Permeabilization of the mitochondrial membrane has been extensively associated with necrotic and apoptotic cell death. Similarly to what had been previously observed for B16F10-Nex2 murine melanoma cells, PdC (palladacycle compounds) obtained from the reaction of dmpa (N,N-dimethyl-1-phenethylamine) with the dppe [1,2-ethanebis(diphenylphosphine)] were able to induce apoptosis in HTC (hepatoma, tissue culture) cells, presenting anticancer activity in vitro. To elucidate cell site-specific actions of dmpa:dppe that could respond to the induction of apoptosis in cancer cells in the present study, we investigated the effects of PdC on isolated RLM (rat liver mitochondria). Our results showed that these palladacycles are able to induce a Ca2+-independent mitochondrial swelling that was not inhibited by ADP, Mg2+ and antioxidants. However, the PdC-induced mitochondrial permeabilization was partially prevented by pre-incubation with CsA (cyclosporin A), NEM (N-ethylmaleimide) and bongkreic acid and totally prevented by DTT (dithiothreitol). A decrease in the content of reduced thiol groups of the mitochondrial membrane proteins was also observed, as well as the presence of membrane protein aggregates in SDS/PAGE without lipid and GSH oxidation. FTIR (Fourier-transform IR) analysis of PdC-treated RLM demonstrated the formation of disulfide bonds between critical thiols in mitochondrial membrane proteins. Associated with the mitochondrial permeabilization, PdC also induced the release of cytochrome c, which is sensitive to inhibition by DTT. Besides the contribution to clarify the pro-apoptotic mechanism of PdC, this study shows that the catalysis of specific protein thiol cross-linkage is enough to induce mitochondrial permeabilization and cytochrome c release.

INTRODUCTION

Mitochondrial dysfunction has been implicated in apoptotic and necrotic cell death, and the general mechanisms described include inner membrane permeabilization, disruption of electron transport or ATP production, changes in the cellular redox state and release of pro-apoptotic proteins into the cytosol [1]. MPT (mitochondrial permeability transition) is described as a Ca2+-dependent and CsA (cyclosporin A)-sensitive process triggered by the opening of a PTP (permeability transition pore). The PTP opening leads to a transitory and non-selective diffusion of solutes with molecular mass of up to 1500 Da through the inner mitochondrial membrane and promotes large amplitude swelling, mitochondrial depolarization and uncoupling of oxidative phosphorylation [2]. Although the role of Ca2+ in MPT is not fully understood, a previous study showed that this cation increases the mitochondrial ROS (reactive oxygen species) generation associated with the MPT [3]. The oxidation of critical thiol groups during oxidative stress conditions seems to induce a cross-linkage of mitochondrial membrane protein thiol groups and sensitizes mitochondria to Ca2+-induced MPT [46].

Although the PTP opening can be regulated by Ca2+, Mg2+, CsA, trifluoperazine, thiol oxidants, ROS, matrix pH, and members of the Bcl-2 family of proteins [711], the biochemical nature of the proteins assembled to form the PTP has been the focus of extensive research, but specific protein components of the pore have not been elucidated. Proposed models suggest that the likely PTP structure consists of a complex formed by apposition of VDAC (voltage-dependent anion channel) and ANT (adenine nucleotide translocator) at contact sites between the mitochondrial outer and inner membranes together with matrix CyP-D (cyclophilin D) [5,12].

On the other hand, several studies have described permeabilization processes of the mitochondrial membranes that are distinct from the MPT [13,14]. There is evidence that the PTP have a regulated and an unregulated opening conductance mode. Regulated PTP opening requires Ca2+ loaded into the matrix space and a chemical inducer and is inhibited by calcium chelators, Mg2+, CsA and BkA (bongkreic acid). Unregulated PTP is Ca2+-independent and, consequently, it is not blocked by calcium chelators and Mg2+ as well as by CsA and BkA [15,16]. It was also proposed that MOMP (mitochondrial outer membrane permeabilization) may occur as a consequence of MPT or may be directly induced by chemicals promoting mitochondrial matrix swelling [14,17].

PdC (palladacycle compounds) obtained from the reaction of dmpa (N,N-dimethyl-1-phenethylamine) with diphenylphosphine derivatives exhibited, in different studies, promising potential as a chemotherapeutic agent. Cyclopalladated complexes derived from dppe [1,2-ethanebis(diphenylphosphine)] showed antitumour activity in vitro and in vivo against B16F10-Nex2 murine melanoma cells of low immunogenicity implanted subcutaneously in mice. Also, these compounds caused a collapse of respiratory activity with an abrupt decrease of extracellular acidification, followed by DNA degradation [18]. In addition, cyclopalladated complexes derived from dppf [1,1′-bis(diphenylphosphine)ferrocene] were able to induce lysosomal permeabilization in K562 human leukaemia cells, which resulted in apoptosis [19]. These results pointed out that the antitumour activity of PdC results from organelle-specific actions. Furthermore, in different sites, PdC should target specific biomolecules, and thus the comprehension of the therapeutic mechanism requires studies focusing on the function of isolated organelles and their molecular components. In the present study, we elucidated the ability of PdC to induce mitochondrial permeabilization and cytochrome c release as a consequence of specific catalytic activity on membrane protein thiol groups. These events may be related to the pro-apoptotic action exhibited by PdC.

METHODS

Materials

All reagents were commercial products of the highest purity grade available, and aqueous solutions were prepared with deionized water (mixed bed ion exchanger, Millipore).

Palladacycles synthesis

The palladacycles used in this study were derived from dmpa complexed to dppe and synthesized as described in [18]. Four compounds were evaluated and the effects of the R1:1 derivative, [Pd(C2,N-(R(+)dmpa)(dppe)].Cl, were presented here as it was the most effective.

