.64, 5.56, 3.67, and 3.44 [4 m, 1H each, J(195Pt,1H) 37, 39, 74, and 72 Hz, respectively, olefinic cod protons], 2.5–1.9 (m, 8H overall, methylene cod protons). 13C{1H} (298 K, CD2Cl2): d 149.71 (C7a), 144.91 (C3a), 139.59 [2J(195Pt,13C) 27 Hz, C2], 124.92 (C7), 125.48 [3J(195Pt,13C) 20 Hz, C3], 124.69 (C6), 123.27 (C5), 121.15 (C4), 111.91 and 111.46 [1J(195Pt,13C) 46 Hz, olefin cod protons cis to Pt], 92.13 and 90.94 [1J(195Pt,13C) 215 and 210 Hz, respectively, olefin cod protons trans to Pt], 48.97 [1J(195Pt,13C) 428 Hz, C1], 32.00, 30.84, 28.62, and 28.04 [J(195Pt,13C) 17, 20, 18, and 17 Hz, respectively, methylene cod protons].
Synthesis of 1
Electrochemical apparatus and procedures
To a toluene solution of 3 (0.30 g, 0.66 mmol, 50 mL) dppe (0.26 g, 0.66 mmol) in toluene (50 mL) was added dropwise at 273 K. Instantaneous reaction occurred and the initial yellow solution became turbid. After 3 h the suspension was allowed to settle and the solution was filtered. The filtrate was dried and washed with cold toluene, yielding a green solid. The crude product was extracted
All manipulations of complexes were performed in an oxygen-free and moisture-free atmosphere; THF was purified by distillation from sodium/benzophenone under an argon atmosphere and then deoxygenated with vacuum line techniques just before use. Ferrocene (Sigma-Aldrich) was purified by crystallization before use. The supporting electrolyte was prepared by exchange reaction of NaBF4
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and n-Bu4NHSO4 in aqueous solution; the precipitate was dissolved in CH2Cl2, dried with Na2SO4, filtered off, and dried by evaporation. The salt n-Bu4NBF4 was then purified by crystallization from ethyl acetate/petroleum ether. Electrochemical experiments were performed in an airtight three-electrode cell connected to a vacuum/argon line. The reference electrode was a standard calomel electrode (SCE; Tacussel ECS C10) separated from the solution by a bridge compartment filled with the same solvent/supporting electrolyte solution as used in the cell. The counter electrode was a platinum spiral with an apparent surface area of approximately 1 cm2. The working electrodes were disks obtained from the cross section of gold wires of different diameters (0.5, 0.125, and 0.025 mm) sealed in glass. Between successive cyclic voltammetry (CV) scans the working electrodes were polished on alumina according to standard procedures and sonicated before use. The E1/2 value of ferrocene is 0.60 V versus SCE in THF/0.2 mM n-Bu4NBF4, 293 K. An EG&G PAR-175 signal generator was used. The currents and potentials were recorded with a Lecroy 9310L oscilloscope. The potentiostat was homebuilt with positive-feedback loop for compensation of the ohmic drop [23]. Absolute electron stoichiometry determination The chronoamperometric diffusion current at a 0.5-mmdiameter disk electrode for a step duration of 0.2 s and the steady-state current at a 12.5-lm-radius gold microdisk electrode (potential scan rate 10 mV s-1) were measured for 3 mM solutions of 1–4, I?, and III? and for the standard ferrocene [D = (1.3 ± 0.2) 9 10-5 cm2 s-1] under strictly similar condition, i.e., THF/0.2 M n-Bu4NBF4. These data allowed determination of the absolute consumption of electrons [24] and of the diffusion coefficients reported in Table 2. Simulations Digital simulations of the voltammograms were performed with the program Antigona for applied electrochemistry (chronoamperometry and voltammetry) written by Loı¨c Mottier and based on the Crank–Nicholson algorithm with a space exponential grid [25] (the program and its running codes can be provided by Loı¨c Mottier, e-mail address
[email protected]). Cell cultures HL-60 (human myeloid leukemic cells) and JR8 (human melanoma cells) were grown in RPMI 1640 (Sigma) supplemented with 15 and 10% heat-inactivated fetal calf
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serum (Biological Industries), respectively. HeLa (human cervix adenocarcinoma cells) were grown in nutrient mixture F-12 (Ham) (Sigma) supplemented with 10% heat-inactivated fetal calf serum (Biological Industries). To both media, penicillin (100 U mL-1), streptomycin (100 lg mL-1), and amphotericin B (0.25 lg mL-1) (Sigma) were added. The cells were cultured at 310 K in a moist atmosphere of 5% carbon dioxide in air. Inhibition growth assays HL-60 cells (4 9 104) were seeded into each well of a 24-well cell culture plate. After incubation for 24 h, various concentrations of the test compounds were added in complete medium and incubated for a further 72 h. HeLa and JR8 cells (4 9 104) were seeded into each well of a 24-well cell culture plate. After incubation for 24 h, the medium was replaced with an equal volume of fresh medium and various concentrations of the test compounds were added. The cells were then incubated in standard conditions for a further 72 h. Stock solutions were made in ethanol (complexes 1 and 3) or in dimethyl sulfoxide (DMSO) (complexes 2 and 4), and later were diluted with complete medium in such a way that the final amount of solvent in each well did not exceed 0.5%. NMR measurements of 2 and 4 in DMSO-d6 showed that no ligandexchange processes occur in this solvent. For the experiments in the presence of CsA, after incubation for 24 h, HeLa cells were treated with 2 lM CsA for 30 min; the medium was replaced with an equal volume of fresh medium, 1.5 lM test agent was added, and the cells were incubated for a further 72 h. A trypan blue assay was performed to determine cell viability. Data were expressed as IC50 values, i.e., the concentration of the test agent inducing 50% reduction in cell number compared with control cultures. Nucleic acid Salmon testes DNA was purchased from Sigma. Its hypochromicity, determined according to the method of Marmur and Doty [26], was more than 35%. The DNA concentration was determined using the molar absorption coefficient e = 6,600 M-1 cm-1 at 260 nm. Flow linear dichroism Linear dichroism measurements were performed with a JASCO J500A circular dichroism spectropolarimeter converted for linear dichroism experiments and equipped with an IBM PC and a JASCO J interface. Linear dichroism is defined as
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LDðkÞ ¼ A==ðkÞ A?ðkÞ where Ak and A\ correspond to the absorbances of the sample when polarized light is oriented parallel or perpendicular to the flow direction, respectively. The orientation is produced by a device designed by Wada and Kozawa [27] at a shear gradient of 500–700 rpm and each spectrum was accumulated four times. A solution of salmon testes DNA (1.5 9 10-3 M) in 10 mM NaClO4 was used. Concentrated stock solutions of test compounds in ethanol were used in such a way that the final amount of solvent did not exceed the 1%. Spectra were recorded at 298 K at different drug-to-DNA concentration ratios.
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and coolant flow rates set at 1, 0.5, and 14 L min-1, respectively. Calibration was carried out by preparing 15 multielement standard solutions containing platinum and phosphorous in the concentration range 0–800 mg L-1 (ppm). Standard solutions were prepared by diluting phosphorous and platinum stock solutions of 1,000 mg L-1 (Spectrascan standards from Teknolab, Norway) with 1% HCl– water solution. The concentrations reported for platinum and phosphorous as a function of incubation time are the mean values of three different experiments. The error bars in the plots correspond to the standard deviations determined by averaging the results of three different experiments.
