Copper·Dopamine Complex Induces Mitochondrial Autophagy Preceding Caspase-independent Apoptotic Cell Death
2009; Elsevier BV; Volume: 284; Issue: 20 Linguagem: Inglês
10.1074/jbc.m900323200
ISSN1083-351X
AutoresIrmgard Paris, Carolina Perez-Pastene, Eduardo Couve, Pablo Caviedes, Susan P. LeDoux, Juan Segura‐Aguilar,
Tópico(s)Alzheimer's disease research and treatments
ResumoParkinsonism is one of the major neurological symptoms in Wilson disease, and young workers who worked in the copper smelting industry also developed Parkinsonism. We have reported the specific neurotoxic action of copper·dopamine complex in neurons with dopamine uptake. Copper·dopamine complex (100 μm) induces cell death in RCSN-3 cells by disrupting the cellular redox state, as demonstrated by a 1.9-fold increase in oxidized glutathione levels and a 56% cell death inhibition in the presence of 500 μm ascorbic acid; disruption of mitochondrial membrane potential with a spherical shape and well preserved morphology determined by transmission electron microscopy; inhibition (72%, p < 0.001) of phosphatidylserine externalization with 5 μm cyclosporine A; lack of caspase-3 activation; formation of autophagic vacuoles containing mitochondria after 2 h; transfection of cells with green fluorescent protein-light chain 3 plasmid showing that 68% of cells presented autophagosome vacuoles; colocalization of positive staining for green fluorescent protein-light chain 3 and Rhod-2AM, a selective indicator of mitochondrial calcium; and DNA laddering after 12-h incubation. These results suggest that the copper·dopamine complex induces mitochondrial autophagy followed by caspase-3-independent apoptotic cell death. However, a different cell death mechanism was observed when 100 μm copper·dopamine complex was incubated in the presence of 100 μm dicoumarol, an inhibitor of NAD(P)H quinone:oxidoreductase (EC 1.6.99.2, also known as DT-diaphorase and NQ01), because a more extensive and rapid cell death was observed. In addition, cyclosporine A had no effect on phosphatidylserine externalization, significant portions of compact chromatin were observed within a vacuolated nuclear membrane, DNA laddering was less pronounced, the mitochondria morphology was more affected, and the number of cells with autophagic vacuoles was a near 4-fold less. Parkinsonism is one of the major neurological symptoms in Wilson disease, and young workers who worked in the copper smelting industry also developed Parkinsonism. We have reported the specific neurotoxic action of copper·dopamine complex in neurons with dopamine uptake. Copper·dopamine complex (100 μm) induces cell death in RCSN-3 cells by disrupting the cellular redox state, as demonstrated by a 1.9-fold increase in oxidized glutathione levels and a 56% cell death inhibition in the presence of 500 μm ascorbic acid; disruption of mitochondrial membrane potential with a spherical shape and well preserved morphology determined by transmission electron microscopy; inhibition (72%, p < 0.001) of phosphatidylserine externalization with 5 μm cyclosporine A; lack of caspase-3 activation; formation of autophagic vacuoles containing mitochondria after 2 h; transfection of cells with green fluorescent protein-light chain 3 plasmid showing that 68% of cells presented autophagosome vacuoles; colocalization of positive staining for green fluorescent protein-light chain 3 and Rhod-2AM, a selective indicator of mitochondrial calcium; and DNA laddering after 12-h incubation. These results suggest that the copper·dopamine complex induces mitochondrial autophagy followed by caspase-3-independent apoptotic cell death. However, a different cell death mechanism was observed when 100 μm copper·dopamine complex was incubated in the presence of 100 μm