Cell culture and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] viability assay

HTC (hepatoma tissue culture) cells were grown in DMEM (Dulbecco's modified Eagle's medium) (Sigma Chemical Co.) supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin in a 5% CO2 atmosphere at 37 °C (Sanyo MCO-20AIC, Japan). For experiments, cells were washed twice with CMF-BSS (calcium- and magnesium-free buffered saline solution), detached from the flasks with trypsin/EDTA, and suspended in the grown media. The cells (2×105) were added to 96-well microplates (0.2 ml final volume) in the presence of different concentrations of R(+) dmpa:dppe (1:1) PdC and incubated for 24 h. After addition of 0.25 mg/ml MTT and incubation for 4 h, plates were read at 630 nm (Microplate Reader Biotek ELX 800, BioTek Instruments) and cell viability was determined in relation to control, performed in the absence of PdC and considered as 100%. Results are presented as the mean±S.D. of three independent experiments.

Annexin V-FITC/PI double-staining and flow cytometry analysis

After treatment with 7.5 μM PdC for 24 h as described above, HTC cells were harvested, washed with cold PBS and suspended in binding buffer (0.01 M Hepes, pH 7.4, 0.14 M NaCl and 2.5 mM CaCl2) at a concentration of 1×106 cells/ml. The suspensions were transferred to 5-ml tubes, and 5 μl of annexin V-FITC and 5 μg/ml PI (propidium iodide) were added. The cells were incubated at 25 °C for 20 min and, after the addition of 0.3 ml of binding buffer, the analysis was performed in a FACSCalibur flow cytometer using the CellQuest software (10000 events were collected per sample). Control cells were treated only with the medium. Results were presented as the mean±S.D. of the triplicates.

Isolation of RLM (rat liver mitochondria)

Mitochondria were isolated by conventional differential centrifugation [20] from adult rats. All work with animals was previously approved and undertaken as required by COBEA (Brazilian College of Animal Experiments), assuring that animals did not suffer unduly during the experimental procedure. Male Wistar rats, weighing approx. 180 g, were killed by cervical dislocation and the liver was immediately removed and homogenized in 250 mM sucrose, 1 mM EGTA and 10 mM Hepes/KOH buffer (pH 7.2) with a Potter–Elvehjem homogenizer. Homogenates were centrifuged at 770 g for 5 min and the resulting supernatant was further centrifuged at 9800 g for 10 min. Pellets were suspended in the same medium containing 0.3 mM EGTA and centrifuged at 4500 g for 15 min. The final pellet was resuspended in 250 mM sucrose and 10 mM Hepes/KOH buffer (pH 7.2) to a final protein concentration of 80–100 mg/ml. All studies with mitochondria were performed within 3 h and mitochondrial protein content was determined by the Biuret reaction [21].

Mitochondrial swelling

RLM (0.4 mg of protein) was added in a standard medium containing 125 mM sucrose, 65 mM KCl, 10 mM Hepes/KOH, pH 7.4, at 30 °C plus 5 mM potassium succinate, 2.5 μM rotenone and 10 μM CaCl2 (final volume 1.5 ml) in the presence or absence of palladacycles. The mitochondrial swelling was estimated from the decrease in the relative absorbance at 540 nm in a Hitachi U-2000 Spectrophotometer (Tokyo, Japan).

Measurements of mitochondrial transmembrane electrical potential (ΔΨ)

Mitochondrial ΔΨ was estimated by changes in the rhodamine 123 fluorescence (0.4 μM), recorded on a Hitachi F-2500 Spectrofluorimeter (Hitachi, Tokyo, Japan) operating at 505/525 nm excitation/emission wavelengths respectively, with a slit width of 5 nm. The results was expressed as percentage of dissipation in relation to uncoupled mitochondria [CCCP (carbonyl cyanide m-chlorophenylhydrazone) 1.0 μM].

Mitochondrial respiration

Mitochondria (1 mg of protein/ml) were incubated in a medium containing 125 mM sucrose, 65 mM KCl, 0.5 mM EGTA, 10 mM K2HPO4 and 10 mM Hepes/KOH, pH 7.2, at 30 °C. Mitochondrial respiration was measured in a PC-interfaced Hansatech oxygraph equipped with a Clark-type electrode with magnetic stirring (Hansatech Instruments, Norfolk, U.K.). State 4 respiration was initiated by addition of 5 mM potassium succinate (plus 2.5 μM rotenone) and state 3 respiration was initiated by adding 400 nmol of ADP. RCR (respiratory control ratio) was determined according to [22].

Determination of protein-thiol content

Mitochondrial membrane thiol groups were measured using DTNB [5,5′-dithiobis(2-nitrobenzoic)acid, Ellman's reagent] as described in [23]. After 10 min incubation under swelling conditions, the mitochondrial suspension was submitted to three subsequent freeze–thawing procedures to release matrix proteins, and then centrifuged for 15 min at 6000 g. The pellet was treated with 0.2 ml of 6% trichloroacetic acid and centrifuged at 6000 g for 15 min to precipitate the mitochondrial membrane proteins. The final pellet was suspended with 1 ml of 0.5 M potassium phosphate buffer, pH 7.6, containing 0.4% SDS. After addition of 0.1 mM DTNB, absorbance was determined at 412 nm and the amount of thiol groups was calculated from ε=13600 M−1 [23].

SDS/polyacrylamide slab gel electrophoresis

The same sample obtained in the protein thiol content assay by freeze–thawing procedures was used for electrophoresis. Samples were incubated in the reaction medium and boiled for 2 min in 250 mM Tris/HCl, pH 7.4, 20% SDS and 50 mM EDTA according to [24]. An aliquot (0.4 μg) of the samples was applied to the electrophoresis gel and the SDS/PAGE was performed in a discontinuous system as described in [25]. After separation at a voltage of 20 mA, the gel (3.5% stacking gel and 10% running gel in acrylamide) was stained with silver nitrate as described by [26].

IR spectroscopy

The samples of mitochondrial membrane proteins obtained by freeze–thawing procedures were dried and used to prepare the KBr pellets. The pellets were analysed by FTIR (Fourier-transform IR) spectroscopy on a Spectrum 100 FTIR Spectrometer (Perkin–Elmer). IR spectra were recorded in the range of 4000–400 cm−1 with 2 cm−1 resolution, and the water effects were eliminated by spectral subtraction. Spectral difference calculations of quantified samples were performed and used for the interpretation of results.