Circular dichroism Immunoblot analysis Circular dichroism spectra were recorded on a JASCO J500A spectropolarimeter equipped with an IBM PC and a Jasco interface. A solution of salmon testes DNA (2.1 9 10-4 M) in 10 mM NaClO4 was used. Concentrated stock solutions of test compounds in ethanol were used in such a way that the final amount of solvent did not exceed 1%. Spectra were recorded at 298 K at different drug-toDNA concentration ratios after 24 h of incubation at 310 K. Quantitative platinum analysis An aqueous solution of salmon testes DNA (1 mM; Sigma) in 10 mM tris(hydroxymethyl)aminomethane (Tris), 1 mM EDTA, pH 7.4 (TE) buffer was incubated at 310 K with test compounds to achieve a DNA-to-drug concentration ratio of 10. Concentrated stock solutions of test compounds in ethanol were used in such a way that the final amount of solvent did not exceed 1%. At a fixed incubation time, aliquots of exact volume were collected and DNA was precipitated with sodium acetate (up to 0.3 M concentration) and cold ethanol (2 vol). The precipitated DNA was washed with 70% ethanol, dried, and then dissolved in a measured volume of TE buffer. The samples were then mineralized by treating them with 3:1 HCl–HNO3 (70%) for at least 24 h. Finally, the samples were resuspended in dilute HCl to determine the phosphorous and platinum content. The analyses of phosphorous and platinum were performed by ICP-AES at emission lines k(P) = 178.290 nm and k(Pt) = 214.423 nm. A Spectroflame Modula sequential and simultaneous ICP-AES spectrometer equipped with a capillary cross-flow nebulizer was used (Spectro Analytical). Analytical determinations were performed using a plasma power of 1.2 kW, a radio-frequency generator of 27.12 MHz, and an argon gas flow with nebulizer, auxiliary,
HeLa cells (5 9 105) were plated and after incubation for 24 h, the medium was replaced with an equal volume of fresh medium and 50 lM concentration of the test compound was added. Concentrated stock solutions of the test compounds in ethanol were used in such a way that the final amount of solvent did not exceed 0.5%. The cells were then incubated in standard conditions for a further 24 h. Detached cells were collected by centrifugation and adherent cells were washed once in phosphate-buffered saline (PBS), lysed, scraped in RIPA buffer containing 1% PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 2 mM phenylmethylsulfonyl fluoride, and pooled with the detached cell pellet. The lysate was centrifuged (10,000g for 10 min at 277 K), the supernatant was recovered, and the protein content was quantified by the method of Bradford. Equal amounts of protein from each sample were loaded on 10% SDS– polyacrylamide gel and the separated proteins were transferred onto a nitrocellulose membrane using a wet transfer apparatus (Bio-Rad Laboratories). ColorBurst electrophoresis marker (Sigma) was used as a molecular mass standard. Following transfer, the membrane was blocked with 3% bovine serum albumin for 30 min in 2 mM Tris–HCl, 13.7 mM NaCl, pH 7.6 (TBS) buffer and then incubated with p53 mouse monoclonal primary antibody (Santa Cruz Biotechnology) overnight at 277 K. Washes with TBS with Tween 20 were carried out before incubation (room temperature, 30 min) with the appropriate horseradish peroxidase labeled secondary antibody (PerkinElmer). The antibody-reactive bands were revealed by a chemiluminescence-based detection method using the ECL Western Blotting Detection Reagents chemiluminescent substrate (GE Healthcare). Equal protein loading was ensured by reprobing the membrane with mouse antibody anti-b-actin.
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Mitochondrial isolation and standard incubation procedures RLM were isolated by conventional differential centrifugation [28] in a buffer containing 250 mM sucrose, 5 mM N-(2-hydroxyethyl)piperazine-N0 -ethanesulfonic acid (HEPES) (pH 7.4) and 1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid (EGTA) as follows. The cell debris, nuclei, and other heavy components were removed by sedimentation at 755g for 5 min in a Sorvall RC-5B centrifuge. The supernatant was filtered through glass wool and the mitochondria were then sedimented at 10,800g for 10 min. The supernatant was discarded, and the mitochondrial pellet was carefully resuspended in ice-cold isolation medium, sedimented again at 15,800g for 5 min, and then resuspended in the final medium. EGTA was omitted from the final wash and resuspension solution. Protein content was measured by the biuret method with bovine serum albumin as a standard [29]. The resulting mitochondrial suspension was maintained on ice in washing medium (isolation medium without EGTA) at a protein concentration of approximately 40 mg mL-1. Mitochondria (1 mg protein mL-1) were incubated in a water-jacketed cell at 293 K. The standard medium contained 250 mM sucrose, 10 mM HEPES (pH 7.4), 5 mM succinate, and 1.25 lM rotenone. Stock solutions were made in ethanol (complexes 1 and 3) or in DMSO (complexes 2 and 4), and later were diluted with standard medium in such a way that the final amount of solvent in each well did not exceed 0.5%. Variations and other additions are given with the description of each experiment Detection of cytochrome c and AIF release The mitochondria (1 mg protein mL-1) were incubated for 15 min at 293 K in standard medium with the appropriate additions. The reaction mixtures were then centrifuged at 13,000g for 10 min at 277 K to obtain mitochondrial pellets. The supernatant fractions were concentrated using a PAGEprepTM protein cleanup and enrichment kit (Pierce, Rockford, IL, USA). Aliquots of 20 lL of the concentrated supernatants were subjected to 15 and 10% SDS polyacrylamide gel electrophoresis for cytochrome c and AIF, respectively. Proteins were then transferred to nitrocellulose and immunoblotting was carried out with TBS, followed by analysis by Western blotting using mouse anticytochrome c and rabbit anti-AIF antibodies (Pharmingen). Cytochrome c and AIF antibody complexes bound to nitrocellulose were detected by enhancement chemiluminescence using antimouse and antirabbit IgG linked to horseradish peroxidase (ECL kit, Amersham).
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Determination of mitochondrial functions Mitochondrial swelling was determined by measuring the apparent absorbance change of mitochondrial suspensions at 540 nm in a 3-mL cuvette using a Kontron Uvikon model 922 spectrophotometer equipped with a magnetic stirrer and thermostatic control. Mitochondria were suspended as indicated earlier and, upon stabilization of the absorbance trace, swelling was assessed after additions of other compounds as described in the figure legends. The determination of protein sulfhydryl groups and mitochondrial glutathione oxidation was carried out using the mitochondrial suspension of the different incubations utilized for determining the mitochondrial swelling. In brief, at the end of incubation (12 min), the total suspension (1 mg mL-1) was placed in Eppendorf 4515c tubes and centrifuged for 1 min at 12,000g, then the supernatant was discarded and the pellet utilized for both measurements. Sulfhydryl group oxidation assay was performed after solubilization of the pellet with 1 mg of solubilization medium (10 mM EDTA, 0.2 M Tris, 1% SDS, pH 8.3), using 5,50 -dithiobis(2-nitrobenzoic acid) at 412 nm in a Kontron Uvikon model 922 spectrophotometer, according to the method of Santos et al. [30]. Glutathione oxidation was assessed by deproteinization of the pellet with 3% metaphosphoric acid and subsequent centrifugation to separate the supernatant on which the determination of oxidized glutathione was performed by the method of Tietze [31]. Determination of mitochondrial membrane potential on whole HeLa cells The mitochondrial membrane potential was evaluated by using the fluorescent lipophilic cationic probe tetramethylrhodamine methyl ester (TMRM; Molecular Probes) [32]. HeLa cells grown to near confluence were treated with 15 and 30 lM 1 in DMSO from a concentrated stock solution prepared in such a way that the final amount of solvent did not exceed 0.5%, and then they were incubated for 24 h at 310 K. After treatment, both floating and adherent cells were collected, resuspended in PBS (106 cells mL-1) and incubated with TMRM (25 lg mL-1) for 10 min at 310 K. After they had been washed with PBS, samples were analyzed with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) equipped with a 15-mW, 488-nm, air-cooled argon ion laser. The fluorescence emission was collected through a 580-nm band-pass filter. At least 10,000 events per sample were acquired in log mode. The results are shown as fluorescence histograms (TMRM signal on the x-axis). The
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reported numbers represent the percentages of cells with depolarized mitochondrial membrane, calculated by using the CellQuest program (Becton-Dickinson), and are the mean of three independent experiments. Evaluation of apoptotic cell death by annexin V–fluorescein isothiocyanate staining To detect phosphatidylserine translocation from the inner face to the outer surface of plasma membrane in the initialing step of apoptosis, an annexin V–fluorescein isothiocyanate (FITC) kit (MBL, Medical & Biological Laboratories, Japan) was used [33]. About 50 phosphatidylserine monomers are estimated to be bound per annexin V molecule. HeLa cells grown to near confluence were treated with 15 and 30 lM 1 in DMSO from a concentrated stock solution prepared in such a way that the final amount of solvent did not exceed 0.5%, and then they were incubated for 24 h at 310 K. After treatment, both floating and adherent cells were collected and resuspended in binding buffer (10 mM HEPES–NaOH, pH 7.5, 140 mM NaCl, and 2.5 mM CaCl2). Cell suspensions (of about 5 9 105) were then incubated with annexin V–FITC (1 lg mL-1) and with propidium iodide (PI; 1 lg mL-1) for 10 min at room temperature in the dark. Samples were then analyzed with the FACScan flow cytometer [33]. At least 10,000 events per sample were acquired in log mode. Data are presented as dot plots (annexin fluorescence on the x-axis; PI fluorescence on the y-axis). The numbers shown in the four quadrants represent the percentage of viable (lower left), necrotic (upper left), early apoptotic (lower right), and late apoptotic (upper right) cells evaluated using CellQuest. The values reported are the mean of three independent experiments.