dicoumarol, an inhibitor of NAD(P)H quinone:oxidoreductase (EC 1.6.99.2, also known as DT-diaphorase and NQ01), because a more extensive and rapid cell death was observed. In addition, cyclosporine A had no effect on phosphatidylserine externalization, significant portions of compact chromatin were observed within a vacuolated nuclear membrane, DNA laddering was less pronounced, the mitochondria morphology was more affected, and the number of cells with autophagic vacuoles was a near 4-fold less. A possible role of copper in the neurodegeneration of dopaminergic neurons is supported by the fact that patients with neurological Wilson disease, a copper deposition disorder, display a number of extrapyramidal motor symptoms, including Parkinsonism. The cerebral manifestations in neurological Wilson disease are expressed as bradykinesia, rigidity, tremor, dyskinesia, and dysarthria (1Oder W. Prayer L. Grimm G. Spatt J. Ferenci P. Kollegger H. Schneider B. Gangl A. Deecke L. Neurology. 1993; 43: 120-124Crossref PubMed Google Scholar). It has been proposed that neurological Wilson disease can be assigned to the group of secondary Parkinsonian syndromes (2Barthel H. Hermann W. Kluge R. Hesse S. Collingridge D.R. Wagner A. Sabri O. Am. J. Neuroradiol. 2003; 24: 234-238PubMed Google Scholar). Interestingly, young workers who worked in the copper smelting industry also developed Parkinsonism (3Caviedes P. Segura-Aguilar J. Neuroreport. 2001; 12: A25Crossref PubMed Scopus (11) Google Scholar). Studies performed in rats showed copper (Cu2+)-induced degeneration of dopaminergic neurons in the nigrostriatal system. Likewise, it was described that copper neurotoxicity in rat substantia nigra and striatum is dependent on NAD(P)H dehydrogenase inhibition (4Diaz-Veliz G. Paris I. Mora S. Raisman-Vozari R. Segura-Aguilar J. Chem. Res. Toxicol. 2008; 21: 1180-1185Crossref PubMed Scopus (17) Google Scholar, 5Yu W.R. Jiang H. Wang J. Xie J.X. Neurosci. Bull. 2008; 24: 73-78Crossref PubMed Scopus (40) Google Scholar). All of these results support a possible role for copper in the neurodegeneration of dopaminergic neurons. The general mechanism of toxicity, induced by the reduced form of copper (Cu+) catalyzing the formation of hydroxyl radicals in the presence of hydrogen peroxide through the Fenton reaction, cannot explain the specific degeneration of dopaminergic neurons in Parkinsonism induced in neurological Wilson disease, or in miners working in the copper smelting industry. The selective action of copper can be explained by the ability of copper to form a complex with dopamine, allowing this metal to be transported by cells that have the ability to take up dopamine (6Paris I. Dagnino-Subiabre A. Marcelain K. Bennett L.B. Caviedes P. Caviedes R. Olea-Azar C. Segura-Aguilar J. J. Neurochem. 2001; 77: 519-529Crossref PubMed Scopus (108) Google Scholar). This specific neurotoxic action of copper in neurons with dopamine uptake is dependent on (i) the ability of copper to form a complex with dopamine (Cu·DA) 2The abbreviations used are: DA, dopamine; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone; GFP-LC3, green fluorescent protein-light chain 3; PBS, phosphate-buffered saline; ANOVA, analysis of variance. 2The abbreviations used are: DA, dopamine; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone; GFP-LC3, green fluorescent protein-light chain 3; PBS, phosphate-buffered saline; ANOVA, analysis of variance. (6Paris I. Dagnino-Subiabre A. Marcelain K. Bennett L.B. Caviedes P. Caviedes R. Olea-Azar C. Segura-Aguilar J. J. Neurochem. 2001; 77: 519-529Crossref PubMed Scopus (108) Google Scholar, 7Kiss T. Gergely A. J. Inorg. Biochem. 