Cytochrome c release

Cytochrome c released from isolated mitochondria was determined by an enzyme immunoassay technique using an Immunoassay Kit (Biosource International, CA, U.S.A.). Mitochondria (0.4 mg of protein/ml) were incubated with the drug under the conditions of the swelling assays for 10 min and centrifuged afterwards at 16000 g for 10 min. The supernatant (50 μl) was added to wells and incubated with a biotin-conjugated monoclonal anti-cytochrome c for 2 h at 25 °C. After five washes, streptavidin–HRP (horseradish peroxidase) solution was added and the plate was incubated for 30 min. The reaction was stopped and absorbance was determined at 450 nm using a microplate reader BioTek ELX800 (BioTek Instruments). Sample concentrations were determined based on a standard curve within a 0.08 to 2.5 ng/ml concentration range (ε=0.595 ng−1·ml).

Statistical analyses

The statistical analyses were performed by one-way ANOVA followed by Dunnett's post test by using GraphPad Prism version 3.00 for Windows (GraphPad Software).

RESULTS

We evaluated the ability for cell death induction and the effects on mitochondrial functions of the R(+) and S(−) enantiomers of the organopalladium(II) complexes derived from dmpa, containing dppe as a ligand, and as they exhibited closely similar effects, only the results of the most potent enantiomer, the R(+) dmpa:dppe derivative (1), have been presented.

Structure of R(+) dmpa:dppe (1:1) PdC

Similarly to what had been previously observed for B16F10-Nex2 cells, R(+) dmpa:dppe (1:1) PdC was able to promote HTC cell death in a dose-dependent manner. The EC50 value for the decrease of the cell viability by R(+) dmpa:dppe (1:1) PdC (MTT reduction test) was approx. 7.5 μM (Figure 1A). Cytometry analysis using annexin V-FITC/PI double-staining revealed that PdC induces predominantly apoptotic cell death in HTC cells (Figure 1B).

Viability of HTC cells after treatment with R(+) dmpa:dppe (1:1) PdC for 24 h

Figure 1
Viability of HTC cells after treatment with R(+) dmpa:dppe (1:1) PdC for 24 h

(A) Cell viability was evaluated by the MTT reduction test. Cells (2×105 per well) were incubated with different concentrations of drug and, after addition of MTT, the absorbance at 630 nm was measured. The percentage of decrease in cell viability induced by R(+) dmpa:dppe (1:1) PdC was expressed in relation to control, which was performed in the absence of PdC and considered as 100% of viability. Results presented are means±S.D. of three independent experiments. (B) The type of cell death was evaluated by flow cytometry analysis using Annexin V-FITC/PI double-staining as described in the Methods section. Results are presented as percentage of viable, apoptotic and necrotic cells (mean±S.D.) of three independent experiments. Letter pairs indicate statistical difference (P<0.05).

Figure 1
Viability of HTC cells after treatment with R(+) dmpa:dppe (1:1) PdC for 24 h

(A) Cell viability was evaluated by the MTT reduction test. Cells (2×105 per well) were incubated with different concentrations of drug and, after addition of MTT, the absorbance at 630 nm was measured. The percentage of decrease in cell viability induced by R(+) dmpa:dppe (1:1) PdC was expressed in relation to control, which was performed in the absence of PdC and considered as 100% of viability. Results presented are means±S.D. of three independent experiments. (B) The type of cell death was evaluated by flow cytometry analysis using Annexin V-FITC/PI double-staining as described in the Methods section. Results are presented as percentage of viable, apoptotic and necrotic cells (mean±S.D.) of three independent experiments. Letter pairs indicate statistical difference (P<0.05).

In order to identify cell site-specific actions of dmpa:dppe that could respond to the induction of apoptosis in HTC cancer cells, the effects of PdC on isolated RLM were investigated. Figure 2(A) shows that the incubation of the R(+) dmpa:dppe (1:1) PdC with RLM energized with 5 mM succinate plus 2.5 μM rotenone induced a large amplitude mitochondrial swelling assessed by the decrease in the relative absorbance (turbidity) at 540 nm. The dose-dependent MP (mitochondrial permeabilization) attained the maximum effect at an extremely low concentration of PdC, 2.5 μM. This effect is specific for these dmpa:dppe (1:1) PdC compounds, since the same concentrations of PdCl2 or the ligand alone were unable to induce the MP (results not shown). To characterize the MP induced by dmpa:dppe (1:1) PdC, the effects of several MPT modulators were evaluated: CsA, EGTA, NEM (N-ethylmaleimide), BkA and DTT (dithiothreitol), on the MP induced by 2.5 μM of the drug. The pre-incubation of mitochondrial suspension with CsA, a classical MPT inhibitor, promoted only 18% inhibition of the PdC-induced mitochondrial swelling that was also insensitive to the Ca2+ chelator EGTA. These results indicated that the MP induced by R(+) dmpa:dppe (1:1) PdC is not a classical MPT process. BkA is a MPT modulator that binds to ANT and stabilizes it in the m-state conformation, inhibiting the PTP opening [26]. However, although partial, BkA had a significant effect on dmpa:dppe (1:1) PdC-induced mitochondrial swelling. This MPT modulator decreased by ∼38% the R(+) dmpa:dppe (1:1) PdC-induced MP. On the other hand, the hydrophobic monofunctional thiol reagent NEM inhibited more than 70% of the palladacycle-induced swelling, and DTT, a thiol reducing agent, suppressed it completely. These results point to the involvement of the critical thiol group of mitochondrial membrane proteins in the PdC-induced mitochondrial permeabilization (Figure 2B).