Results and discussion Synthesis and NMR characterization The new complex 1 was prepared by reaction of equimolar dppe and 3 [18] in toluene at 273 K. Instantaneous substitution of the diolefin by the diphosphine occurred as confirmed by the 31P{1H} NMR spectrum of 1 consisting of two doublets at d 42.52 and d 44.51 [2J(P,P) 5.2 Hz] that exhibit well-defined satellites due to 195Pt. The one-bond 195 Pt–31P couplings are at 4,223 and 2,077 Hz, respectively. In general, the presence of a chlorine atom trans to a phosphorous nucleus noticeably increases the extent of the Pt–P coupling, so we attribute the 31P resonance at d 42.52 to P(A). The 1H NMR spectrum of an acetone-d6 solution of 1 recorded at 298 K shows the presence of seven
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well-separated signals for the indenyl protons, only partially overlapped in the region from d 8 to d 7.2 by the signals of dppe phenyl ring protons. In particular, the resonance due to the H1 nucleus appears at d 4.99 as a doublet with a 17.4-Hz coupling with P(B) (as confirmed by the heterocorrelated 1H,31P COSY), and the resonances of H2 and H3 appear as multiplets at d 6.83 and d 5.81, respectively. The five-membered ring protons H1, H2, and H3 show satellites due to 195Pt, and the coupling constants are 136.4, 19.1, and 12.5 Hz, respectively. The resonances of the indenyl six-membered ring appears as multiplets at d 7.68 (H7), d 6.79 (H6), d 6.73 (H5), and d 6.68 (H4). The assignments were obtained through a NOESY experiment; in particular, the signal of H7 appears at a considerably low field becasue of the deshielding effect of the platinum atom. A very complex resonance pattern of the dppe methylene protons appears at d 2.31 and d 2.18 (CH2-B), and at d 2.08 and d 1.99 (CH2-A). Also, in this case the assignments were made possible by the respective couplings with 31P(B) and 31P(A), respectively, observed in the heteronuclear correlation experiment. The 13C resonances, detected in the 1D measurement, were assigned by 2D heteronuclear multiple quantum correlation and 2D heteronuclear multiple bond correlation. The absence of any equivalence between protons (and carbon) nuclei is indicative of an asymmetric indenyl moiety, which confirms its g1-coordination to platinum. A very similar spectral pattern, and then an g1-indenyl coordination, is observed for 3. Our NMR analysis (apart from small variations arising from the use of a different solvent) differs to a some extent from that already reported [34]. In particular, on the basis of NOESY measurements, we obtained a reversed assignment for the six-membered ring protons (and of the corresponding carbon atoms, Structure 1). In fact, we assigned the more deshielded proton resonance (d 7.56) to H7, that at d 7.42 to H4, and the resonances at d 7.17 and d 7.14 to H5 and H6, respectively, confirming the deshielding effect of the platinum atom. Finally, the trans effect of a halogen on the values of the coupling constants with 195Pt allowed us to attribute the more deshielded signals to the olefin cod protons labeled A and B, viz., to those trans to the chlorine atom. Exchange spectroscopy (EXSY) measurements (see the electronic supplementary material) showed that in the phase-sensitive NOESY measurements a series of very intense positive cross peaks, in phase with the diagonal of the 2D map, correlates the H1 and H3 indenyl resonances. A similar correlation is observed between the H4 and H7 protons, as well as between the H5 and H6 protons, so a metal shift between the 1-position and 3-position atoms of the indenyl moiety must be invoked. This behavior occurring in an g1-indenyl–iridium complex has been previously reported by some of us [34]. EXSY
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measurement [35–37] is a powerful tool suitable for detecting chemical exchange processes occurring even in the region of slow motion, i.e., before line broadening occurs. In indenyl systems, these exchange processes likely take place through an intermediate situation where the fivemembered ring coordinates the metal atom in an g3-mode, so avoiding the unstable 20-electron g5 coordinative shell of platinum. As an indication of the relative activation barrier of the exchange process, from variable-temperature measurements between 298 and 328 K, we calculated the values of the rate constants (Table 1) by integration of the NMR diagonal and correlation peaks of the exchanging protons in the 2D map [38]. The DH= (37 ± 5 and 83 ± kJ mol-1) and DS= (-132 ± 16 and 6 ± 11 J mol-1 K-1) values were obtained for 1 and 3, respectively (Table 1). The lower DH= value calculated for 1 suggests that the g3 situation is energetically more accessible in 1 than in 3. In addition, the rate constants of 0.19 ± 0.06 and 0.03 ± 0.01 s-1 at 298 K for 1 and 3, respectively, indicate that the reactivity of 1 is markedly higher at the temperature where the biological activity was checked. The noticeably negative DS= value found for 1, compared with that of 3 (DS= & 0), may be attributed to the higher donor capability of dppe with respect to cod. This effect should induce a partial cleavage of the Pt–Cl bond with consequent charge separation and strong solvent reorganization around the metal in the transition state of 1. Finally, attempts to prepare complex solutions in mixtures of dimethylformamide-d6 or acetone-d6 with D2O in order to check the reactivity of 1 in the presence of water, such as exchange of Cl-, or changes of the g1 binding mode or the appearance of metal-free indene ligand, were unsuccessful. In fact, in the presence of D2O, even at a low percentage, the complex solubility was too low to allow us to obtain suitable NMR measurements. Conversely, in acetone-d6,
dimethylformamide-d6, or CD2Cl2 the solutions were stable for days. Electrochemical data Electrochemical methods can be used to gain insight into the electronic and structural properties of the metal centers and the coordinated ligands in organometallic compounds [39]. In particular, the existence of a structure–potential relationship is crucial for the well-tuned design of new complexes useful in catalytic processes and for better understanding the role of metallodrugs in biological applications [40]. In this perspective, we investigated the redox properties of complexes 1 and 3 (Structure 1) in the attempt to correlate their different biological activities with their electrochemical characteristics. The reduction of the complexes (Table 2) was studied by CV experiments in THF/0.2 M n-Bu4NBF4 in the range of potential scan rates 0.1 \ v \ 200 V s-1, and the cyclic voltammograms obtained at 0.5 V s-1 are reported in Fig. 1. Electrochemical reduction of 1 The voltammetric reduction of 1 at 0.5 V s-1 (Fig. 1a) displays a wave (R1) in the Ep range from -1.97 to -2.21 V versus the SCE depending on the potential scan rates (v = 0.05–200 V s-1) which is chemically irreversible over all the potential scan rates investigated. The peak current of the reduction wave is not proportional to v1/2 as would be expected for a simple reversible process [41]. The value of the current function, i.e., the apparent number of
Table 2 Electrochemical data Complex
Ep1 (V)
1
-2.02 -1.63
3 Table 1 Kinetic data for the dynamic process obtained from exchange spectroscopy measurements
?
I III?
