From the
Department of Experimental Pathology and Oncology and
Department of Anatomy, Histology, and Forensic
Medicine, University of Florence, 50134-Florence, and the
¶Department of Ophthalmology and Visual Sciences,
University-Hospital San Raffaele of Milan, 20132 Milan, Italy
Received for publication, March 5, 2003 , and in revised form, April 18, 2003.
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ABSTRACT |
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INTRODUCTION |
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The mitochondrial permeability transition pore (PTP) is a complex, large
conductance channel that plays a pivotal role in triggering apoptosis
(12). The opening of PTP is
responsible for disruption of the mitochondrial transmembrane electrochemical
gradient ( from 180 to 0 mV). The oligomerization of
apoptotic members of the bcl-2 family follows, thus forming
mitochondrial transmembrane channels through which a number of apoptotic
factors are released into the cytoplasm to trigger the mitochondrion-dependent
(intrinsic) pathway of apoptosis execution
(1315).
This is orchestrated by caspase cascade activation, which leads to cleavage of
cellular substrates. Mitochondrion-dependent (intrinsic) and membrane death
receptor-dependent (extrinsic) apoptotic pathways are known to share the same
caspases with two exceptions: caspase 9 for the former
(16) and caspase 8 for the
latter (17). Factors and
mechanisms involved in PTP formation are still unclear. Nevertheless, three
proteins, the adenine nucleotide translocator, the voltage-dependent anion
channel, and cyclophilin-D, appear to play the main structural role
(1315,
18). Besides a number of
ancillary structural/regulative proteins, such as benzodiazepine peripheral
receptor, creatine kinase, hexokinase, and Bax, that are recruited by the
voltage-dependent anion channel-adenine nucleotide translocator-cyclophilin-D
complex, several molecules are positive (Bax, Ca2+, ROS,
and atractyloside) or negative (Bcl-2, ATP, bongkrekic acid, and cyclosporin
A) determinants of PTP opening
(15,
18).
Some authors (1924) demonstrated in isolated mitochondria that the PTP harbors a ubiquinone-binding site and is regulated by complex I of the mitochondrial respiratory chain. Irrespective of the method used to induce permeability transition, the opening of PTP was strongly inhibited by some ubiquinone analogues, including Ub0, decyl-Ub, and Ub10, whereas other tested quinones were ineffective or had antithetic effects (23). Specific structural features have been demonstrated to be necessary for regulation of PTP by ubiquinone analogues, independently of their ability to decrease mitochondrial ROS production to the same extent. These results supported the idea that PTP regulation by ubiquinones is exerted through binding to a common site rather than through redox reactions (23). Interestingly, the natural CoQ10 has a very strict structural analogy with the synthetic Ub10 (CoQ2); as shown in Fig. 1, their difference is restricted to the number of side isoprenoid units within the side chain, which has been reported to not influence substantially the folding of the molecule (25). These considerations prompted us to hypothesize that what was observed with ubiquinone analogues applied to isolated mitochondria (1924) could underlie a natural phenomenon operating in the whole cell. In line with this possibility, the respiratory chain complex I has been suggested to be a constituent of mitochondrial PTP (26). Armstrong and co-workers (27, 28) have designed a new model for mitochondrial PTP, in which the ROS producer respiratory chain complex III is functionally linked to the permeability transition. The ability of complex III to generate ROS and to open mitochondrial PTP could override the antiapoptotic function of Bcl-2. Noteworthy, both respiratory chain complexes I and III share CoQ10 as electron acceptor or electron donor, respectively. These observations led us to explore the possibility that, besides its free radical scavenging property, CoQ10 may be endowed with antiapoptotic activity as a modulator of PTP opening.