Mitochondrial swelling induced by R(+) dmpa:dppe (1:1) PdC

Figure 2
Mitochondrial swelling induced by R(+) dmpa:dppe (1:1) PdC

Mitochondria (0.25 mg of protein/ml) were incubated in a medium containing 125 mM sucrose, 65 mM KCl and 10 mM Hepes-KOH, pH 7.4, in the presence of 5 mM succinate, 2.5 μM rotenone and 10 μM CaCl2, at 30 °C. (A) Swelling was initiated by the addition of 1.0, 1.5, 2.0 or 2.5 μM R(+) dmpa:dppe (1:1) PdC as indicated in the Figure. (B) The effects of 0.1 mM DTT, 1.0 μM CsA, 5.0 μM BkA and 0.5 mM EGTA on mitochondrial swelling induced by 2.5 μM R(+) dmpa:dppe (1:1) PdC. The arrows indicate the addition of drugs and the traces are representative of at least three experiments with different mitochondrial preparations.

Figure 2
Mitochondrial swelling induced by R(+) dmpa:dppe (1:1) PdC

Mitochondria (0.25 mg of protein/ml) were incubated in a medium containing 125 mM sucrose, 65 mM KCl and 10 mM Hepes-KOH, pH 7.4, in the presence of 5 mM succinate, 2.5 μM rotenone and 10 μM CaCl2, at 30 °C. (A) Swelling was initiated by the addition of 1.0, 1.5, 2.0 or 2.5 μM R(+) dmpa:dppe (1:1) PdC as indicated in the Figure. (B) The effects of 0.1 mM DTT, 1.0 μM CsA, 5.0 μM BkA and 0.5 mM EGTA on mitochondrial swelling induced by 2.5 μM R(+) dmpa:dppe (1:1) PdC. The arrows indicate the addition of drugs and the traces are representative of at least three experiments with different mitochondrial preparations.

Considering that PTP opening causes dissipation of ΔΨ due to non-selective diffusion of solutes with molecular mass of up to 1500 Da through the inner mitochondrial membrane, we investigated the effects of different concentrations of R(+) dmpa:dppe (1:1) PdC on the ΔΨ, state 4 respiration rate and the mitochondrial calcium release. Under the experimental conditions of swelling assays, PdC induced the dissipation of ΔΨ assessed by rhodamine 123 (Figure 3A) and safranin O (results not shown) in a dose-dependent manner and the maximum effect, compared with CCCP, an uncoupler of oxidative phosphorylation, was obtained at 2.5 μM concentration. Similar to that observed in the swelling assays, DTT abrogated totally the disruption of ΔΨ induced by R(+) dmpa:dppe (1:1) PdC, showing that this effect is a consequence of the mitochondrial swelling. In parallel, we observe an increase in the state 4 mitochondrial respiration rate (Figure 3B) and the release of Ca2+ (Figure 3C) induced by the same R(+) dmpa:dppe (1:1) PdC concentrations.

Effect of R(+) dmpa:dppe (1:1) PdC on the mitochondrial transmembrane potential (ΔΨ), oxygen consumption and calcium efflux

Figure 3
Effect of R(+) dmpa:dppe (1:1) PdC on the mitochondrial transmembrane potential (ΔΨ), oxygen consumption and calcium efflux

Mitochondria (0.25 mg of protein/ml) were incubated in the medium described in the legend of Figure 1 at 30 °C. (A) For ΔΨ measurement, 0.4 μM rhodamine 123 was added, and the formation of ΔΨ was stimulated by the addition of 5 mM succinate (plus 2.5 μM rotenone). The concentrations of R(+) dmpa:dppe (1:1) PdC are expressed in mM and the percentage of dissipation induced by PdC was calculated considering as 100% the difference between the control experiment (absence of drugs) and the addition of 1.0 μM CCCP. *P<0.05, compared with control. (B) The stimulatory effect of R(+) dmpa:dppe (1:1) PdC on the state 4 respiration of isolated rat liver mitochondria energized with 5.0 mM succinate (plus 2.5 μM rotenone) was calculated in relation to the rate of control that was normalized to the value of 1.0. The inset is a representative trace of succinate-supported isolated rat liver mitochondrial respiration showing the coupling of the mitochondrial preparations (RCR ∼6.7) and the stimulatory effect of 2.5 μM R(+) dmpa:dppe (1:1) PdC on the succinate-supported state 4 respiration. The additions are indicated by the arrows and the numbers are the oxygen consumption rate in (ng atoms of O)/min per mg of protein. Respiratory control was calculated by the ratio between state 3 and state 4 respiration rates. Results presented in (A) and (B) are the means±S.D. of three independent experiments and the state 4 respiration rate of the control was 66.3 nmol of O2/ml per min. (C) Calcium release measured by the absorbance of arsenazo III (675–687 nm); arrow indicates the addition of 5 mM succinate and the traces are representative of at least three experiments with different mitochondrial preparations.

Figure 3
Effect of R(+) dmpa:dppe (1:1) PdC on the mitochondrial transmembrane potential (ΔΨ), oxygen consumption and calcium efflux

Mitochondria (0.25 mg of protein/ml) were incubated in the medium described in the legend of Figure 1 at 30 °C. (A) For ΔΨ measurement, 0.4 μM rhodamine 123 was added, and the formation of ΔΨ was stimulated by the addition of 5 mM succinate (plus 2.5 μM rotenone). The concentrations of R(+) dmpa:dppe (1:1) PdC are expressed in mM and the percentage of dissipation induced by PdC was calculated considering as 100% the difference between the control experiment (absence of drugs) and the addition of 1.0 μM CCCP. *P<0.05, compared with control. (B) The stimulatory effect of R(+) dmpa:dppe (1:1) PdC on the state 4 respiration of isolated rat liver mitochondria energized with 5.0 mM succinate (plus 2.5 μM rotenone) was calculated in relation to the rate of control that was normalized to the value of 1.0. The inset is a representative trace of succinate-supported isolated rat liver mitochondrial respiration showing the coupling of the mitochondrial preparations (RCR ∼6.7) and the stimulatory effect of 2.5 μM R(+) dmpa:dppe (1:1) PdC on the succinate-supported state 4 respiration. The additions are indicated by the arrows and the numbers are the oxygen consumption rate in (ng atoms of O)/min per mg of protein. Respiratory control was calculated by the ratio between state 3 and state 4 respiration rates. Results presented in (A) and (B) are the means±S.D. of three independent experiments and the state 4 respiration rate of the control was 66.3 nmol of O2/ml per min. (C) Calcium release measured by the absorbance of arsenazo III (675–687 nm); arrow indicates the addition of 5 mM succinate and the traces are representative of at least three experiments with different mitochondrial preparations.

