Attenuation of UV-Induced Apoptosis by Coenzyme Q10 in Human ...

Attenuation of UV-Induced Apoptosis by Coenzyme Q10 in Human Cells Harboring Large-Scale Deletion of Mitochondrial DNA CHENG-FENG LEE,a,c CHUN-YI LIU,a,c SHU-MEI CHEN,a MARIANNA SIKORSKA,b CHEN-YU LIN,a TZU-LING CHEN,a AND YAU-HUEI WEIa aDepartment

of Biochemistry and Molecular Biology, and Center for Cellular and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan

bInstitute

for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada

ABSTRACT: Chronic progressive external ophthalmoplegia (CPEO) syndrome is one of the mitochondrial diseases caused by large-scale deletions in mitochondrial DNA (mtDNA) that impair the respiratory function of mitochondria and result in decreased production of ATP in affected tissues. In order to investigate whether CPEO-associated mtDNA mutations (i.e., 4,366-bp and 4,977bp large-scale deletions) render human cells more vulnerable to apoptosis, we constructed cybrids carrying the deleted mtDNA. Assays for cell viability, DNA fragmentation, cytochrome c release, and caspase 3 activation revealed that UV irradiation at 20 J/m2 triggered apoptosis in all the cybrids. This treatment also produced elevated intracellular levels of reactive oxygen species (ROS). The rate of UV-induced cell death was more pronounced in the cybrids harboring mtDNA deletions than in the control cybrid with wild-type mtDNA. Subsequently, we evaluated the effect of coenzyme Q10 on the UV-triggered apoptosis. The results showed that after pretreatment of the cybrids with 100 ␮M coenzyme Q10 the UV-induced cell damage (i.e., ROS production and activation of caspase 3) was significantly reduced. Taken together, these findings suggest that large-scale deletions of mtDNA increased the susceptibility of human cells to the UV-triggered apoptosis and that coenzyme Q10 mitigated the damage; hence, it might potentially serve as a therapeutic agent to treat mitochondrial diseases resulting from mtDNA deletions. KEYWORDS: mitochondrial DNA; mutation; apoptosis; UV irradiation; coenzyme Q10

c C-F.L. and C-Y.L. contributed equally to this work. Address for correspondence: Professor Yau-Huei Wei, Department of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan. Voice: +886-2-28267118; fax: +886-2-2826-4843. [email protected]

Ann. N.Y. Acad. Sci. 1042: 429–438 (2005). © 2005 New York Academy of Sciences. doi: 10.1196/annals.1338.036 429

430

ANNALS NEW YORK ACADEMY OF SCIENCES

INTRODUCTION In the past four decades more than 100 mitochondrial diseases have been described; most of them affect selective populations of cells in the neuromuscular and central nervous systems. Mitochondrial diseases are often caused by point mutation or deletion of mtDNA and are associated with a broad spectrum of clinical manifestations.1 There is also strong evidence suggesting that mitochondrial function defects, resulting from the qualitative and quantitative alterations of mtDNA, contribute to the development of neurodegenerative diseases.2,3 The abnormalities in mtDNA affect directly the oxidative phosphorylation process and result in the reduction of ATP level and increased production of reactive oxygen species (ROS) in the affected tissues. Indeed, mitochondria not only generate most of the intracellular ROS, but also serve as a key player in the initiation and execution of apoptotic cell death. This, in turn, can worsen the pathophysiology and clinical outcome of the mitochondrial diseases. Although great progress has been made in the fundamental understanding of several mitochondrial disorders, at present there are essentially no effective treatments for these diseases. The general strategy for selecting pharmacological therapies is to use agents that can interfere with the pathological process, or preferably agents that can modify respiratory chain function and reduce the level of cytotoxic metabolites, including ROS. One agent that falls into this category is coenzyme Q10. Coenzyme Q10 is a well-characterized electron carrier of the respiratory chain, which is mainly localized in the inner mitochondrial membrane where it serves as a highly mobile carrier of electrons and protons between the flavoproteins and the cytochrome system.4 Several studies demonstrated that the fully reduced form of ubiquinone (ubiquinol) might function as an antioxidant.5,6 These studies indicate that ubiquinol may prevent both the initiation and propagation phases of lipid peroxidation, because of its location in the hydrophobic region of the membrane lipid bilayer where lipid peroxidation often takes place. In order to evaluate the effects of mtDNA mutations on cellular behavior under stress, we have established cybrids harboring large-scale mtDNA deletions (4,366 bp and 4,977 bp) using skin fibroblasts from patients with clinically proven CPEO syndrome as the mtDNA donors.7 The cybrids were challenged with UV irradiation, a well-known trigger of apoptosis,8 in the presence or absence of coenzyme Q10. We hypothesized that the mutated mtDNA would render the cybrids more vulnerable to the UV-induced cell death and that coenzyme Q10, by acting as an effective antioxidant, might protect them from the UV irradiation-triggered cellular damage.