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In this study we demonstrate that CoQ10 provides effective
protection against apoptosis of rabbit keratocytes not only in response to the
free radicals generated by UVC irradiation but also in response to three
apoptotic stimuli generally known to act independently of free radical
generation such as chemical hypoxic drug antimycin A
(29), the
C2-ceramide, a cell-permeable analogue of natural lipidic apoptotic
messenger ceramide (30), and
survival factor withdrawal (serum starvation)
(18). This evidence emerged
from examination of two classes of different parameters: 1) ROS, MDA, and
superoxide dismutase (SOD) activity levels for assessment of the effect of our
treatments on free radical generation; and 2) cellular morphology, number of
living and apoptotic cells, ATP levels, and DNA status for assessment of the
effect of our treatments on cell life and death. In contrast, another well
known free radical scavenger, vitamin E (-tocopherol)
(3132),
whose effect in association with CoQ10 has been described in our
previous work (5), provides
effective protection against apoptosis only in response to UVC
irradiation.
The antiapoptotic activity of CoQ10 was mediated by hindering mitochondrial depolarization, cytochrome c release to cytoplasm, and procaspase 9 activation. This suggests that the mechanism by which CoQ10 prevents apoptosis may involve inhibition of mitochondrial PTP opening that triggers the intrinsic execution pathway of apoptosis. This possibility and the hypothesis that this novel functional activity may underlie physical localization of CoQ10 in the PTP, possibly in association with other components of the complex, is now under study.
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EXPERIMENTAL PROCEDURES |
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TreatmentsEach of the four damaging agents was applied at
doses experimentally established to induce apoptosis: UVC irradiation (254 nm)
at 15 mJ/cm2, the respiratory chain blocker antimycin A at 200
µM concentration, the apoptotic signaling lipid
C2-ceramide (a synthetic cell-permeable analogue of endogenous
ceramides) at 20 µM concentration, and FBS restriction to 0.5%.
Treatments with 10 µM CoQ10 or with 10
µM vitamin E (-tocopherol), both dissolved in 0.04%
Lutrol F127 used as vehicle to ensure cellular uptake of this hydrophobic
molecule (5,
6), were commenced 2 h prior to
application of apoptotic stimuli. Vehicle alone-treated cells were used as
controls. In some experiments vitamin C (ascorbic acid) was also used.
Identification of Apoptosis on the Basis of Cellular Morphology and DNA End Labeling by Klenow-FragELTM AssayApoptotic RCE cells were identified by light microscopy following end labeling of DNA fragments by Klenow-FragELTM kit (Oncogene Research Products, Boston). Briefly, following apoptotic treatments, the keratocytes were detached from substrate and fixed in 4% formaldehyde and 80% ethanol at a concentration of 106 cells/ml. Prior to detection, the cells (3 x 105) were affixed to glass slides by cytocentrifugation, air-dried, and processed according to the manufacturer's instructions. In this assay Klenow binds to exposed ends of DNA fragments and catalyzes the template-dependent addition of biotin-labeled deoxyribonucleotides, which are then detected using streptavidin-horseradish peroxidase conjugate. Diaminobenzidine reacts with the labeled fragmented DNA of apoptotic cell nuclei, generating a dark brown insoluble chromogen that contrasts with counterstained methyl green cytoplasms. In contrast, viable cells appear uniformly green or even unstained.