1985; 25: 247-259Crossref PubMed Scopus (21) Google Scholar), (ii) uptake of Cu·DA complex by dopamine transporters, (iii) oxidation of dopamine to aminochrome, and (iv) one-electron reduction of aminochrome by inhibiting NAD(P)H dehydrogenase (6Paris I. Dagnino-Subiabre A. Marcelain K. Bennett L.B. Caviedes P. Caviedes R. Olea-Azar C. Segura-Aguilar J. J. Neurochem. 2001; 77: 519-529Crossref PubMed Scopus (108) Google Scholar). These findings may explain the selective neurotoxic action of copper in the brain, but they do not explain the cell death mechanism. Currently, cell death is divided into three categories: apoptosis, autophagy, and necrosis. At the current time, only apoptosis and autophagic cell death are generally accepted as being legitimate forms of programmed cell death. Alternative models of cell death have therefore been proposed, including para-apoptosis, mitotic catastrophe, oncosis, and pyroptosis (8Wang Y. Li X. Wang L. Ding P. Zhang Y. Han W. Ma D. J. Cell Sci. 2004; 117: 1525-1532Crossref PubMed Scopus (166) Google Scholar, 9Bröker L.E. Kruyt F.A. Giaccone G. Clin. Cancer Res. 2005; 11: 3155-3162Crossref PubMed Scopus (782) Google Scholar, 10Fink S.L. Cookson B.T. Infect. Immun. 2005; 73: 1907-1916Crossref PubMed Scopus (1403) Google Scholar, 11Bredesen D.E. Stroke. 2007; 38: 652-660Crossref PubMed Scopus (38) Google Scholar, 12Krysko D.V. Vanden Berghe T. D'Herde K. Vandenabeele P. Methods. 2008; 44: 205-221Crossref PubMed Scopus (516) Google Scholar). Necrosis is characterized mostly by the absence of caspase activation, cytochrome c release, and DNA oligonucleosomal fragmentation. Apoptotic cells are characterized by the formation of blebs, chromatin condensation, DNA oligonucleosomal fragmentation, and exposure of phosphatidylserine on the external membrane. This mode of cell death can be dependent or independent of activation of caspases (13Leprêtre C. Scovassi A.I. Shah G.M. Torriglia A. Int. J. Biochem. Cell Biol. 2008; 41: 1046-1054Crossref PubMed Scopus (10) Google Scholar). On the other hand, autophagy can be distinguished from apoptosis by sequestration of bulk cytoplasm and organelles in double or multimembrane autophagic vacuoles that then fuse with the lysosomal system. Some of these described mechanisms are related to neurological diseases such as Parkinson disease (14Dagda R.K. Zhu J. Kulich S.M. Chu C.T. Autophagy. 2008; 4: 770-782Crossref PubMed Scopus (226) Google Scholar, 15Vogiatzi T. Xilouri M. Vekrellis K. Stefanis L. J. Biol. Chem. 2008; 283: 23542-23556Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar). Cells can use different methods to activate their own destruction, and more than one death program may be activated at the same time (16Rickmann M. Vaquero E.C. Malagelada J.R. Molero X. Gastroenterology. 2007; 132: 2518-2532Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17Hwang S.O. Lee G.M. Biotechnol. Bioeng. 2008; 99: 678-685Crossref PubMed Scopus (95) Google Scholar). The purpose of this study was to examine the Cu·DA complex-induced cell death mechanism in RCSN-3 cells, a cell line that expresses dopamine, norepinephrine, and serotonin transporters (18Paris I. Martinez-Alvarado P. Perez-Pastene C. Vieira M.N. Olea-Azar C. Raisman-Vozari R. Cardenas S. Graumann R. Caviedes P. Segura-Aguilar J. J. Neurochem. 2005; 92: 1021-1032Crossref PubMed Scopus (41) Google Scholar, 19Paris I. Lozano J. Cardenas S. Perez-Pastene C. Saud K. Fuentes P. Dagnino-Subiabre A. Raisman-Vozari R. Shimahara T. Kostrzewa J.P. Chi D. Kostrzewa R.M. Caviedes P. Segura-Aguilar J. Neurotox. Res. 2008; 13: 221-230Crossref PubMed Scopus (17) Google Scholar). Chemicals—Dopamine, dicoumarol, menadione, Dulbecco's modified Eagle's medium/Ham's F-12 nutrient mixture (1:1), Hanks' solution, ascorbic acid, tert-butylhydroxytoluene, phosphotungstic acid, thiobarbituric acid, n-butanol, 