MATERIALS AND METHODS Cell Culture and Experimental Treatments A series of cybrid clones harboring different proportions of mtDNA with largescale deletions were made by fusing enucleated skin fibroblasts from two CPEO patients with mtDNA-null human osteosarcoma (ρ0) cells.9 The proportion of mtDNA with large-scale deletions in different cybrid clones was quantified by the Southern hybridization method developed in our laboratory.7

LEE et al.: COENZYME Q10 REDUCES APOPTOSIS OF HUMAN CYBRIDS

431

Cybrids were grown in DMEM supplemented with 5% FBS, 100 mg/mL pyruvate, and 50 mg/mL uridine in humidified 5% CO2/95% air and were exposed to 20 J/m2 UV irradiation when 70% confluent was reached. The cells were analyzed 25 h later. In some experiments, prior to the UV exposure, the cybrids were pretreated for 24 h with 100 µM coenzyme Q10 added directly to the culture media from a watersoluble formulation prepared as described below. Cell viability was measured by trypan blue exclusion assay. In brief, cells were harvested by trypsinization, resuspended in phosphate-buffered saline (PBS, pH 7.3), stained with 0.4% trypan blue, and counted using a hemocytometer. Analysis of DNA Fragmentation DNA fragmentation was analyzed by electrophoresis on a 2% agarose gel as described by Herrmann et al.10 Assay of Caspase 3 Activation Caspase 3 activity was measured using a fluorescent substrate, Ac-DEVD-AFC.11 Fluorescence intensity of the cleaved substrate was determined using a Hitachi F-3000 Spectrofluorometer (Hitachi, Ltd., Tokyo) set at an excitation wavelength of 380 nm and an emission wavelength of 508 nm. Western Blotting Cell lysate was separated by electrophoresis on 12% SDS-PAGE, blotted onto a Hybond-P+ membrane (Amersham Biosciences, Uppsala, Sweden) and immunoblotted with anti–caspase 3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Immunofluorescent Staining of Cytochrome c Cells were first incubated with 100 nM MitoTracker Red and then immunostained for cytochrome c with an anti–cytochrome c antibody (Santa Cruz Biotechnology) and FITC-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA). The coverslips were examined under a confocal microscope.12 Measurement of Intracellular ROS After incubation with 80 µM 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Molecular Probes, Eugene, OR)13 at 37°C for 20 min, cells were harvested and resuspended in 0.5 mL HEPES buffer, and fluorescence intensity was measured with a flow cytometer (model EPICS XL-MCL, Beckman Coulter, Miami, FL). Preparation of Water-Soluble Formulation of Coenzyme Q10 Water-soluble formulation of coenzyme Q10 described by Sikorska et al.14 was made using polyoxyethanyl 600 tocopheryl sebacate (PTS-600) as a carrier, prepared according to U.S. patent 6,045,826.15

432


TABLE 1. Summary of biological effects of UV irradiation with and without coenzyme Q10 pretreatment on cybrid clones harboring mutated mtDNA mtDNA mutation Cybrid clones Genotype Mutant mtDNA(%) Viability (%) DNA ladder Control UV Caspase 3 activity Control CoQ10 UV CoQ10 + UV

4,366-bp deletion Lin 0 wild type undetectable 50.2 ± 27.7

Lin 2 mutant 14.3 42.1 ± 17.8

1-3-16 wild type undetectable 35.4 ± 3.4

– +

−

–

−

++

+

++

14.2 ± 5.7 9.9 ± 4.2 260.2 ± 23.3 223.5 ± 44.6

17.2 ± 2.0 14.3 ± 2.9 280.4 ± 25.9 206.6 ± 32.1

8.8 ± 2.7 9.1 ± 4.0 190.8 ± 5.5

150.3 ± 40.1

11.3 ± 2.3 9.1 ± 2.7 222.9 ± 13.3 158.4 ± 14.5

102.1 ± 8.1 86.9 ± 3.3 152.8 ± 11.1 120.7 ± 8.2

ND ND ND ND

ND ND ND ND

Relative intracellular level of H2O2 (%) 100.0 ± 0.0 Control

CoQ10 UV CoQ10 + UV

4,977-bp deletion

76.2 ± 18.6 133.9 ± 19.2 110.4 ± 21.5

51-10 mutant 79.8 14.8 ± 2.4

Note: Caspase 3 activity was assayed 25 h after exposure to UV irradiation at 20 J/m2. The activity of caspase 3 is expressed in arbitrary units. The intracellular level of H2O2 is expressed as a relative value with respect to a control cybrid harboring wild-type mtDNA. ND, not determined.