Measurement of ROS and MDA-4-HNE Levels and of SOD Activity The extent of free radical generation by cultured cells exposed to UVC, antimycin A, C2-ceramide, or FBS restriction to 0.5% was measured by means of three independent indexes: reactive oxygen species (ROS), lipid peroxidation level, and superoxide dismutase activity. Generation of ROS was assessed immediately after treatments, using 2',7'-dichlorofluorescein diacetate (Sigma) (33), by a confocal laser scanning microscope (Bio-Rad MRC 1024 ES scanning microscope) equipped with a krypton/argon laser source. The emitted fluorescence was monitored at 488 and 568 nm wavelengths with a Nikon plan Apo x 60 oil-immersion objective. To this purpose, series of optical sections (512 x 512 pixels) were taken through the depth of cells with a thickness of 1 µm at intervals of 0.8 µm. Twenty optical sections for each samples examined were projected as a single composite image by superimposition. To monitor the rate of lipid peroxidation, the levels of typical end products of the process, which are MDA plus 4-hydroxy-2-alkenals, exemplified by 4-hydroxy-2-nonenal (4-HNE), were determined in the cellular extracts prepared and analyzed at 2 h following treatments. Measurements were made using a colorimetric method at 586 nm, according to the reaction of a chromogenic reagent, N-methyl-2-phenylindole, with MDA and 4-HNE in the presence of methanesulfonic acid at 45 °C (34). SOD activity was determined at 2 h following treatments using a spectrophotometric assay (Bioxytech, Portland, OR). For this purpose, RCE cells were lysed in 4 volumes of water added to 1 volume of cells, and the cellular lysates were extracted with ethanol/chloroform 62.5: 37.5 (v/v) and vortexed for 30 s. Cellular membranes were separated by centrifugation at 3000 x g at 4 °C for 10 min. The upper phase was collected to determine SOD activity. The absorbance of samples was measured spectrophotometrically at 525 nm.
Quantification of Living and Apoptotic CellsThe number of living cells was evaluated by the MTT (C,N-diphenyl-N'-4,5-dimethyl thiazol-2-yl tetrazolium bromide) colorimetric assay (reduction of tetrazolium salt to formazan as described previously (35)). The cumulative apoptotic events were scored by the time-lapse videomicroscopy using a Zeiss inverted phase contrast microscope equipped with a 10x objective, Panasonic CCD cameras, and JVC BR9030 time-lapse video recorders. After cell detachment from the substrate, an apoptotic event was counted the moment the cell had shrunk completely and blebbing started (36).
Analysis of ATP LevelsCellular levels of ATP were evaluated 24 h following apoptotic stimuli, using untreated cells as control as reported previously (36). The cells (23 x 105) were pelletted, resuspended in distilled water, and boiled for 35 min. Samples were then cooled to room temperature and stored frozen at 20 °C for later measurements. ATP in the extracts was quantified by a bioluminescence assay with an ATP determination kit (Molecular Probes), using a liquid scintillation analyzer (Camberra Packard) for bioluminescence analysis, according to the manufacturer's instructions.
Detection of Change in Mitochondrial Transmembrane Potential
()The change in
occurring during
apoptosis was detected by fluorescence-based assay. The RCE cells were
cultured on coverslips in Dulbecco's modified Eagle's medium containing the
lipophilic cationic probe 5,5',
6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimidazol-carbocyanine
iodide (JC-1, 5 mg/ml, Molecular Probes, Eugene, OR) for 15 min at 37 °C.
This dye has a unique feature: at hyperpolarized membrane potentials (to
140 mV) it forms a red fluorescent J-aggregate, whereas at depolarized
membrane potentials (to 100 mV) it remains in the green fluorescent
monomeric form. Prior to detection, cells were washed in phosphate-buffered
saline and placed in an open slide-flow loading chamber that was mounted on
the stage of a confocal scanning microscope (Bio-Rad) equipped with a
krypton/argon laser source. The emitted fluorescence was monitored at 488 and
568 nm wavelengths with a Nikon plan Apo x60 oil-immersion objective
(37). Imaging acquisition of
the optical sections through the cell was performed as described above.
Western Blot Analysis of Cytoplasmic Cytochrome cRCE cells were evaluated 24 h following application of apoptotic stimuli. Cytosolic fractions were prepared as reported previously (38). Proteins in the cytosolic extracts were quantified by the BCA Protein Assay Reagent (Pierce). Proteins (25 µg/lane) were electrophoresed through SDS-polyacrylamide 12.5% gel and electroblotted onto nitrocellulose membrane (Schleicher & Schuell) using a transblotter (Bio-Rad). The nonspecific signals were blocked with blocking buffer (5% w/v instant nonfat milk powder in phosphate-buffered saline) and incubated overnight at 4 °C with 1 µg/ml of anti-cytochrome c monoclonal antibody (Pharmingen). The membrane was washed and subsequently incubated with goat anti-mouse IgG horseradish peroxidase conjugate (Sigma). Detection was carried out using a commercial chemiluminescence procedure (Amersham Biosciences).