1,1,3,3-tetramethoxypropane, metaphosphoric acid, and copper sulfate were purchased from Sigma. Calcein AM, ethidium homodimer-1, Annexin V Alexa Fluor 488, JC1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) and Rhod-2AM were purchased from Molecular Probes (Eugene, OR). Cyclosporine, Z-VAD-FMK inhibitor, and DEVD-CHO, o-phthalaldehyde, and N-ethylmaleimide were purchased from Calbiochem. Caspase-3 antibody was purchased from Cell Signaling Co. (Beverly, MA). IgG anti-rabbit biotinylated and Cy-3-conjugated Streptavidin were purchased from Vector Co. Vector green fluorescent protein-light chain 3 (GFP-LC3) was a gift from Dr. Zsolt Tallocky, Columbia University Medical Center. FuGENE HD was purchased from Roche Applied Science. In all experiments performed in this study, 100 μm Cu·DA complex was used. To obtain 100 μm Cu·DA in cell culture medium containing amino acids, which chelate Cu2+ (20Chikira M. Inoue M. Nagane R. Harada W. Shindo H. J. Inorg. Biochem. 1997; 66: 131-139Crossref PubMed Scopus (22) Google Scholar), it was necessary to incubate 100 μm dopamine with 1 mm CuSO4 for 2 min (6Paris I. Dagnino-Subiabre A. Marcelain K. Bennett L.B. Caviedes P. Caviedes R. Olea-Azar C. Segura-Aguilar J. J. Neurochem. 2001; 77: 519-529Crossref PubMed Scopus (108) Google Scholar). Cell Culture—The RCSN-3 cell line grows in monolayers, with a doubling time of 52 h, a plating efficiency of 21%, and a saturation density of 56,000 cells/cm2 in normal growth media composed of Dulbecco's modified Eagle's medium/Ham's F-12 (1:1), 10% bovine serum, 2.5% fetal bovine serum, 40 mg/liter gentamicin sulfate. The cultures were kept in an incubator at 37 °C with 100% humidity, and the cells grew well in atmospheres of both 5% or 10% CO2 (6Paris I. Dagnino-Subiabre A. Marcelain K. Bennett L.B. Caviedes P. Caviedes R. Olea-Azar C. Segura-Aguilar J. J. Neurochem. 2001; 77: 519-529Crossref PubMed Scopus (108) Google Scholar, 19Paris I. Lozano J. Cardenas S. Perez-Pastene C. Saud K. Fuentes P. Dagnino-Subiabre A. Raisman-Vozari R. Shimahara T. Kostrzewa J.P. Chi D. Kostrzewa R.M. Caviedes P. Segura-Aguilar J. Neurotox. Res. 2008; 13: 221-230Crossref PubMed Scopus (17) Google Scholar, 21Dagnino-Subiabre A. Marcelain K. Arriagada C. Paris I. Caviedes P. Caviedes R. Segura-Aguilar J. Mol. Cell. Biochem. 2000; 212: 131-134Crossref PubMed Google Scholar). Incubations—In all experiments performed in this study, we incubated RCSN-3 cells with 100 μm Cu·DA complex or 100 μm Cu·DA complex in the presence of 100 μm dicoumarol. For control conditions, we used 100 μm dopamine, 1 mm CuSO4, or 100 μm dicoumarol incubated alone. Cell Death—For Cu·DA complex cell death experiments, cells were incubated with cell culture medium but in the absence of bovine serum and phenol red for 0.5, 1, 2, 3, 4, 7, and 12 h. Cell death was measured by counting live and dead cells after staining with 0.5 μm Calcein AM and 5 μm ethidium homodimer-1 for 45 min at room temperature in the dark (LIVE/DEAD viability/cytotoxicity kit, Molecular Probes). Calcein AM is a marker for live cells, and ethidium homodimer-1 intercalates into the DNA of dead cells. Cells were counted with a phase-contrast microscope equipped with fluorescence using the following filters: Calcein AM: 450–490 nm (excitation) and 515–565 nm (emission); ethidium homodimer-1: 510–560 nm (excitation) and LP-590 nm (emission). GSH/Oxidized Glutathione—The measurement of glutathione was carried out using a fluorometric method (22Hissin P.J. Hilf R. Anal. Biochem. 1976; 74: 214-226Crossref PubMed Scopus (3642) Google Scholar). The cells were treated with trypsin to 0.3% for 15 min at 37 °C and were washed with PBS. The cells were resuspended in a solution containing 0.1 m phosphate buffer, pH 8, 5 mm EDTA, and 25% metaphosphoric acid and centrifuged at 4000 rpm for 15 min. Glutathione in the supernatant was measured using a fluorometric method (22Hissin P.J. Hilf R. Anal. Biochem. 