The inhibitory effect of DTT on the PdC-induced mitochondrial swelling and associated processes suggest that this drug was able to mediate the MP via alterations in the thiol redox state of the mitochondrial membrane proteins. To investigate the mechanism of R(+) dmpa:dppe (1:1) PdC-induced MP in isolated rat liver mitochondria, the reduced thiol content of mitochondria was measured by using DTNB. Figure 4(A) shows that, similarly to the well-known pro-oxidant t-BOOH, PdC significantly decreased the reduced thiol content of mitochondria, at the same conditions of swelling measurements, and the pre-incubation with DTT also completely inhibited this effect. In this experiment, we used a freeze–thawing method [27] to measure only thiols of the mitochondrial membrane proteins, although a similar result was obtained with total mitochondrial suspensions (results not shown). This result indicates just the disappearance of reduced thiol groups but it supplies poor information about the chemical modifications occurred with these -SH groups. However, the decrease in the mitochondrial thiol content promoted by R(+) dmpa:dppe (1:1) PdC was also accompanied by proportional cross-linkage of mitochondrial membrane proteins, as shown by the SDS/PAGE electrophoresis (Figure 4B) and, again, DTT completely inhibited the formation of mitochondrial protein aggregates. These results suggest that the disappearance of thiol groups induced by PdC probably results in SH oxidation followed by the formation of disulfide bonds.

Effect of R(+) dmpa:dppe (1:1) PdC on the protein thiol groups of the mitochondrial membrane

Figure 4
Effect of R(+) dmpa:dppe (1:1) PdC on the protein thiol groups of the mitochondrial membrane

Mitochondria were incubated under the conditions described in the legend of Figure 1 and samples were prepared by the freeze–thawing technique (see the Methods section for details). (A) The reduced thiol content of the mitochondrial membrane proteins was measured using DTNB. C, control (absence of drugs); t-BOOH, 0.6 mM t-butyl hydroperoxide, as positive control; 1.0, 1.5, 2.0 and 2.5, different concentrations of R(+) dmpa:dppe (1:1) PdC in μM; DTT+2.5, 0.1 mM DTT plus 2.5 μM R(+) dmpa:dppe (1:1) PdC. *P<0.05, compared with control. (B) SDS/polyacrylamide slab gel electrophoresis of the membrane proteins isolated from mitochondrial suspensions submitted to the following conditions: (b) 0.1 mM DTT plus 2.5 μM R(+) dmpa:dppe (1:1) PdC; (c) 0.1 mM DTT; (d) 2.5 μM R(+) dmpa:dppe (1:1) PdC and (e) control (absence of drug). Lane (a) is the Kaleidoscope molecular-mass standard (10–220 kDa),

Figure 4
Effect of R(+) dmpa:dppe (1:1) PdC on the protein thiol groups of the mitochondrial membrane

Mitochondria were incubated under the conditions described in the legend of Figure 1 and samples were prepared by the freeze–thawing technique (see the Methods section for details). (A) The reduced thiol content of the mitochondrial membrane proteins was measured using DTNB. C, control (absence of drugs); t-BOOH, 0.6 mM t-butyl hydroperoxide, as positive control; 1.0, 1.5, 2.0 and 2.5, different concentrations of R(+) dmpa:dppe (1:1) PdC in μM; DTT+2.5, 0.1 mM DTT plus 2.5 μM R(+) dmpa:dppe (1:1) PdC. *P<0.05, compared with control. (B) SDS/polyacrylamide slab gel electrophoresis of the membrane proteins isolated from mitochondrial suspensions submitted to the following conditions: (b) 0.1 mM DTT plus 2.5 μM R(+) dmpa:dppe (1:1) PdC; (c) 0.1 mM DTT; (d) 2.5 μM R(+) dmpa:dppe (1:1) PdC and (e) control (absence of drug). Lane (a) is the Kaleidoscope molecular-mass standard (10–220 kDa),