Analysis of Caspase 9 ActivityCaspase 9 activity was determined by the Caspase-9 Colorimetric Protease Assay (BioSource Europe, S.A., Nivelles, Belgium). Cytosolic extracts, prepared by lysing cells with Cell Lysis Buffer provided in the kit, were incubated with the colorimetric substrate Leu-Glu-His-Asp (LEHD) conjugated to the chromophore p-nitroanilide, in 50 µl of 2x Reaction Buffer containing 10 mM dithiothreitol. After a 2-h incubation at 37 °C, the absorbance of samples was measured at A405 nm in Bio-Rad enzyme-linked immunosorbent assay reader.
Analysis of DNA (Nucleosomal Laddering)Apoptotic internucleosomal DNA fragmentation was evaluated by classical assay, detecting electrophoretically a separated ladder of fragmented DNA. The genomic DNA was extracted from RCE cells as described by Blankenberg et al. (39). The fragments were separated by gel electrophoresis in 0.8% agarose containing ethidium bromide (0.2 µg/ml), UV-visualized, and photographed.
Statistical AnalysisThe statistical evaluation of the data was performed with the two-tailed Student's t test for unpaired values. Differences were considered statistically significant when p < 0.05. The data are reported as percentage of the maximal value.
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RESULTS |
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Free Radical Generation Was Restricted to UVC-IrradiationWe have then analyzed the effects of the apoptotic doses of UVC irradiation, antimycin A, C2-ceramide and serum starvation on free radical generation. For this purpose, three different parameters, which are cellular levels of ROS, MDA-4-HNE levels, and SOD activity, were analyzed after application of apoptotic stimuli (Fig. 2). As shown in Fig. 2A, UVC irradiation was the only apoptotic stimulus able to generate ROS as visualized, immediately after treatment, by confocal microscopy following ROS labeling with the fluorescent indicator (2', 7'-dichlorofluorescein diacetate) as described by Formigli et al. (36). In keeping with this observation, Fig. 2B shows that only UVC irradiation markedly enhanced MDA-4-HNE levels (left panel) and SOD activity (right panel), evaluated at the 2nd h after treatment, compared with untreated cells (from 5.6 nmol/mg of protein to 19.4 nmol/mg of protein, and from 4.8 units/mg of protein to 17.2 units/mg of protein, respectively). Increase of ROS, MDA-4-HNE, and SOD activity induced by UVC irradiation was substantially lowered by a 2-h pretreatment with either 10 µM CoQ10 or vitamin E (Fig. 2C).
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CoQ10 but Not Vitamin E Increased Cell Survival in Response to Free Radical Independent Apoptotic StimuliOnce ascertained that apoptosis by antimycin A, C2-ceramide and serum starvation was not consequent to free radical generation, the effects of a 2-h pretreatment with 10 µM CoQ10 or vitamin E on cell survival at the 24th h following application of apoptotic stimuli was evaluated by the MTT assay. As shown in Fig. 3, UVC irradiation, antimycin A, C2-ceramide, and serum starvation induced a dramatic decrease in the number of living cells compared with untreated controls, which was significantly attenuated by pretreatment with CoQ10 (upper panel: from 82 to 49%, from 56 to 29%, from 61 to 24%, and from 51 to 80%, respectively). On the contrary, pretreatment with vitamin E was able to attenuate significantly this decrease only in response to UVC irradiation (lower panel: from 82 to 58%).