-1.13 -1.10
Ep2 (V) – -1.92 – -1.92
D 9 106b (cm2 s-1)
nappa 2.05 ± 0.15c
8.0 ± 0.5c
d
8.5 ± 0.9d
e
4.5 ± 0.3e 4.6 ± 0.3f
1.00 ± 0.10
1.95 ± 0.10 0.95 ± 0.10f
Complex
T (K)
k (s-1)
DH= (kJ mol-1)
DS= (J mol-1 K-1)
1
298
0.19 ± 0.01
37 ± 5
-132 ± 16
308
0.40 ± 0.02
318
0.62 ± 0.03
328
0.83 ± 0.04
298
0.03 ± 0.01
b
Diffusion coefficient [Dferrocene = (1.3 ± 0.2) 9 10-5 cm2 s-1]
308
0.13 ± 0.01
c
Wave R1 in Fig. 1a
318 328
0.35 ± 0.02 0.75 ± 0.04
d
Wave R1 in Fig. 1c Wave r1 in Fig. 1a
3
a Absolute electron number for the characteristic time h = 0.2 s; v B RT/Fh (see the electronic supplementary material)
83 ± 7
See Structure 1 for the structures of the complexes
123
The solvent was tetrahydrofuran, scan rate 0.5 V s-1; all potentials are in volts relative to a standard calomel electrode; T = 293 K. Supporting electrolyte 0.2 M n-Bu4NBF4. Digital simulations of the cyclic voltammograms according to the proposed mechanism were performed with the program Antigona (see ‘‘Materials and methods’’)
6 ± 11
e f
Wave r1 in Fig. 1c
J Biol Inorg Chem (2011) 16:695–713
703
(a)
R1
1 + I
r1
o1 O2
O3
O1
0
-1
-2
Potential (V vs SCE)
(b) +
-1
I v = 5 Vs + -1 I v = 0.5 Vs
0
-1
-2
Potential (V vs SCE)
(c)
the potential scan rate displays a slope of d(Ep)/d(log v) = 60 mV, whereas the half-peak width is (Ep - Ep/2) = 100 mV in the scan rate range. These values allow the electron transfer coefficient a = 0.48 (T = 293 K) to be estimated by means of Eqs. 1 and 2, which describe the variation of the peak potential and the half-peak width for a totally irreversible electron transfer (na = 1) (see the electronic supplementary material) [41]: Ep ¼ ð1:15RT=ana F Þlog v þ constant, ð1Þ Ep Ep=2 ¼ 1:857RT=aF:
In the general case in which the electron transfer is Nernstian at low scan rate and irreversible at high scan rate, the current function normalized to its value at the highest scan rate is expected to reach the maximum factor of 0.446/ 0.495a1/2 [41]. In the case of 1, the calculated factor is 1.3, much lower than the observed value of 1.9, indicating that the kinetics of the electron transfer alone does not justify the variation of the current function. These data together with the determination of the absolute electron stoichiometry [24, 42] (Table 2, napp = 2.05 at scan rate v \ 0.2 V s-1) indicate that at sufficiently low scan rate the reduction of 1 is a two-electron process and it becomes a one-electron process at high scan rate as expected for an E1CE2 mechanism with E01 E02 (Eqs. 3–5): [PtCl(η1-C9H7)L2]
3 (exp) 3 (calcd) + III (exp) + III (calcd)
R2
[PtCl(η -C9H7)L2] 1
3
R1
[Pt(η -C9H7)L2]
r2
ð2Þ
•
+
[PtCl(η1-C9H7)L2] •
e−
[PtCl(η -C9H7)L2]
•
3
+
-
e
[Pt(η -C9H7)L2] 3
(3) •
[Pt(η -C9H7)L2] 3
•
−
−
+ Cl
(4) (5)
1: L2=dppe
r1
o2 0
-1
-2
Potential (V vs SCE) Fig. 1 Experimental cyclic voltammograms of a 3 mM 1 (black line) and 3 mM I? (red line) in tetrahydrofuran (THF)/0.2 mM n-Bu4NBF4 (T = 293 K, scan rate 0.5 V s-1) at a 0.5-mm-diameter gold disk electrode and of b 3 mM I? at a 0.125-mm-diameter gold disk electrode (scan rate 5 V s-1) (red line) and at a 0.5-mm-diameter gold disk electrode (scan rate 0.5 V s-1) (black dash-dotted line). c Experimental (line) and calculated (circles) cyclic voltammograms in THF/0.2 mM n-Bu4NBF4 at a 0.5-mm-diameter gold disk electrode (T = 293 K, scan rate 0.5 V s-1) of 3 mM 3 (black line) and 3 mM III? (red line). SCE standard calomel electrode
electrons consumed, napp ¼ ðip v1=2 Þ=ðip v1=2 Þ1 ; decreases upon increasing the potential scan rate and tends to 1 for v [ 200 V s-1. For v [ 5 V s-1, the plot of Ep versus
The first reduction step (Eq. 3) produces the radical anion 1-, which undergoes a chemical reaction (Eq. 4) giving a species which is reduced at more positive potential than 1 (Eq. 5). After potential scan reversal at v = 0.5 V s-1, the reduction is followed by the appearance of the anodic waves O1 (-0.90 V), O2 (-0.46 V), and O3 (0.26 V), which arise from the oxidation of reduction products. When the scan rate is increased, the intensity of wave O1 is almost the same, whereas the intensities of waves O2 and O3 decrease, indicating that the corresponding species arise from the decomposition of the intermediate which corresponds to wave O1. To assess the reduction mechanism of 1, to characterize the nature of the reaction C, and, consequently, to attribute the oxidation waves O1–O3, we compared the cyclic voltammogram of 1 with the cyclic voltammograms of authentic samples of I?, obtained by abstraction of Clwith AgPF6. In fact, as previously reported for the related complex [RuCl(g5-C9H7)(dppe)] [43], the reduction of metal–halide complexes usually induces the cleavage of the M–X bond. Moreover, in indenyl complexes the energy
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barrier between the 19-electron and 17-electron radical intermediates, accompanied by variation of the coordination mode, is generally very low [44–49]. As shown in Fig. 1b, at higher potential scan rates (v = 5 V s-1) the reduction of I? generates a partially reversible wave r1 in the Ep range from -1.10 to -1.28 V versus the SCE. The constant value of the apparent number of electrons consumed napp ¼ ip v1=2 = ip v1=2 1 , the result of the determination of the absolute electron stoichiometry [24, 42] (napp = 1.95 ± 0.10 at scan rate v B 0.1 V s-1), and the slope of the plot of Ep versus the potential scan rate [d(Ep)/d(log v) = 30 mV] (see the electronic supplementary material) are in favor of a twoelectron reversible process [41] at low scan rate giving rise to the anion [Pt(g3-C9H7)(dppe)]- (I-). These results allow us to suggest the formation of the g3-17-electron radical [Pt(g3-C9H7)(dppe)] (I) as the species responsible for the second electron transfer at more positive potential than that of the neutral complex 1. Scheme 1a depicts the proposed overall mechanism for the reduction of 1 and I?.
(a)
As shown in Eq. 4 and Scheme 1, the Pt–Cl bond cleavage does not explain by itself the behavior of the voltammetric reduction of 1. In fact, on the basis of the observed chemical irreversibility of wave R1 up to the scan rate investigated (v = 200 V s-1), a very fast reaction such as the g1–g3 coordinative rearrangements in Eq. 4 must precede the rate-determining chemical step corresponding to the chloride dissociation from [PtCl(g3-C9H7)(dppe)]-. At high scan rate, when wave R1 is monoelectronic and still irreversible, wave O1 is still present. This indicates that it corresponds to the oxidation of the 19-electron species [PtCl(g3-C9H7)(dppe)]-, which regenerates the starting complex 1. After scan reversal at v = 0.5 V s-1, only oxidation wave O1 at 0.32 V is present when starting with I? and it can be ascribed to the oxidation of species arising from rearrangement or degradation of I-. However, this wave is absent in the anodic scan reversal after reduction of 1. This finding is justified by the presence in solution of Cl- that participates in the back reaction of the second reversible
(b) Reduction 1
Reduction 1 + e-
+ ePh
Ph P
Cl
Pt P 1
Ph
Ph Ph
Ph P
Cl
Pt P
Pt
Ph Ph
1.
Cl
Cl
Pt
3.
3
Cl Cl
Pt Ph P Ph
P
Pt
Ph Ph
- Cl-
- Cl-
Reduction 2 -
+e
+e Pt Ph P Ph
I+
P
Ph Ph
Ph Ph P
I.
Pt P
Ph Ph
Reduction 2 -
-
+e
Pt Ph P Ph
I-
P
Ph Ph
Pt
Pt
III+
III.