Several studies have demonstrated that MPT induced by Ca2+ plus pro-oxidants is associated with the production of protein aggregates due to cross-linkage of thiol groups resulting from the formation of disulfide bonds [6]. In fact, the increased free radical production is able to oxidize thiol groups of the mitochondrial membrane proteins, resulting in cross-linkage and opening of the PTP [28]. In this regard, we investigated whether the thiol oxidation induced by PdC could be mediated by free radicals. The pre-incubation of mitochondrial suspension with several antioxidants [α-tocopherol, BHT (butylhydroxytoluene), uric acid and ascorbate] did not present any inhibitory effect on the mitochondrial swelling elicited by R(+) dmpa:dppe (1:1) PdC (results not shown). Also, R(+) dmpa:dppe (1:1) PdC did not increase the free radical generation in isolated mitochondria assessed by DCF (2′,7′-dichlorofluorescein) fluorescence and did not induce the lipid oxidation of the mitochondrial membranes, assessed as TBA (thiobarbituric acid)-reactive substances (results not shown), suggesting that PdC-induced MP occurs in the absence of oxidative stress. In parallel, a decrease of GSH content in R(+) dmpa:dppe (1:1) PdC-treated RLM was not observed, showing that the reactivity of these palladacycles with thiol groups is restricted to the thiol of membrane proteins. In fact, the structure of R(+) dmpa:dppe (1:1) PdC is compatible with a relative lipophilicity of the drug and consequent preferential partitioning into the biological membranes. The formation of protein cross-linkage mediated by R(+) dmpa:dppe (1:1) PdC leading to MP could be rationalized by the reactivity of these compounds towards nucleophiles such as the thiol group. It is possible that R(+) dmpa:dppe (1:1) PdC reacts with thiols in two possible pathways, leading the protein thiol group to form an intermediate in which the thiol group became ‘palladade’ (2). The nucleophilic attack of a thiol group on the Pd2+ ion of R(+) dmpa:dppe (1:1) PdC could lead to the opening of the palladade ring of the drug due to concomitant protonation of the amino group co-ordinating with the metal (path a) or could lead to the abstraction of Pd-co-ordinated chloride converted into HCl after abstracting a proton from the nucleophile (path b). The proposed intermediates a or b could follow other two possible pathways: nucleophilic attack of a second thiol group on the Pd ion (grey arrow) concomitant with the displacement of the second leaving group bonded to Pd, or the nucleophilic attack at the palladade sulfur atom (black arrow), displacing the Pd moiety concomitant with the formation of a sulfur bridge. This second pathway was named here a redox pathway, since it involves changes in the redox state of both Pd and sulfur atoms. To investigate these hypotheses, FTIR analyses were performed of mitochondrial membrane proteins obtained by acid precipitation in the same conditions as swelling experiments. The vibrational spectra of mitochondrial membranes precipitated by acid corroborated the redox pathway as the mechanism responsible for the membrane protein cross-linkage and consequent MP was promoted by R(+) dmpa:dppe (1:1) PdC. Figure 5(A) shows that mitochondrial samples treated with R(+) dmpa:dppe (1:1) PdC exhibits an absorption band at 474 cm−1 (line b) compatible with a disulfide stretching (S–S) that was not present in the samples pre-treated with DTT before the R(+) dmpa:dppe (1:1) PdC (line a). Due to the complexity and diversity of biomolecules composing mitochondrial membranes, it was important to establish whether the effects of R(+) dmpa:dppe (1:1) PdC on mitochondria were a consequence of exclusive attack on thiol groups. Figure 5(B), line a, shows that there is no significant IR spectral difference between mitochondria control samples and that treated with R(+) dmpa:dppe (1:1) PdC in the presence of DTT, suggesting that the reducing agent prevented the modification of mitochondrial components by the palladacycle. The subtraction of the spectrum of mitochondria treated with R(+) dmpa:dppe (1:1) PdC plus DTT from that obtained from samples treated with R(+) dmpa:dppe (1:1) PdC alone resulted in a spectrum with a absorption band at 2347 cm−1 (Figure 5B, line b), assigned to the normal vibrational mode of the S-H stretching [30]. The spectral difference promoted by R(+) dmpa:dppe (1:1) PdC in mitochondria was corroborated by the subtraction of the vibrational spectrum exhibited by R(+) dmpa:dppe (1:1) PdC-treated mitochondria from that obtained from non-treated mitochondria (Figure 5B, line c), since the subtraction resulted in a negative band at 2347 cm−1. These results demonstrated that R(+) dmpa:dppe (1:1) PdC has a specific catalytic action on critically reduced thiol groups of mitochondrial membrane proteins, promoting their cross-linkage to disulfide bonds, which leads to MP.

Possible reaction mechanisms for R(+) dmpa:dppe (1:1) PdC with thiols

Scheme 2
Possible reaction mechanisms for R(+) dmpa:dppe (1:1) PdC with thiols

See text for details.

Scheme 2
Possible reaction mechanisms for R(+) dmpa:dppe (1:1) PdC with thiols

See text for details.

FTIR spectra of the mitochondrial membrane proteins

Figure 5
FTIR spectra of the mitochondrial membrane proteins

Mitochondria were incubated under the conditions described in the legend of Figure 1. (A) Mitochondrial suspension incubated with 0.1 mM DTT plus 2.5 μM R(+) dmpa:dppe (1:1) PdC (a) or mitochondrial suspension incubated with 2.5 μM R(+) dmpa:dppe (1:1) PdC alone (b). (B) Difference between vibrational spectra obtained from mitochondrial suspension incubated with 0.1 mM DTT minus control (a), mitochondrial suspension incubated with R(+) dmpa:dppe (1:1) PdC minus 0.1 mM DTT plus 2.5 μM R(+) dmpa:dppe (1:1) PdC (b), and mitochondrial suspension incubated with 2.5 μM R(+) dmpa:dppe (1:1) PdC minus control (c).

Figure 5
FTIR spectra of the mitochondrial membrane proteins

Mitochondria were incubated under the conditions described in the legend of Figure 1. (A) Mitochondrial suspension incubated with 0.1 mM DTT plus 2.5 μM R(+) dmpa:dppe (1:1) PdC (a) or mitochondrial suspension incubated with 2.5 μM R(+) dmpa:dppe (1:1) PdC alone (b). (B) Difference between vibrational spectra obtained from mitochondrial suspension incubated with 0.1 mM DTT minus control (a), mitochondrial suspension incubated with R(+) dmpa:dppe (1:1) PdC minus 0.1 mM DTT plus 2.5 μM R(+) dmpa:dppe (1:1) PdC (b), and mitochondrial suspension incubated with 2.5 μM R(+) dmpa:dppe (1:1) PdC minus control (c).

MP has been related to the induction of apoptosis in cells throughout the release of pro-apoptotic proteins, including cytochrome c [7,31]. Considering that R(+) dmpa:dppe (1:1) PdC was able to promote cell death in a melanoma cell line [18], and the present study revealed that the palladacycle-promoted MP was not the classical Ca2+-dependent and CsA-sensitive MPT, it was interesting to know whether the PdC-induced mitochondrial swelling was accompanied by cytochrome c release. As expected, PdC induced the cytochrome c release in isolated rat liver mitochondria that, similarly to the swelling assays, was completely abolished by DTT, but not by CsA (Figure 6). This result suggests that the cytochrome c release by R(+) dmpa:dppe (1:1) PdC is a consequence of the mitochondrial permeabilization promoted by palladacycle-promoted thiol oxidation and disulfide bonds. The R(+) dmpa:dppe (1:1) PdC-induced cytochrome c release points to the possibility of the involvement of other mitochondrial proapoptotic factors such as AIF (apoptosis-inducing factor) [32] and Smac/DIABLO [direct IAP (inhibitor of apoptosis protein)-binding protein with low pI] [33] in the apoptosis promoted by the palladacycle, a possibility that remains to be elucidated.