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The effects of the 2-h pretreatment with 10 µM CoQ10 or vitamin E on cell apoptosis induced by UVC irradiation, antimycin A, C2-ceramide, and serum starvation were then evaluated using light microscopy and ultramicroscopy (not shown) as reported previously (3, 6, 36). Identification of apoptotic cells with fragmented DNA was also carried out by end labeling with the Klenow-FragELTM, by which apoptotic cells are easily recognized by the presence of a dark brown stain in contrast to viable cells that appear green or even unstained. At the 24th h following treatment with UVC irradiation, antimycin A, C2-ceramide, and serum starvation, a significant number (about 3060% depending on type of apoptotic stimulus) of RCE cells contained brown-stained fragmented DNA (apoptotic cells). Treatment with 10 µM CoQ10 2 h before application of apoptotic stimuli dramatically reduced the number of brown-stained cells, so that the number of cells stained green (viable cells) overcame 90% of all cells. Treatment with 10 µM vitamin E significantly reduced brown-stained (apoptotic) cells in response to UVC irradiation but not in response to the other, free radical unrelated, apoptotic stimuli. Results obtained with UVC and serum starvation, either pretreated or not with CoQ10 or vitamin E, are shown in Fig. 4. Quite similar results have been obtained using 10 µM vitamin C (ascorbic acid) as antioxidant (not shown).
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CoQ10 Decreased the Number of Apoptotic Events in Response to Free Radical Independent Apoptotic StimuliThe possibility that CoQ10 increased cell survival by inhibiting apoptosis was then evaluated by time-lapse videomicroscopy at 24 h following UVC irradiation or treatment with antimycin A, C2-ceramide, or serum starvation. Following all treatments, a high number of apoptotic RCE cells was observed in cultured plates, which dramatically decreased if cells were pretreated with CoQ10 (not shown). Corresponding quantitative data are reported in Fig. 5. When compared with untreated or CoQ10-pretreated controls, the number of cumulative apoptotic events scored by time-lapse videomicroscopy markedly increased, following application of all apoptotic stimuli, but to a significantly lower extent if the cells were pretreated with CoQ10 for 2 h before the induction of apoptosis. These results clearly indicated that CoQ10 was able to increase cell survival by preventing apoptosis even in response to stimuli that do not generate free radicals.
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CoQ10 Attenuated Lowering of ATP Levels in Response to All Apoptotic StimuliExecution of the apoptotic death program requires massive consumption of ATP and, consequently, is accompanied by a dramatic reduction in ATP cellular levels. As shown in Fig. 6, UVC irradiation, antimycin A, C2-ceramide, and serum starvation markedly lowered ATP cellular levels as compared with untreated controls. Nevertheless, this lowering was significantly attenuated by a 2-h pretreatment with CoQ10 (from 65 to 28%, from 76 to 41%, from 81 to 51%, and from 60 to 27%, respectively, as compared with respective controls). In further experiments the effects of CoQ10 in response to UVC irradiation were omitted.
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CoQ10 Counteracted Mitochondrial
Depolarization, Cytochrome c Release, Caspase 9 Activation, and DNA
Fragmentation in Response to All Apoptotic StimuliThe effects of
antimycin A, C2-ceramide, and serum starvation on mitochondrial
membrane charge was quantified by the uptake of JC-1
(Fig. 7). The shift in membrane
charge was observed as disappearance of fluorescent red-orange-stained
mitochondria (large negative ) and an increase in fluorescent
green-stained mitochondria (loss of
). Following application of
apoptotic stimuli most cells underwent mitochondrial membrane depolarization;
indeed, cells with red-orange-stained mitochondria dropped to
20% of
total cells. Treatment with CoQ10 before application of apoptotic
stimuli prevented significantly mitochondrial membrane depolarization, because
more than 60% of cells examined maintained red-orange-stained mitochondria.
CoQ10 was most efficient in serum-starved cells and had less effect
in response to antimycin A or C2-ceramide.
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Cytochrome c, caspase 9 activity, and DNA status were analyzed as indexes of apoptosis execution by means of the intrinsic (mitochondrion-dependent) pathway (14) (Fig. 8). Fig. 8A shows the release of cytochrome c to cytoplasm, evaluated by Western blotting analysis; at 24 h after application of all free radical unrelated apoptotic stimuli, cytoplasmic levels of cytochrome c markedly increased but remained substantially unaffected if treatments were preceded by CoQ10 administration. Similarly, Fig. 8B shows that caspase 9 activity undergoes a 68-fold enhancement at 24 h after application of apoptotic stimuli. This enhancement was dramatically lower when apoptotic stimuli application was preceded by CoQ10 administration. Fig. 8C shows that pretreatment with CoQ10 also prevented DNA internucleosomal fragmentation elicited by all free radical unrelated apoptotic stimuli (40).