2 + 2e-
Pt
Pt
2
2
III2-
Scheme 1 Proposed mechanism for the reduction of 1 and I? (left), 3 and III? (right)
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705
step (Eq. 4), that the radical anion [PtCl(g3-C9H7)(dppe)]is restored. Finally, wave O2 matches the oxidation wave of the indenyl anion as demonstrated by comparison with an authentic sample of indenyl potassium and previously observed in the same experimental conditions for the complexes [Ru(g5-C9H7)Cl(PPh3)2] [43] and [Rh(g5C9H7)(cod)] [45]. This evidence indicates that PtCl(g3C9H7)(dppe)]- undergoes partial degradation producing the indenyl anion (O2) and the fragment Pt(dppe) (O3). Electrochemical reduction of 3 The voltammetric reduction of 3 (Fig. 1c) displays cathodic characteristics completely different from those found for the dppe derivative 1. In fact, at 0.5 V s-1 it displays two waves R1 (-1.63 V) and R2 (-1.92 V). Wave R1 is chemically irreversible and the apparent number of elec trons consumed, napp ¼ ip v1=2 = ip v1=2 1 , remains constant over all the range of scan rates investigated (see the electronic supplementary material). These data, together with the determination of the absolute electron stoichiometry [24, 42] (napp = 1.00 ± 0.10 at scan rate v \ 0.2 V s-1), indicate that the first reduction of 3 is a one-electron process (Eq. 3, L2 is cod). Conversely, the current function of wave R2 decreases as the scan rate increases, and disappears at scan rate v C 1,000 V s-1; moreover, the ip value increases as the concentration of 3 increases. Finally, the reduction wave observed after consumption of 1 faraday per mole at the controlled potential of the first wave matches exactly the second wave R2 of 3 (see the electronic supplementary material). Taken as a whole, these results are clear evidence for an EC reduction mechanism in which a one-electron transfer is followed by a second-order homogeneous reaction (Eqs. 3, 6, and 7). [PtCl( 1-C9H7)L2]
[PtCl( 3-C9H7)L2]
5
5
2 [Pt( -C9H7)L2]
[Pt( 5-C9H7)L2]2
[Pt( 5-C9H7)L2]
[Pt( -C9H7)L2]2
+ 2e−
+ Cl−
(6) (7)
{[Pt( 5-C9H7)L2] 2}2−
(8)
3: L2=cod
Interestingly, wave R2 at -1.92 V is present in the cathodic scan of [Pt(g-C9H7)(cod)]? (III?), also obtained from 3 by abstraction of Cl- with AgPF6, after the primary monoelectronic reduction (R1) (Fig. 1c). The high potential difference between the two waves of III? allows the appearance of the anodic counterpart (O2) of the second cathodic process, revealing its chemically reversible nature. The intensity of the return peak is lower than that of the reduction peak since the electron transfer coefficient for the reoxidation process is much smaller (aox = 1 - ared = 0.2) than that of the reduction process (ared = 0.8, determined by
digital simulation). In fact, the smaller the electron transfer coefficient is, the more stretched the wave is [41]. In conclusion, as postulated for the reduction of the dppe derivative, the Pt–Cl bond cleavage occurs preceded by a fast reaction, the latter accounting of the irreversibility of the first wave (Eq. 6). However, in contrast to what is observed with dppe, the radical [Pt(g5-C9H7)(cod)] formed is not prone to the second reduction but undergoes a second-order reaction with respect to the starting substrate. The digital simulation of the available data allowed us to qualitatively assess the suggested reduction mechanism. The results of the calculations revealed that the chemical reaction (Eq. 7) is the bimolecular dimerization of the radical [Pt(g5-C9H7)(cod)] with rate constant kdim = 7 9 105 M-1 s-1 and that the dimer is reduced at higher potential in a two-electron process (Eq. 8). The overall mechanism for the reduction of 3 and III? is illustrated in Scheme 1b. The competition between a homogeneous reaction and a heterogeneous electron transfer process is always in favor of the latter when the potential of the radical reduction is more positive than that of the reaction product (i.e., the dimer). This implies that [Pt(g5-C9H7)(cod)] is reduced at more negative potential than the corresponding dppe intermediate, in apparent contrast with the higher p-electron-donor nature of the diphosphine. However, from analysis of the reduction potential difference between 1 and 3 reported in Fig. 1, it appears that the electron donor difference between dppe and cod is -390 mV, a value very close to that found for the first reduction of 2 and 4 (-410 mV), but much more negative than that found between I? and III? (-30 mV). The reason resides in the relationship between the redox potential and the structure of the complexes. In both neutral 1 and 3, platinum exhibits an g1 hapticity affording a 16-electron metal coordination sphere, so the higher potential reduction of 1 is mainly due to the better donor properties of dppe than those of cod. Conversely, I? and III? are reduced at very similar potentials in spite of the different nature of their ancillary ligands, indicating that the cations possess a different indenyl– platinum bonding mode. In fact, the electrochemical data, together with the electronic properties of cod and dppe, indicate that III? is a 18-electron [Pt(g5-C9H7)(cod)]? species, whereas in I? the coordination slips toward the g3-allyl, 16-electron species [Pt(g3-C9H7)(dppe)]?, so the reduction is anticipated by approximately 360 mV. Similar structural features should be present in the corresponding radical species [Pt(g3-C9H7)(dppe)] (I) and [Pt(g5-C9H7)(cod)], in agreement with the different reduction mechanism. In fact, [Pt(g3-C9H7)(dppe)] is more easily reduced at the same potential as the starting complex 1 in an overall two-electron reduction. In contrast, [Pt(g5C9H7)(cod)] is not reduced in the potential window scanned and dimerizes, indeed giving rise to a species which is reduced at more negative potential (less than -2.3 V).
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Biological activity The capacity of 1–4 to inhibit cell growth was evaluated on three human tumor cell lines: HeLa (cervix adenocarcinoma), JR8 (scalp melanoma), and HL-60 (promyelocytic leukaemia). After incubation of the cells for 72 h in the presence of test derivatives, we determined the IC50 value, i.e., the concentration (lM) of compound able to cause 50% of cell death with respect to the control culture. The well-known drug cisplatin was tested in the same experimental conditions and was used as a reference compound. The data obtained are reported in Table 3. The indenyl derivatives 1 and 3 show the capacity to inhibit cell growth, whereas for 2 and 4, characterized by the presence of a chloride instead of the indenyl substituent, no effect was evidenced. In detail, compound 1 exerts a notable antiproliferative activity and, indeed, it shows IC50 values comparable to those obtained for the reference drug on all cell lines taken into account. Complex 3 appears to have some cytotoxicity on both JR8 and HL60 cells, but this effect is more than 1 order of magnitude lower than that of 1. On the other hand, 3 appears unable to exert any effect on HeLa cells up to 50 lM concentration. These overall results point out that the indenyl moiety is an essential requirement for the occurrence of the antiproliferative capacity, to which the dppe substituent contributes more significantly than the cod substituent. Interaction with DNA The ability of 1 and 3 to exert antiproliferative activity on cells prompted us to investigate the intracellular target responsible for the cytotoxicity of these derivatives. It was already well established that DNA constitutes the biological target of platinum-based drugs [50] and, furthermore, nuclear chromatin was postulated as a cellular target for a lipophilic phosphine-coordinated gold complex
Table 3 Cell growth inhibition in the presence of test compounds and cisplatin as a reference drug Compound
IC50 (lM)a of cell lines HeLa
JR8
HL-60
1
1.70 ± 0.30
0.73 ± 0.12
0.47 ± 0.06
2
[50
[50
[50
3
[50
32.8 ± 3.4
7.5 ± 0.3
4
[50
[50
[50
Cisplatin
0.84 ± 0.06
0.73 ± 0.10
1.60 ± 0.08
a
The mean values ± the standard deviation of at least four experiments are reported
123
[7]; thus, the macromolecule could also participate in the biological mechanisms of 1 and 3. In this connection, the observation that for these derivatives the planar moiety of indenyl plays a crucial role in the antiproliferative activity (Table 3) suggested the possibility that a complexation through an intercalative mode of binding with DNA could take place. To verify this, flow linear dichroism experiments in the presence of 1 and 3 were performed. Nevertheless, in the presence of test compounds no linear-dichroism-induced signal was evidenced (see Fig. S5a). This behavior indicates that 1 and 3 do not give rise to a molecular complex with the macromolecule and excludes a contribution of the planar indenyl group to the antiproliferative effect through an intercalative mode of binding between base pairs. It has already been demonstrated that the binding of some platinum drugs to DNA induces changes in circular dichroism spectra, which are indicative of conformational alterations [51]. The presence of 1 or 3 does not cause any significant variation in the circular dichroism spectrum of DNA (see Fig. S5b), thus indicating that 1 and 3 do not induce detectable conformational changes in the macromolecule. Nevertheless, the possibility that derivatives 1 and 3, similarly to cisplatin, coordinate to DNA was investigated. For this purpose, we determined DNA platination by ICPAES and, in particular, the evolution of DNA complexation of 1 and 3 was studied at different incubation times, by analyzing the total amount of phosphorus and of platinum bound to DNA, after exposure to test derivatives. The analysis of phosphorus allowed us to determine, in accordance with previous studies and using Eq. 9, the number of possible coordination sites provided by each DNA double strand [52, 53]: ½P ½P ½TS0 ¼ lim ½DS ¼ 1 ð9Þ 2 ½DS!1 2 where [TS]0 is the total coordination site concentration, [P] is the total average phosphorus concentration, and [DS] is the concentration of the double strands. The ratio [Pt]b/[TS]0 between the concentration of the platinum bound per double strand, [Pt]b, and the total number of coordination sites present per double strand, [TS]0, corresponds to the number of platinum derivatives coordinated per total number of coordination sites of DNA double strand. Figure 2 shows the dependence on time of [Pt]b/[TS]0 for 1, 3, and cisplatin. The reactivity toward double strands decreases in the order cisplatin [ 3 [ 1. In addition, the data shown in Fig. 2 indicate that the binding of 1 and 3 occurs in two steps. The first process takes place at t \ 80 9 103 s, whereas the second occurs at t [ 80 9 103 s. It has been suggested that the binding of ligands to DNA owing to the jth reaction event, with overall rate constant
J Biol Inorg Chem (2011) 16:695–713
707