1976; 74: 214-226Crossref PubMed Scopus (3642) Google Scholar). Phosphatidylserine Determination—RCSN-3 cells were incubated as described above for 2 h at 37 °C. Phosphatidylserine externalization was determined by staining the cells with Alexa Fluor 488-labeled annexin V (5 μl of annexin V/100 μl of solution, Vybrant™ Apoptosis Assay Kit #2, Molecular Probes) and 5 μm ethidium homodimer-1 in Annexin Binding Buffer (50 mm HEPES, 700 mm NaCl, 12.5 mm CaCl2, pH 7.4). The cells were incubated at room temperature for 35 min. After the incubation period, the cells were washed with 1× Annexin Binding Buffer, and the samples were kept in darkness. The cells were counted in a phase-contrast microscope equipped with fluorescence. First the cells were counted using fluorescence and then using the following filters: Alexa Fluor 488: 450–490 nm (excitation) and 515–565 nm (emission); ethidium homodimer-1: 510–560 nm (excitation) and LP-590 nm (emission). Feulgen Staining—The cells were incubated as described above in the absence of bovine serum and phenol red for 3, 4, 7, and 12 h. Cells were fixed in 1 ml of ethanol:acetic acid (3:1) at 4 °C for 1 h and washed with 70% ethanol and distilled water three times for 5 min. The samples were hydrolyzed for 5 min in 5 n HCl at 20 °C and stained by the Feulgen procedure using Schiff reagent (Merck) for 1 h at room temperature in the dark. The samples were rinsed in acid-metabisulfite solution three times for 10 min and two times in water for 5 min before being dehydrated with alcohol (70, 95, and 100%) and air-dried. The cells were analyzed in a phase-contrast microscope equipped with fluorescence using the following filters: 510–560 nm (excitation) and LP-590 nm (emission). Caspase 3 Activation—RCSN-3 cells were incubated with Cu·DA complex as described above for 7 h were washed and fixed at –20 °C with 1 ml methanol (100%) for 30 min. After washing with PBS and incubation with 1.5% milk for 40 min at room temperature, the cells are incubated with 500 μl of antibodies against activated caspase 3 diluted 1:250 in PBS containing 1% bovine serum albumin and 0.02% sodium azide overnight at 4 °C. After washing, the samples were incubated with biotinylated IgG anti-rabbit, diluted 1:500 for 90 min at room temperature under dark conditions. The samples were washed and incubated with Cy-3-conjugated Streptavidin at 1.5 μg/ml diluted in PBS for 1 h. To determine the specificity of caspase 3 activation, we used 50 μm Z-VAD-FMK and 100 μm DEVD-CHO preincubated for 1 h before the cells were stained with caspase 3. DNA Laddering—RCSN-3 cells were incubated as described above for 1, 2, 3, 4, 7, and 12 h. The medium was removed, and the cells were be incubated in 90 μl of 10% SDS and 810 μl of buffer TE (10 mm Tris-HCl, 1 mm EDTA, pH 8) for 2–5 min at room temperature. As a positive control, 100 μm menadione was used. The extracted cells were placed in a tube containing 900 μl of saturated phenol and centrifuged at 14,000 rpm for 10 min at 4 °C. The aqueous phase was extracted by addition of 900 μl of saturated phenol and centrifuged at 14,000 rpm for 10 min at 4 °C. This procedure was performed twice. The aqueous phase was mixed with 900 μl of 24:1 chloroform:isoamilic ethanol and centrifuged at 14,000 rpm for 10 min 4 °C. The aqueous phase was removed and resuspended in 90 μl of 3 m sodium acetate, pH 5.2, and isopropanol to fill the tube, incubated at –20 °C for 24 h, and centrifuged at 14,000 rpm for 15 min at 4 °C. The pellet was resuspended in 70% ethanol and centrifuged at 14,000 rpm for 10 min at 4 °C. The precipitate was dried at room temperature