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The ability of CoQ10 to prevent cytochrome c release and caspase 9 activation in response to free radical unrelated apoptotic stimuli, two events that are triggered by PTP opening, suggests that, independently of its free radical scavenging property, CoQ10 inhibits apoptosis by directly maintaining mitochondrial PTP in the closed conformation. Finally, prevention by CoQ10 of DNA internucleosomal fragmentation indicates that blocking of intrinsic apoptotic pathway by pretreatment with CoQ10 suffices to prevent the "ignition" of the entire apoptotic machinery triggered by apoptotic stimuli.
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DISCUSSION |
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By using the same keratocyte cell line (RCE), we show here that CoQ10 prevents apoptosis also in response to apoptotic stimuli that do not generate free radicals, which are antimycin A, C2-ceramide, and serum starvation. UVC irradiation, a well known free radical-generating damaging agent, has been used as control. We also demonstrate that the mechanism underlying the free radical scavenging independent antiapoptotic properties of CoQ10 is inhibition of mitochondrial depolarization.
Multiple and often interrelated mechanisms have been described to account
for the effects of a single apoptotic stimulus. The damaging effect of UVC
irradiation is consequent to direct alteration of molecular substrates, such
as proteins and nucleic acids, or mediated by generation of free radicals,
which are mainly reactive oxygen species
(41). Antimycin A is a
chemical hypoxic drug that has been reported to prevent free radical
generation (27,
4247).
Antimycin A induces apoptosis by poisoning the mitochondrial respiratory chain
complex III (29) but also by
mimicking a cell death-inducing Bcl-2 homology domain
(48). C2-ceramide
is a cell-permeable analogue of physiological ceramides, a small group of
membrane sphingolipids involved in cell growth, differentiation, and apoptosis
(30,
49,
50). Ceramide-induced
apoptosis is mainly consequent to its ability to collapse mitochondrial
(51), either by
direct inhibition of mitochondrial respiratory chain complex III
(52) or by formation of large
transmembrane channels that raise mitochondrial permeability
(53,
54). Withdrawal of survival
factors, achieved in cultured cells by serum starvation, commit cells to
apoptosis by different mechanisms, such as mitogen-activated protein kinase
induction (55), ceramide
release (56), and COX-2
activation (57), whose common
target is triggering the mitochondrion-dependent apoptotic pathway
(18). On the other hand, some
authors have reported that treatments with antimycin A
(58,
59), C2-ceramide
(60), and serum starvation
(11) can also lead to ROS
generation. Because apoptosis execution is accompanied by ROS generation
(27,
28,
41,
61,
62), whether a ROS increase
during apoptosis is a cause or an effect of this phenomenon is not easy to
establish. Because this point is very crucial for our study, in which it was
critical to exclude antimycin A, C2-ceramide, and serum starvation
as directly generating free radicals, we quantified three different free
radical generation indexes: ROS, MDA, and SOD activity levels. Quantification
was performed early after application of apoptotic stimuli, which is prior to
commencement of apoptosis execution as monitored by time-lapse
videomicroscopy. A marked increase in ROS, MDA, and SOD activity levels
occurred in response to UVC irradiation but not to treatment with
antimycinAorC2-ceramide or serum starvation, which indicated that,
at least in our experimental system, the three latter apoptotic stimuli do not
generate free radicals.