The fitting parameters determined by Eq. 10 are reported in Table 4. In particular, it should be noted that, in contrast to cisplatin, for 1 and 3 a two-step reaction is observed. Moreover, the maximum concentration of 1 and 3 bonded per unit concentration of DNA coordination sites ½Ptmax b =½TS0 is less than a half that of cisplatin. In detail, for derivative 1, ½Ptmax b =½TS0 is 48% for step 1 and 52% for step 2. Furthermore, for 1, 3, and cisplatin, . ½TS0 has increasing values in the order 48% \ ½Ptmax b;1 . 85% \ 100%, respectively, whereas in 1 and 3, ½Ptmax b;2
100
[Pt]b/[TS]0 x10-3
80 cisplatin 1 3 fit
60
40
20
0
0
40
80
120
160
3
Time / s x10
Fig. 2 Binding of platinum in 1, 3, and cisplatin to the coordination sites (S) of DNA as a function of incubation time
obs kj;T ;
is the result of a number of elementary ith parallel binding processes, which depend on the type of ith site present in the nucleic acid, the frequency of the appearance of each ith site along the chain (fi), the binding constant associated with each interaction process (Ki), and the firstorder reaction rate constant (ki) associated with each type of site [52, 53]. In this connection, the curves in Fig. 2 were satisfactorily fitted using the following equation: 2 n o X ½Ptb ½Ptmax b ¼ bj 1 ent ; ð10Þ j ½TS0 ½TS0 j¼1 where [Pt]b/[TS]0 is the total concentration of the platinum derivative that has undergone coordination reaction with double-helix DNA owing to two different (jth) reaction max events at time t. bj ¼ ½Ptmax is the maximum b;j =½Pt b fraction of the platinum derivative which reacted in both P obs jth events, and nj ðs1 Þ ¼ ½TS0 ni¼1 fi Ki;j ji;j ¼ ½TS0 kj;T is the overall experimental rate constant for the jth reaction event of the ligand with the substrate. ½Ptmax is the total b concentration of DNA sites coordinated by platinum ½Ptmax
b derivatives. Finally, ½Ptmax b;j =½TS0 ¼ bj ½TS . 0
½TS0 exhibits decreasing values in the order 52% [ 15%, respectively. As regards the overall experimental binding constant, nj(j = 1, 2) of the jth reaction pathway, both n1 (s-1) and n2 (s-1) of 1 are about 10 times lower than those of 3. This result is in accordance with the higher intrinsic reactivity of 3 with respect to 1, which is responsible for the higher . max ½Ptmax ½TS. b;1 =½TS and also for the low ½Pt b;2 This evidence indicates that the reaction mechanism responsible for the biological activity exhibited by 1 and 3 is different from that of cisplatin, which involves coordination reactions and suggest that binding step 2 is likely crucial for the antiproliferative activity of the new platinum complexes. In summary, we concluded that (1) the antiproliferative activity in this case does not seem to be dependent on events involving binding to DNA and (2) the binding studies strongly suggest that a molecular target different from DNA could be responsible for the antiproliferative effect exerted by 1. Effect on p53 induction To support the previous hypothesis, we determined the effect of 1 on p53 expression in HeLa cells. The p53 protein can induce several responses in cells, including cell cycle arrest and apoptosis, as a consequence of the occurrence of many types of stress, including DNA damage. In particular, we employed Western blot analysis to examine the p53 status after treatment of cells with 1 and cisplatin, used as a reference compound. Figure 3 illustrates that the treatment of HeLa cells with 1 induces a practically negligible effect on p53 expression, whereas, as
Table 4 Parameters for the binding reaction of 1, 3, and cisplatin with DNA Compound
a ½Ptmax b =½TS0
1
2.93 9 10-2 ± 9 9 10-4 -2
2.71 9 10
3 Cisplatin a
-2
7.77 9 10
½Ptmax b;1 =½TS0
-4
± 7 9 10
-3
± 2 9 10
1.40 9 10-2 ± 6.5 9 10-4 -2
2.30 9 10
–
½Ptmax b;2 =½TS0
n1 (s-1)
-4
± 7.5 9 10
5.75 9 10-5 ± 6 9 10-6 -4
1.20 9 10
-5
5.50 9 10
± 1.5 9 10
-5
± 4.6 9 10
-6
n2 (s-1)
1.53 9 10-2 ± 3 9 10-4 -3
4.06 9 10
± 1 9 10
–
-4
5.60 9 10-5 ± 1 9 10-6 5.57 9 10-4 ± 2 9 10-5 5.50 9 10-5 ± 4.6 9 10-6
Maximum concentration of platinum bonded to the complexing sites of DNA ([TS]0)
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1
cisplatin
p53
β-actin
Fig. 3 Effect of 1 and cisplatin on p53 expression in HeLa cells. When present, 50 lM 1 and 50 lM cisplatin
expected, cisplatin provokes an evident increase in p53 level. Effect on mitochondria Mitochondria are cellular organelles which play a central role in the apoptosis process by releasing some apoptogenic signal molecules, such as AIF and cytochrome c [54]. An important process that induces mitochondria to release these proapoptotic factors into the cytosol is the occurrence of the MPT. This phenomenon is regulated by the opening of a large nonselective channel known as the permeability transition pore. Opening of this pore provokes the dissipation of the inner transmembrane potential (DW), matrix swelling, and outer membrane disruption with the consequent release of AIF and cytochrome c, which trigger the caspase-independent and caspase-dependent pathways, respectively [55]. The induction of the MPT is a phenomenon strictly linked to the redox state of mitochondria and is often recognized as the result of an oxidative stress which occurs in these organelles. In this regard, the subsequent experiments were performed on RLM to verify if the most active compound, 1, can be involved in the opening of the transition pore. The results reported in Fig. 4a show that RLM, energized by succinate oxidation in the presence of rotenone, if incubated in standard medium with compound 1, undergo an apparent decrease in the absorbance of the suspension, of about 1 U, in 20 min of incubation, indicative of the occurrence of matrix colloid-osmotic swelling. In the absence of Ca2?, 1 is completely ineffective. It is noteworthy that 2 and 4, at 10 lM concentration, in the presence of Ca2?, are ineffective in inducing mitochondrial swelling. Cisplatin does not exhibit a significant effect. If RLM are incubated with 1 in the presence of a typical MPT inhibitor (CsA or bongkrekic acid) or with the Ca2? chelator EGTA or the alkylant N-ethylmaleimide (NEM), mitochondrial swelling is almost completely prevented, suggesting that it is the result of the transition pore opening. The observation that NEM strongly inhibits the swelling raises the hypothesis that the effect of 1 is linked to an oxidative stress taking place in the presence of Ca2?. As
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can be seen in the histogram in Fig. 4b, Ca2? (control) or 1 in the absence of Ca2? is able to induce a decrease in the number of reduced thiols of about 13 and 8%, respectively. In contrast, 1 and Ca2? together, further decrease the number of reduced thiols by about 30%. A more accentuated effect of 1 and Ca2?, alone or together, is apparent at the level of the redox state of glutathione. In fact, Ca2? and 1, each alone, decrease the glutathione content by about 15.5 and 8.5%, respectively, whereas when they are present together, the glutathione content is reduced by about 35.5%. The results reported in the immunoblot in Fig. 5 show the correlation between the induction of the MPT by 1 and its potential effect as a proapoptotic agent by the release of cytochrome c and AIF. As evident in the immunoblot 10 lM 1, in the presence of Ca2?, is able to induce a strong efflux of cytochrome c (lane d), much higher than that noticed in the presence of Ca2? alone (lane c). With 20 lM 1, the release of cytochrome c in the presence of Ca2? is further increased (lane e), whereas the presence of CsA strongly inhibits this effect (lane f). Similar results can be observed in the immunoblot showing AIF efflux. In conclusion, the release of the proapoptotic factors cytochrome c and AIF induced by 1, in the presence of Ca2?, was consistent with the activation of both the caspasedependent and the caspase-independent signaling pathways, potentially leading to apoptosis. This was confirmed by the significant inhibition of both factors exhibited by CsA (lane f). The observed release of cytochrome c, by Ca2? alone, is most likely due to an alteration of the phospholipid architecture of the outer membrane which breaks the network of electrostatic bindings which normally stabilizes cytochrome c in the intermembrane space and favors its release. However, as also previously reported [56], the amount of cytochrome c released by Ca2? is not sufficient to trigger the apoptotic pathway. It should be noted that 1, alone, does not induce any amount of proapoptotic factor. Evaluation of mitochondrial membrane potential on HeLa cells To obtain information on the mitochondrial activity, a flow-cytometric study was carried out using the fluorescent lipophilic cationic probe TMRM (Fig. 6). This probe, bearing a delocalized positive charge, enters the negatively charged mitochondria, where it accumulates in an innermembrane potential-dependent manner. When mitochondria undergo a depolarization event, TMRM no longer accumulates inside the mitochondria and becomes distributed throughout the cytosol. When it is dispersed in this manner, overall cellular fluorescence levels drop dramatically and this event can easily be visualized by
J Biol Inorg Chem (2011) 16:695–713
(a) -Ca2+ 1+ CsA / BKA / EGTA / NEM control 2 or 4 cisplatin
control or 1 alone
(mV)
180
A 540 0.2
10 mV 150
4 min
2 min
100
1
10
(b) glutathione and reduced thiol groups (%)
Fig. 4 Effects of 1, 2, 4, and cisplatin on mitochondrial swelling (a). All incubations were carried out with standard incubation procedures in the presence of 20 lM Ca2?, except where indicated otherwise (-Ca2?). When present, 10 lM 1,10 lM 2, 10 lM 4, 10 lM cisplatin, 1 lM cyclosporin A (CsA), 10 lM N-ethylmaleimide (NEM), 5 lM bongkrekic acid (BKA), and 1 mM ethylene glycol bis(2-aminoethyl ether)N,N,N0 ,N0 -tetraacetic acid (EGTA). The assays were performed at least four times with similar results. A downward deflection indicates absorbance decrease. The inset shows the effect of 1 alone or in the presence of Ca2? on electrical potential (DW). DE is the electrode potential. b Oxidation of mitochondrial glutathione (black bars) and thiol groups (gray bars) induced by 1. Experimental conditions as in a. The data are expressed as a percentage of the glutathione or thiol reduction, and represent the mean ± the standard deviation (SD) from four independent experiments
709
100
80
60
40
20
1
)
2+
a
co n
a
b
20 μ M Ca2+
−
+
+
+
+
+
10 μ M 1
−
−
−
+
−
−
20 μ M 1
−
−
−
−
+
+
1 μ M CsA
−
+
−
−
−
+
1
(-C
-C
a
tro
l
2+
0
Fig. 5 Release of cytochrome c (cyt c) and apoptosis-inducing factor (AIF) induced by 1 in the presence of Ca2? and CsA. The result of the Western blotting of the supernatant fraction is shown. Rat liver mitochondria were incubated with standard incubation procedures
fluorescence microscopy or quantitated by flow cytometry. Treatment with 15 and 30 lM 1 for 24 h clearly induced an evident increase of cell population with reduced
c
d
e
f
fluorescence (from 8.4% in control samples to more than 80% in treated ones), suggesting a depolarization of mitochondrial membrane.