and resuspended in TE. The amount of DNA was measured spectrophotometrically, and 4 μg was run on an agarose gel. Electrophoresis was carried out in 2.5% agarose gels in TBE buffer (45 mm Tris-base, 45 mm boric acid, and 1.6 mm EDTA). Determination of Mitochondrial Membrane Potential—RCSN-3 cells were incubated as described above for 2 h at 37 °C. The cells were incubated in the dark for 40 min at room temperature with freshly prepared 1.5 μg/ml JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) in PBS and then washed three times with PBS. The cells were observed immediately after labeling using a confocal microscope to quantify the intensity of fluorescence simultaneously for the green monomeric form at 488 nm (excitation) and 510–525 nm (emission) and for red fluorescent aggregates-JC1 at 543 nm (excitation) and 570 nm (emission). Background images were obtained from a cell-free section of the coverslip. A ratio image generated by dividing the fluorescence intensity at 590 nm by the fluorescence intensity at 527 nm is reported as a relative mitochondrial membrane electric potential value (23Reers M. Smith T.W. Chen L.B. Biochemistry. 1991; 30: 4480-4486Crossref PubMed Scopus (864) Google Scholar). Transmission Electron Microscopy—Cells incubated as described above were pelleted and fixed with 3% glutaraldehyde in 0.1 m cacodylate buffer, pH 7.4, for 120 min, washed three times and post-fixed in osmium tetroxide 1% for 60 min. The cells were dehydrated in an ascending ethanol battery ranging from 20% to 100%, and were later placed in 3:1, 2:1, 1:1, 1:2, and 1:3 ratios of propylene oxide or epom-812 resin for 1 h at room temperature, respectively. Ultrathin sections of 70 nm were made and impregnated with 2% uranyl acetate and Reynold's lead citrate. The sections were visualized in a Zeiss EM-900 transmission electron microscope at 50 kV and photographed. The negatives were scanned at 600 × 600 dpi resolution, and the images obtained were analyzed later with a PC-compatible computer using customized software. GFP-LC3 Plasmid Transfection—Cells were grown in culture medium in 24-well plates for 48 h after being transfected with GFP-LC3 plasmid (gift from Dr. Zsolt Tallocky). To form the transfection complex, we used 2 μl of FuGENE HD transfection reagent (Roche Applied Science) and 0.5 μg of GFP-LC3 plasmid DNA in 25 μl of total volume of medium for each plate, kept in darkness the for 15 min. Thereafter, the transfection complex in 150 μl of culture medium was added to each plate, and the mixture was incubated for 1 h. Then, 350 μl of culture medium was added to each plate and cultured for 2 days. Determination of GFP-LC3 and Rodamine-2AM Colocalization—GFP-LC3-transfected RCSN-3 cells were grown to 60% confluence on coverslips (25-mm diameter) and incubated with cell culture medium alone or in the presence of 3 μm Rhodamine-2AM for 1.5 h. The cells were visualized on a confocal microscope (LSM-410 Axiovert-100, Zeiss). GFP-LC3 fluorescence was determined by using an excitation wavelength of 488 nm and an emission wavelength of 510–525 nm. Rhodamine-2AM fluorescence was determined using 543 and 570 for excitation and emission, respectively. Data Analysis—All data are expressed as mean ± S.E. values. Statistical significance was assessed using ANOVA for multiple comparisons and Student's t test for comparison between two given groups. Cell Death—A rapid linear increase in the cell death of RCSN-3 cells was observed when the cells were incubated with 100 μm Cu·DA complex in the presence of 100 μm dicoumarol for 4 h (82 ± 2% cell death, p < 0.001) This was followed by a slower increase in cell death between 4 and 12 h. Similarly, a slower cell death was induced by the incubation of cells with 100 μm Cu·DA complex alone, in