Cytochrome c release induced by R(+) dmpa:dppe (1:1) PdC

Figure 6
Cytochrome c release induced by R(+) dmpa:dppe (1:1) PdC

Mitochondria were incubated under the conditions described in the legend of Figure 1 and the amount of cytochrome c (cit c) released was quantified in an ELISA-based method. Values are means±S.D. of two different experiments, each one performed in triplicate. *P<0.05 compared with control. PdC, 2.5 μM R(+) dmpa:dppe (1:1) PdC; DTT, 0.1 mM DTT; CsA, 1μM CsA.

Figure 6
Cytochrome c release induced by R(+) dmpa:dppe (1:1) PdC

Mitochondria were incubated under the conditions described in the legend of Figure 1 and the amount of cytochrome c (cit c) released was quantified in an ELISA-based method. Values are means±S.D. of two different experiments, each one performed in triplicate. *P<0.05 compared with control. PdC, 2.5 μM R(+) dmpa:dppe (1:1) PdC; DTT, 0.1 mM DTT; CsA, 1μM CsA.

DISCUSSION

MP plays an important role in necrotic or apoptotic cell death [1,31] and it may result from cross-linkage between unspecific mitochondrial membrane proteins and not by a pore constituted of pre-defined proteins [34]. Although all forms of permeabilization of the mitochondrial membranes have been the focus of extensive research in the last three years, the molecular nature of the proteins assembled to form the mitochondrial PTP remains not fully elucidated. In the case of Ca2+-dependent and CsA-sensitive PTP, there is strong evidence for the participation of the ANT, cyclophilin D, VDAC and, possibly, benzodiazepinic receptor and hexokinase demonstrated by using specific modulators or knockout studies [3537]. However, there are several studies in the literature showing that the assembly of mitochondrial membrane proteins to form the PTP depends on the cross-linkage of thiol groups [4,6,12].

Cyclopalladated complexes derived from dppe showed antitumour activity in vitro and in vivo against B16F10-Nex2 cells of low immunogenicity implanted subcutaneously in mice and the cell death induced by these compounds was related to a collapse of respiratory activity and DNA degradation [18]. Similarly to what had been previously observed for B16F10-Nex2 cells, R(+) dmpa:dppe (1:1) PdC was able to induce cell death of HTC cells, indicating that the potential application of this compound as an chemotherapeutic drug is not restricted to melanoma type tumours. The collapse of respiratory activity observed in melanoma cells [18] suggested the involvement of mitochondria in the palladacycle-induced cell death.

For this reason, in this paper, we studied the effects of an organopalladium(II) complex with anti-tumoral activity on isolated rat liver mitochondria to help in the understanding of the mechanisms of cell death induced by dppe-derived palladacycles. These compounds acted as a potent mitochondrial-targeted thiol reagent and brought new insights into the MP mechanisms, mainly on the requirement of Ca2+ and ROS to the oxidation of protein thiol groups related to MP. According to our results, R(+) dmpa:dppe (1:1) PdC induced MP, assayed as mitochondrial swelling, at extremely low concentrations, reaching the maximum extent with 2.5 μM. Associated with the R(+) dmpa:dppe (1:1) PdC-induced swelling, dissipation of mitochondrial transmembrane potential, uncoupling of the oxidative phosphorylation and mitochondrial calcium release was observed, since DTT was able to prevent all these processes (results not shown). Although the palladium atom may be responsible for the reactivity of these drugs, the induction of mitochondrial permeabilization was shown to be very specific for the organopalladium(II) complex, depending on the entire structure and not on the palladium atom alone, since PdCl2 was not able to promote the permeabilization. Interestingly, even in the presence of EGTA, R(+) dmpa:dppe (1:1) PdC elicited the mitochondrial permeabilization, showing the independence of Ca2+ accumulation in the mitochondrial matrix. An additional proposed role for Ca2+ in the sensitization to PTP opening is related to changes in the fluidity of mitochondrial membranes, exposing thiol groups [6,28,29]. This is different to the organotellurates RT3 and RT4 [38], that similar to Ca2+, also promote changes in the membrane fluidity. R(+) dmpa:dppe (1:1) PdC probably accesses the critical thiol groups independently of changes in the membrane fluidity, since it did not alter the fluorescence emission of ANS (8-anilinonaphthalene-1-sulfonic acid) incorporated into mitochondria (results not shown). Our findings suggest that R(+) dmpa:dppe (1:1) PdC exhibits a high and specific reactivity to thiol groups of the mitochondrial membrane proteins and, probably due to its accessibility to these SH groups, the effects of calcium to expose critical thiols are not relevant to the induction of MP by PdC.

To investigate the nature of the permeability pore formed in the presence of PdC, we evaluated the effects of several modulators of the MPT. CsA, which inhibits the MPT by binding to Cyp D, had a minor effect on the R(+) dmpa:dppe (1:1) PdC-promoted MP. However, BkA, which inhibits the MPT by binding to ANT, had a more significant effect on the R(+) dmpa:dppe (1:1) PdC-induced MP (Figure 2B). These results suggest that the pore formed by R(+) dmpa:dppe (1:1) PdC could have an heterogeneous composition, with the population encompassing ANT higher than that encompassing Cyp D.

According to Zoratti et al. [39], the independence of Ca2+ and the insensitivity to CsA inhibition are one of the defining features of “non-classical” MPT process. It has been reported that high doses of thiol reagents, such as diamide, DIDS (4,4′-di-isothio-cyanostilbene-2,2′-disulfonate) and PhAsO (phenylarsine oxide), are able to promote the permeabilization of mitochondrial membranes, detected as swelling, in these “non-classical” conditions [4,40,41]. In this regard, the inhibition of the R(+) dmpa:dppe (1:1) PdC-induced mitochondrial swelling was total when isolated mitochondria were pre-incubated with DTT, indicating that R(+) dmpa:dppe (1:1) PdC could act as a thiol reagent, promoting the oxidation of protein thiol groups. This effect was corroborated by the decrease in the reduced thiol content in the mitochondrial membrane proteins associated with the presence of protein aggregates distinguishable in the electrophoresis gel. However, R(+) dmpa:dppe (1:1) PdC exhibited maximal efficiency at very low concentrations, different to the thiol reagents cited above, probably due to accessibility to thiol groups buried in the membranes and high reactivity of the Pd centre in this palladacycle. This reactivity of the R(+) dmpa:dppe (1:1) PdC with specific thiol groups present in the hydrophobic membrane environment was confirmed by the potent inhibitory effect of NEM, a monothiol reagent which covalently blocks hydrophobic thiol groups at physiological pH and inhibits MPT [42].