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(a)
(a)
0.2%
4.6%
90.3%
4.8%
FL2-PI
n. cells
8.4%
FL3-TMRM FL1-annexin V-FITC
(b) (b)
FL2-PI
n. cells
82.5%
1.1%
34.0%
27.9%
37.0%
FL3-TMRM
(c) 97.7%
FL1-annexin V-FITC
FL2-PI
n. cells
(c)
FL3-TMRM Fig. 6 Mitochondrial membrane potential assessed by flow cytometry after tetramethylrhodamine methyl ester (TMRM) staining. The results are shown as fluorescence histograms (TMRM signal on the xaxis). a Control HeLa cells, b cells treated with 15 lM, and c cells treated with 30 lM 1. The numbers reported represent the percentages of cells with depolarized mitochondrial membrane. A representative experiment of four experiments is shown
Analysis of apoptosis by annexin V-FITC staining in HeLa cells Apoptosis induction in HeLa cells, after treatment with 1 in DMSO, was evaluated by flow cytometry after annexin V–FITC and PI labeling (Fig. 7), which allows one to distinguish early apoptotic cells (annexin V-positive/PInegative) from late ones (annexin V-positive/PI-positive) and dead cells (PI-positive). Treatment with 15 lM 1 for 24 h caused an increase of both early (from 4.8 to 37%)
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1.7%
87.5%
7.9%
2.9%
FL1-annexin V-FITC Fig. 7 Analysis of apoptosis by flow cytometry after annexin V– fluorescein isothiocyanate (FITC) staining. The data are presented as dot plots [annexin fluorescence on the x-axis; propidium iodide (PI) fluorescence on the y-axis]. a Control Hela cells, b cells treated with 15 lM 1, c cells treated and with 30 lM 1. The numbers shown in the four quadrants represent the percentage of viable (lower left), necrotic (upper left), early apoptotic (lower right), and late apoptotic (upper right) cells. A representative experiment of four experiments is shown
and late (from 4.6 to 34%) apoptotic populations. Higher doses of 1 (30 lM) induced a greater cytotoxic effect, as demonstrated by the increase of the late apoptotic fraction after 24 h treatment (87.5%).
J Biol Inorg Chem (2011) 16:695–713
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cell number (%)
100
80
60
40
20
sA C
1 + 1
co nt ro
co nt ro l l+ C sA
0
Fig. 8 Effects of CsA on cell growth inhibition induced by 1. HeLa cells were incubated for 72 h in the absence or in the presence of 1.5 lM 1 without pretreatment (black bars) and after pretreatment with 2 lM CsA for 30 min (white bars). Values are the mean ± SD of four independent experiments
Effect of CsA on HeLa cells A cell inhibition growth assay in the presence of CsA was performed to correlate the mitochondrial effect of 1 with the apoptotic induction on whole cells. The histograms in Fig. 8 report the results expressed as a percentage of the number of viable cells with respect to untreated culture. In detail, incubation of HeLa cells for 72 h in the presence of 1.5 lM 1 induces, as expected, a decrease of about 37% in cell number (black bars), whereas after pretreatment for 30 min with CsA, 1.5 lM 1 induces a decrease of only about 20% (white bars). The CsA inhibition of the antiproliferative effect induced by 1 supports the hypothesis that mitochondria constitute the cellular target for the cytotoxic effect and that the opening of the transition pore is the critical step for the cell death signaling.
Conclusions The antiproliferative activity of a series of platinum complexes of general formula [PtCl(g1-C9H7)L2] (L2 is dppe 1 or L2 is cod 3) and [PtCl2L2] (L2 is dppe 2 or L2 is cod 4) on three human tumor cell lines has been evaluated. Compound 1 exhibited a very interesting effect, comparable to that of cisplatin. Also, compound 3 showed cytotoxicity, but it was significantly lower with respect to that of 1, whereas 2 and 4 appeared to be ineffective. The intrinsic structural difference in solution of 1 and 3 is strongly supported by NMR measurements. Similarly to g1-allyl metal systems, g1-indenyl complexes typically
undergo an g1–g3–g1 exchange process taking place through a bonding situation in which the g3 coordination prevents the unstable 20-electron g5 coordination. EXSY experiments allowed us to determine the value of the rate constants k and of the activation barrier for 1 and 3. kdppe is significantly higher than kcod at the temperature where the biological activity was checked and DH= is much lower for 1, indicating that the intermediate allyl structure is energetically more accessible in 1 than in 3. Concerning the redox properties as arising from the electrochemical characterization, 1 undergoes two monoelectronic reduction steps, whereas 3, after a single monoelectronic reduction, rapidly dimerizes to form a stable, sterically hindered and chemically reversible species which prevents the occurrence of the second electron transfer. The values of the reduction potentials of [Pt(g3C9H7)dppe]? (I?) and [Pt(g3-C9H7)cod]? (III?) and the proposed reduction mechanism of 1 and 3 clearly indicate that the g3-allyl structure of the key intermediate [Pt(g3C9H7)(dppe)] (I) makes the second electron transfer viable. Conversely, [Pt(g5-C9H7)(cod)] dimerizes, forming a biologically inactive species. The study of the interaction of 1 and 3 with DNA demonstrated for these indenyl complexes a weak ability to react with the macromolecule, which reasonably cannot account for the notable antiproliferative effect exerted by 1. This conclusion is also supported by the low expression level of p53 in HeLa cells after treatment with 1. The reported results of the effects of 1 at the mitochondrial level clearly demonstrate that RLM undergo the phenomenon of MPT. In fact, the results reported in Fig. 4a show that 1 behaves like a typical MPT inducer: the induction of a colloid-osmotic swelling and its inhibition by typical inhibitors of MPT such as CsA, bongkrekic acid, EGTA, and NEM are confirmation that the transition pore opening is the result of the action of 1. The additional observation that NEM inhibits swelling supports the hypothesis that the MPT may be the result of an oxidative stress induced by the compound. This is clearly confirmed by the results in Fig. 4b showing the oxidation of sulfhydryl groups and glutathione provoked by 1 and Ca2?, incubated alone or together. As is well known, for the induction of the MPT it is necessary, first of all, that Ca2?, at supraphysiological concentrations, interacts with a critical site localized on adenine nucleotide translocase [57]. Indeed, two critical thiol groups belonging to two cysteines must be oxidized to form a disulfide bridge [58]. The occurrence of only one of these events does not open the pore. The observation that Ca2? alone, besides interacting with adenine nucleotide translocase, is also able to oxidize 13% of thiols (Fig. 4b) without opening the pore (Fig. 4a) suggests that in this condition the critical cysteines remain reduced. Compound 1, alone, oxidized 8% of sulfhydryl
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groups, five percentage points less than Ca2? (Fig. 4b); however, also in this case the pore remains closed. In conclusion, the pore opens only when the drug is present together Ca2?, that is, when the above-mentioned events take place concomitantly. Further studies are under way to check the hypothesis that apoptosis via the mitochondrial pathway takes place owing to intracellular redox reaction and reactive oxygen species generation by 1. The release of cytochrome c and AIF by 1 in the presence of Ca2? indicates the activation of the caspasedependent and the caspase-independent pathways, respectively, leading to apoptosis. These observations indicate that 1 may be considered as a proapoptotic agent. This possibility is demonstrated by phosphatidylserine exposure on the outer surface of the cytoplasmic membrane clearly showed the onset of the apoptotic process. In addition, the mitochondrial membrane polarization determined using the fluorescent lipophilic cationic probe TMRM showed the percentages of cells with depolarized mitochondrial membrane and conclusively demonstrates that 1 induces apoptosis through the mitochondrial pathway. Finally, the mechanism of action of 1, unusual within the field of platinum-based drugs, along with the noteworthy antiproliferative activity, makes this platinum chloride indenyl complex an interesting lead for the development of new organometallic anticancer drugs. Acknowledgments Massimiliano Imhoff and Annalisa Bisello are gratefully acknowledged for their expert assistance in the synthesis and electrochemical measurements.