The administration of CoQ10 2 h prior to apoptotic stimuli
prevents apoptosis not only in response to UVC irradiation but also to
antimycin A, C2-ceramide, or serum starvation, i.e.
independently of the ability of apoptotic stimuli to trigger or not trigger
free radical generation. This protective effect was clearly demonstrated by
several evidences, including changes of cell morphology detected by light
microscopy and ultramicroscopy, quantification of living and apoptotic cells,
and analysis of ATP cellular levels. Indeed, CoQ10 significantly
enhances the number of living cells evaluated by the MTT analysis and lowers
the number of cumulative apoptotic events scored by time-lapse videomicroscopy
in response to any apoptotic stimulus, whereas the free radical scavenger
vitamin E was effective only against the free radical-generating UVC
irradiation. In line with this assertion, pretreatment with CoQ10
was able to prevent the massive reduction in ATP levels induced by all
apoptotic stimuli, a phenomenon that is typically associated with the
energy-consuming apoptosis execution and in particular with
collapse consequent to PTP opening.
We suggest that the mechanism by which CoQ10 exerts its
antiapoptotic activity is associated with inhibition of PTP opening. This
possibility is supported by a body of literature. First, Fontaine et
al.
(2022)
have demonstrated that Ca2+-dependent opening of PTP in
isolated mitochondria can be prevented by two synthetic quinone analogues,
ubiquinone 0 and the decyl-ubiquinone, whereas the other quinones tested are
ineffective; on this basis, they have proposed a model of mitochondrial PTP
that harbors a ubiquinone-binding site. Second, Walter et al.
(23) have identified the
structural features required for regulation of the mitochondrial PTP by
ubiquinone analogues and defined three functional classes of ubiquinone
analogues, PTP inhibitors, PTP inducers, and PTP-inactive quinones, on the
basis of their ability to keep the mitochondrial PTP open or closed or to
counteract the effects of both inhibitors and inducers. Because all
ubiquinones used decrease mitochondrial ROS production to the same extent,
they claim that their different regulative effect on PTP, when present, is
mediated by their binding to a common site rather than by their free radical
scavenging activity, possibly through secondary changes in the PTP
Ca2+ binding affinity. Our result indicating that
CoQ10, but not vitamin E, prevents mitochondrial depolarization,
cytochrome c release, and caspase 9 activation induced by apoptotic
stimuli strongly support this possibility. Indeed, these three sequential
events are specifically associated with opening of PTP and lead to
mitochondrion-dependent apoptosis execution. The PTP opening-dependent
mitochondrial permeability transition collapses the mitochondrial
and induces a massive ATP hydrolysis, an early event in the apoptotic pathway.
The consequent release of cytochrome c into cytoplasm and caspase 9
activation triggers caspase cascade that culminates in DNA laddering and
apoptosis (12). All these
apoptosis execution-related events occurred in our experimental model and were
substantially prevented by treatment with CoQ10. On the basis of
these considerations, CoQ10 may be a candidate as a functional
analogue of the Bcl-2 protein
(63). Moreover, association of
complexes I and III of the mitochondrial respiratory chain with mitochondrial
PTP (19,
20,
2328)
and the integration of CoQ10 in both complexes suggest the role of
CoQ10 as a structural element and modulator of mitochondrial PTP.
Experiments are now in progress to obtain direct evidence of this possibility
and to "pin-point" localization of CoQ10 in the PTP
complex.
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FOOTNOTES |
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Recipient of a fellowship from the Federazione Italiana Ricerca sul
Cancro.
|| To whom correspondence should be addressed: Dept. of Experimental Pathology and Oncology, University of Florence, Viale G.B. Morgagni 50, 50134-Florence, Italy. Tel.: 39-55-4282308; Fax: 39-55-4282309; E-mail: sergio{at}unifi.it.
1 The abbreviations used are: CoQ10, coenzyme Q10; PTP,
permeability transition pore; ROS, reactive oxygen species; FBS, fetal bovine
serum; MDA, malondialdehyde; 4-HNE, 4-hydroxy-2-nonenal; SOD, superoxide
dismutase; Ub, ubiquinone; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ,
mitochondrial membrane potential.
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ACKNOWLEDGMENTS |
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REFERENCES |
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