Although it is well known that mitochondria have a high content of thiol groups (GSH and cysteine residues of proteins) and that the redox state of these groups influences the mitochondrial function [12,43,44], there is no direct evidence of the S-S formation by thiol reagents in the literature related to MP. Most studies suggest its occurrence indirectly by using the DTNB reaction [38,45,46]. Thus, besides the insights about the role of Ca2+ and the proteins that compose the PTP, the FTIR analysis presented here showed, for the first time, direct evidence of the formation of disulfide bonds associated with mitochondrial permeabilization by the appearance of a characteristic band in the IR region attributed to S-S vibration simultaneously to the disappearance of a -SH vibration. Around this frequency range (∼2500 cm−1), only a limited number of vibrations are known to absorb, of which the S-H of the cysteine thiol is the most abundant in proteins [30,47]. The vibrational spectra do not demonstrate characteristic vibrational bands of S-Pd species, corroborating that the decrease in the reduced thiol content measured with DTNB is due to the formation of disulfide.

Previous studies have demonstrated that the PTP opening in oxidative stress conditions can be associated to oxidation of -SH groups by free radicals [6,28]. It was demonstrated that the permeabilization of the inner mitochondrial membrane by peroxynitrite is independent of Ca2+ and mediated by membrane protein thiol cross-linking and lipid peroxidation [48]. On the other hand, the opening of Ca2+-dependent and Ca2+-independent PTP by organotelluranes that exhibited a high antioxidant activity on the mitochondrial lipid fraction was recently described [38]. Under our experimental conditions, several results suggest that R(+) dmpa:dppe (1:1) PdC, although not exhibiting antioxidant action, did not induce oxidation of thiol groups through the increase in the generation of free radicals: (i) the pre-incubation of mitochondrial suspensions with different antioxidants and radical scavengers did not prevent the mitochondrial swelling, (ii) when mitochondria was incubated with R(+) dmpa:dppe (1:1) PdC, the induction of the oxidation of mitochondrial membrane lipids and GSH was not observed, and (iii) the addition of PdC to the mitochondria did not promote the increase in DCF fluorescence. These results demonstrate that R(+) dmpa:dppe (1:1) PdC is a thiol reagent that directly catalyses the disulfide formation responsible for mitochondrial permeabilization, as proposed in 2. It is worthy of note that the relative lipophilicity of the R(+) dmpa:dppe (1:1) PdC allows its preferential partitioning into the biological membranes and restricts its effects on critical thiol groups of membrane proteins. The reactivity of the organo-palladium derivatives has been previously proposed in a study with another dmpa palladacycle, the dmpa:dppf. In this study, a related palladacycle compound, the dppf exhibited inhibitory effect on cathepsin B activity, a cysteine proteinase with a thiol group in the catalytic site [19].

The release of cytochrome c by the non-classical MP induced by R(+) dmpa:dppe (1:1) PdC suggests that the apoptotic mitochondrial pathway is associated with antitumour activity presented by the palladacycle. However, by considering the lysosomal permeabilization in K562 cells induced by a related palladacycle compound, dppf [19], further investigation is necessary to establish whether R(+) dmpa:dppe (1:1) PdC can also act on lysosomes, and what organelle permeabilization occurs upstream or downstream of this process that culminates with tumoural cell death promoted by this drug.

Conclusions

The results presented here demonstrated that the mitochondrial permeabilization promoted by PdC in isolated rat liver mitochondria is independent of Ca2+ and ROS and occurs due to cross-linkage of vicinal thiols present in the mitochondrial membrane proteins with formation of disulfide bonds catalysed by R(+) dmpa:dppe (1:1) PdC. Since this mitochondrial permeabilization was associated with cytochrome c release by isolated mitochondria, the results may help to explain the pro-apoptotic effect exhibited by these drugs in tumour cells.

BkA was kindly donated by Professor Sergio A. Uyemura (Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Universidade de São Paulo, São Paulo, Brazil). We thank Daniela P. S. Phelippin and Karla L. Fiel for technical assistance.

Abbreviations

     
  • ANT

    adenine nucleotide translocator

  •  
  • BkA

    bongkreic acid

  •  
  • CCCP

    carbonyl cyanide m-chlorophenylhydrazone

  •  
  • CsA

    cyclosporin A

  •  
  • DCF

    2′,7′-dichlorofluorescein

  •  
  • dmpa

    N,N-dimethyl-1-phenethylamine

  •  
  • dppe

    1,2-ethanebis(diphenylphosphine)

  •  
  • dppf

    1,1′-bis (diphenylphosphine)-ferrocene

  •  
  • DTNB

    5,5′-dithiobis(2-nitrobenzoic acid)

  •  
  • DTT

    dithiothreitol

  •  
  • FTIR

    Fourier-transform IR

  •  
  • HTC cells

    hepatoma, tissue culture cells

  •  
  • MP

    mitochondrial permeabilization

  •  
  • MPT

    mitochondrial permeability transition

  •  
  • MTT

    3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide

  •  
  • NEM

    N-ethylmaleimide

  •  
  • PdC

    palladacycle compound

  •  
  • PI

    propidium iodide

  •  
  • PTP

    permeability transition pore

  •  
  • RCR

    respiratory control ratio

  •  
  • RLM

    rat liver mitochondria

  •  
  • ROS

    reactive oxygen species

  •  
  • VDAC

    voltage-dependent anion channel

FUNDING

This work was supported by the Brazilian research funding agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [grant numbers 98/11398-3, 06/00995-9] and A. C. F. C., I. L. N. and T. R. are fellows of the Fundação de Amparo ao Ensino e Pesquisa da Universidade de Mogi das Cruzes (FAEP-UMC).

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