which case a linear increase until 4 h (65 ± 1% cell death, p < 0.001) was also observed. Interestingly, the incubation of cells with 1 mm CuSO4 alone induced only moderate cell death and only after 7-h incubation (35 ± 2% cell death, p < 0.01). No significant cell death was observed when the cells were incubated with 100 μm dopamine or 100 μm dicoumarol alone (Fig. 1A). The oxidized glutathione levels significantly increased when the cells were incubated with 100 μm Cu·DA complex in the absence or presence of 100 μm dicoumarol (187%, p < 0.05 and 211%, p < 0.05, respectively). Likewise, the levels of reduced glutathione decreased significantly when the cells were incubated with 100 μm Cu·DA complex in the presence of 100 μm dicoumarol (67% decrease, p < 0.01) (Fig. 1B). To determine whether the redox state was involved in Cu·DA complex-induced cell death, we incubated the cells in the presence of 500 μm ascorbic acid for 2 h. A significant decrease in the cell death was observed in the presence of ascorbic acid in cells treated with Cu·DA complex in the absence or presence of dicoumarol (56%, p < 0.001 and 69%, p < 0.001, respectively) (Fig. 1C). Phosphatidylserine Externalization—The externalizations of phosphatidylserine and ethidium homodimer-1, as a marker for cell death, were detected in cells incubated for 2 h. The incubation of the cells with 100 μm Cu·DA complex in the presence of 100 μm dicoumarol did not induce a significant increase in the externalization of phosphatidylserine (16 ± 8%) despite the significant cell death (63 ± 9, p < 0.01). These results contrast with a significant increase in phosphatidylserine externalization (64 ± 1%, p < 0.001) with a moderate cell death (29 ± 4%, p < 0.01) induced by Cu·DA complex alone. No significant changes in phosphatidylserine externalization and cell death were observed when the cells were treated with 100 μm dopamine, 100 μm dicoumarol, or 1 mm CuSO4 alone (Fig. 2A). Caspase 3 Activation—The caspase 3 activation was determined in cells incubated for 7 h with Cu·DA complex. We detected caspase 3 activation in cells treated with 100 μm Cu·DA complex in the absence and presence of 100 μm dicoumarol, and in cells treated with 100 μm menadione. However, the activation observed in cells treated with Cu·DA complex was not specific, because both 50 μm Z-VAD-FMK and 100 μm DEVD-CHO only inhibited menadione-induced caspase 3 activation (81%, p < 0.001 and 94%, p < 0.001, respectively). Chromatin Alterations—Chromatin alterations were measured by staining cell nuclei with Feulgen. A significant increase in cells with chromatin alterations was observed when the cells were incubated with 100 μm Cu·DA complex in the absence (34 ± 9%, p < 0.01) and in the presence of 100 μm dicoumarol (35 ± 9%, p < 0.01) at 7 h. No chromatin alterations were observed when the cells were treated with 100 μm dopamine, 100 μm dicoumarol, or 1 mm CuSO4 alone. The positive control of 100 μm menadione exhibited a significant increase of chromatin alterations (90 ± 3%, p < 0.001 (Fig. 3A)). Like to the positive control with menadione, chromatin alterations observed in cells treated with Cu·DA complex in the presence and absence of 100 μm dicoumarol continuously increased from 3 to 12 h (Fig. 3B). DNA Fragmentation—To determine the formation of small fragments resulting from DNA fragmentation, we used a DNA laddering technique after incubation of the cells with 100 μm Cu·DA complex in the absence and presence of 100 μm dicoumarol for 1, 2, 3, 4, 7, and 12 h. No small DNA fragments were observed when the cells were incubated with Cu·DA complex in the absence or presence of dicoumarol for 1, 2, 3, 4, and 7 h (Fig. 4A showed only at 7 h). However, small fragments of DNA were observed in cells incubated for 