References 1. Abu-Surrah AS, Kettunen M (2006) Curr Med Chem 13:1337–1357 2. Sanghamitra NJ, Phatak P, Das S, Samuelson AG, Somasundaram K (2005) J Med Chem 48:977–985 3. Snyder RM, Mirabelli CK, Johnson RK, Sung CM, Faucette LF, McCabe FL, Zimmerman JP, Whitman M, Hempel JC, Crooke ST (1986) Cancer Res 46:5054–5060 4. Berners-Price SJ, Mirabelli CK, Johnson RK, Mattern MR, McCabe FL, Faucette LF, Sung CM, Mong SM, Sadler PJ, Crooke ST (1986) Cancer Res 46:5486–5489 5. Berners-Price SJ, Girard GR, Hill DT, Sutton BM, Jarrett PS, Faucette LF, Johnson RK, Mirabelli CK, Sadler PJ (1990) J Med Chem 33:1386–1392 6. Berners-Price SJ, Johnson RK, Mirabelli CK, Faucette LF, McCabe FL, Sadler PJ (1987) Inorg Chem 26:3383–3387 7. Hoke GD, Rush GF, Bossard GE, James V, McArdle JV, Jensen BD, Mirabelli CK (1988) J Biol Chem 263:11203–11210 8. Lippert B (1999) Cisplatin: chemistry and biochemistry of a leading anticancer drug. Verlag Helvetica Chimica Acta, Zurich, p 563 9. Wang D, Lippard SJ (2005) Nat Rev Drug Discov 4:307–320
123
J Biol Inorg Chem (2011) 16:695–713 10. Chang SG, Jung JC, Rho YS, Huh JS, Kim JI, Hoffman RM (1996) Anticancer Res 16:3423–3428 11. Chang SG, Kim JI, Jung JC, Rho YS, Lee KT, An Z, Wang X, Hoffman RF (1997) Anticancer Res 17:3239–3242 12. Rho YS, Lee KT, Jung JC, Chang SG, Yoon C, An ZL, Hoffman RM, Chang SG (1999) Anticancer Res 19:157–161 13. Chan HL, Ma DL, Yang M, Che CM (2003) Chembiochem 4:62–68 14. Barnes KR, Kutikov A, Lippard SJ (2004) Chem Biol 11:557–564 15. Top S, Kaloun EB, Vessieres A, Leclercq G, Laios I, Ourevitch M, Deuschel C, McGlinchey MJ, Jaouen G (2003) Chembiochem 4:754–761 16. Margiotta N, Ostuni R, Ranaldo R, Denora N, Laquintana V, Trapani G, Liso G, Natile G (2007) J Med Chem 50:1019– 1027 17. Westland AD (1965) J Chem Soc 3060 18. O’Hare D (1987) Organometallics 6:1766–1772 19. Bax A, Subramian S (1986) J Magn Reson 67:565–569 20. Otting G, Wu¨thrich K (1988) J Magn Reson 76:569–574 21. Drobny G, Pines A, Sinton S, Weitekamp DP, Wemmer D (1978) Faraday Symp Chem Soc 13:49–55 22. Bax A, Summers MF (1986) J Am Chem Soc 108:2093–2094 23. Amatore C, Lefrou C, Pflu¨ger F (1989) J Electroanal Chem 270:43–59 24. Amatore C, Azzabi M, Calas P, Jutand A, Lefrou C, Rollin YJ (1990) Electroanal Chem 288:45–63 25. Speiser B (1996) In: Bard AJ, Rubinstein R (eds) Electroanalytical chemistry, a series of advances, vol 19. Marcel Dekker, Basel, pp 1–108 26. Marmur J, Doty P (1962) J Mol Biol 5:109–118 27. Wada A, Kozawa S (1964) J Polym Sci Part A 2:853–864 28. Schneider WC, Hogeboom GH (1950) J Biol Chem 183:123–128 29. Gornall AG, Bardawill CJ, David MM (1949) J Biol Chem 177:751–766 30. Santos AC, Uyemura SA, Lopes JLC, Bazon JN, Minigatto FE, Curti C (1998) Free Radic Biol Med 24:1455–1461 31. Tietze F (1969) Anal Biochem 27:502–522 32. Rasola A, Geuna M (2001) Cytometry 45:151–157 33. van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsperger CP (1998) Cytometry 31:1–9 34. Bellomo S, Ceccon A, Gambaro A, Santi S, Venzo A (1993) J Organomet Chem 453:C4–C6 35. Perrin CL, Dwyer TJ (1990) Chem Rev 90:935–967 36. Venzo A, Bisello A, Ceccon A, Manoli F, Santi S (2000) Inorg Chem Commun 3:1–4 37. Pandolfo L, Seraglia R, Venzo A, Gross S, Kickelbick G (2005) Inorg Chim Acta 358:2739–2748 38. Dwyer TJ, Norman JE, Jasien PG (1998) J Chem Educ 75:1635–1640 39. Pombeiro AJL, Amatore C (2004) Trends in molecular electrochemistry. Marcel Dekker/FontisMedia, Lausanne/New York 40. Jaouen G, Beck W, McGlinchey MJ (2006) In: Jaouen G (ed) Bioorganometallics: biomolecules, labeling, medicine. Wiley, Weinheim 41. Bard J, Faulkner LF (2001) Electrochemical methods, 2nd edn. Wiley, New York 42. Jutand A (2008) Chem Rev 108:2300–2347 43. Santi S, Broccardo L, Bassetti M, Alvarez P (2003) Organometallics 22:3478–3484 44. Stoll ME, Belanzoni P, Calhorda MJ, Drew MGB, Felix V, Geiger WE, Gamelas CA, Gonc¸alves IS, Roma˜o CC, Veiros LF (2001) J Am Chem Soc 123:10595–10606 45. Amatore C, Ceccon A, Santi S, Verpeaux JN (1997) Chem Eur J 3:279–285
J Biol Inorg Chem (2011) 16:695–713 46. Sun S, Sweigart DA (1996) In: Stone FGA, West R (eds) Advances in organometallic chemistry, vol 40. Academic Press, San Diego 47. Geiger WE (1995) Acc Chem Res 28:351–357 48. Tyler DR (1991) Acc Chem Res 24:325–331 49. Trogler WC (1990) Organometallic radical processes, vol 22. Elsevier, Amsterdam 50. Jamieson ER, Lippard SJ (1999) Chem Rev 99:2467–2498 51. Brabec V, Kleinwachter V, Butour JL, Johnson NP (1990) Biophys Chem 35:129–141 52. Di Noto V, Dalla Via L, Gia O, Mochi Onori A, Cellai L, Marciani Magno S (2000) J Phys Chem B 104:4992–4999
713 53. Dalla Via L, Di Noto V, Gia O, Marciani Magno S (2005) J Photochem Photobiol B Biol 79:59–65 54. Sartorius U, Schmitz I, Krammer PH (2001) Chembiochem 2:20–29 55. Kroemer G, Galluzzi L, Brenner C (2007) Physiol Rev 87:99–163 56. Dalla Via L, Marini AM, Salerno S, La Motta C, Condello M, Arancia G, Agostinelli E, Toninello A (2009) Bioorg Med Chem 17:326–336 57. Halestrap AP, Davidson AM (1990) Biochem J 268:153–160 58. McStay GP, Clarke SJ, Halestrap AP (2002) Biochem J 367:541–548
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