12 h with Cu·DA complex, both in the absence or presence of 100 μm dicoumarol (Fig. 4B). However, DNA laddering was more pronounced in cells treated with Cu·DA complex alone. Incubation of the cells with 100 μm dopamine, 100 μm dicoumarol or 1 mm CuSO4 did not result in small DNA fragments (Fig. 4). As a positive control, the cells were incubated with 100 μm menadione, which produced small DNA fragments both at 7 and 12 h. Mitochondrial Membrane Potential Changes—Caspase independent DNA fragmentation is associated with mitochondrial release of proteins related to oligonucleosomal DNA fragmentation (24Widlak P. Li L.Y. Wang X. Garrard W.T. J. Biol. Chem. 2001; 276: 48404-48409Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Therefore, we studied changes in the mitochondrial membrane potential by using JC-1 in cells incubated with 100 μm Cu·DA complex in the absence or presence of 100 μm dicoumarol for 2 h. A significant change in the mitochondrial membrane potential was observed both when the cells were incubated with Cu·DA complex in the absence (1.9 ± 0.2 RF, p < 0.05) and presence of dicoumarol (1.2 ± 0.3 RF, p < 0.01) compared with the control cells (3.3 ± 0.4 RF). No significant changes were observed when the cells were incubated with 100 μm dopamine, 100 μm dicoumarol, or 1 mm CuSO4 alone (Fig. 5). The Effect of Cyclosporine A on Cell Death and Phosphatidylserine Externalization—We studied the effect of cyclosporine A on cell death and phosphatidylserine externalization observed after treatment with Cu·DA complex. A 72% inhibition of phosphatidylserine externalization was observed in the presence of 5 μm cyclosporine A (18.4 ± 2%, p < 0.001) when the cells were incubated with 100 μm Cu·DA complex. However, no effect of cyclosporine A on cell death was observed in cells treated with Cu·DA complex. These results contrast with the lack of effect of cyclosporine A on phosphatidylserine externalization and cell death when the cells were treated with 100 μm Cu·DA complex in the presence of 100 μm dicoumarol (Fig. 2B). Cytoplasm Ultrastructural Analysis—Transmission electron microscopy was performed to examine cellular changes at the ultrastructural level to obtain more information about the mechanism of cell death induced by Cu·DA complex, because caspase-independent cell death has been observed both during apoptosis and autophagy (25Shrivastava A. Tiwari M. Sinha R.A. Kumar A. Balapure A.K. Bajpai V.K. Sharma R. Mitra K. Tandon A. Godbole M.M. J. Biol. Chem.,. 2006; 281: 19762-19771Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 26Knaapen W.M. Davies M.J. De Bie M. Haven A.J. Martinet W. Kockx M.M. Cardiovasc. Res. 2001; 51: 304-312Crossref PubMed Scopus (219) Google Scholar). The ultrastructure of cells in control conditions (Fig. 6A) is characterized by a normal morphology of cytoplasm, mitochondria, and nucleus. No significant morphological changes in relation to control conditions were observed in cells treated with 100 μm dopamine, 1 mm CuSO4, or 100 μm dicoumarol for 2, 4, and 7 h. However, autophagic vacuoles and residual bodies appeared as a main cellular component characterized by a single or double membrane limited compartment that segregated cytoplasmic constituents during different degradation stages in cells treated for 2 h with 100 μm Cu·DA complex in the absence or presence of 100 μm dicoumarol. Enlarged autophagic compartments were observed in a highly vacuolated cytoplasm after 4 h of treatment with 100 μm Cu·DA complex in the absence and presence of 100 μm dicoumarol. Determination of Autophagosomes—We determined the formation the autophagosome vacuoles induced by Cu·DA complex in the absence or presence of 100 μm dicoumarol in GFP-LC3-trans
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