FIGURE 1. Quantification of the proportion of mtDNA with 4,366-bp deletion in the cybrids by Southern hybridization. The upper and lower bands represent the wild-type mtDNA and mtDNA with 4,366-bp deletion, respectively. The relative amount of deleted mtDNA was then determined by a laser-scanning densitometer.


433

RESULTS Two sets of cybrids harboring the 4,366-bp deletion (Lin 2 with the deletion and control Lin 0 with wild-type mtDNA) and the 4,977-bp deletion (51-10 with the deletion and control 1-3-16 with wild-type mtDNA) were used in this study. The Lin 2 cybrid clone contained 14.3% of mtDNA with 4,366-bp deletion (FIG. 1) and the clone 51-10 harbored 79.8% of mtDNA with 4,977-bp deletion (TABLE 1). The cybrids were subjected to UV irradiation at 20 J/m2 and were analyzed 25 h later. The results are summarized in TABLE 1. After UV exposure, the viability of cybrids, especially those with mutated mtDNA, was significantly reduced. The viability of Lin 2 mutant cybrids dropped by nearly 60% after the UV exposure and the same treatment killed 85% of the 51-10 cybrids. Clearly, there was a positive corre-

FIGURE 2. UV-triggered DNA fragmentation in cybrids harboring 4,366-bp–deleted mtDNA. DNA fragmentation was more extensive in cybrids harboring 4,366-bp mtDNA deletion than in cybrids harboring wild-type mtDNA.

434


FIGURE 3. UV-induced caspase 3 activation in the cybrids harboring 4,366-bp– deleted mtDNA. The activation of caspase 3 by proteolytic cleavage was more pronounced in the cybrids harboring the deletion as compared with the cybrids harboring only the wildtype mtDNA. α-Tubulin was used as an internal standard.

lation between the degree of mitochondrial defects and cell death after exposure of cybrids to UV irradiation. The UV-induced DNA fragmentation was very evident in the Lin cybrids (FIG. 2), and the fragmentation was more pronounced in the Lin 2 mutant cybrid. The same was true for the 51-10 and 1-3-16 cybrid clones (TABLE 1). The treatment triggered activation of caspase 3 (FIGS. 3 and 5A, TABLE 1), which was marginally higher in the mutant cybrids than in controls (Lin 2 vs. Lin 0 by 11% and 51-10 vs. 1-3-16 by 17%); it was accompanied by a release of cytochrome c from mitochondria (FIG. 4). Generation of ROS in response to the irradiation was established by measuring intracellular concentration of hydrogen peroxide (FIG. 5B, TABLE 1). The intracellular level of ROS in the mutant cybrids was approximately 14% higher than that in the relevant controls. In a parallel set of experiments, the Lin cybrids were pretreated for 24 h with 100 µM coenzyme Q10 added directly to cell culture media prior to UV irradiation. This treatment lowered the ROS release by approximately 30% (FIG. 5B, P < 0.05) and caspase 3 activation by nearly 40% (P < 0.05) when analyzed 25 h after the UV exposure (FIG. 5, TABLE 1). Clearly, this pretreatment with coenzyme Q10 was able to offset the severity of the apoptotic response in the cybrids. Taken together, our results showed that UV irradiation triggered, in all the cybrids, apoptotic cell death associated with ROS production and mediated by caspase 3 activation and cytochrome c release. The apoptotic markers were more pronounced in cybrids with defective mtDNA, and sensitivity of the cybrids to apoptosis and their survival after the UV exposure was related to the degree of mitochondrial defects. Coenzyme Q10 pretreatment quenched, to a certain degree, the ROS generation and activation of caspase 3, and thus reduced apoptotic response of the cybrids.

DISCUSSION MtDNA mutations are responsible for a heterogeneous group of mitochondrial diseases. Their heterogeneity, however, cannot be fully explained by the proportion of mutated mtDNA. The clinical manifestations of these diseases are thought to be related not only to the severity of defects in energy metabolism, but also to apoptosis


435

FIGURE 4. Cytochrome c release from mitochondria after UV irradiation. The cybrid (Lin 2) harboring 4,366-bp–deleted mtDNA was double stained with MitoTracker Red (upper panel) and anti-cytochrome c–FITC antibodies (middle panel). Under the control condition without exposure to UV (left panel), mitochondria assumed filamentous structure, and cytochrome c was localized in the mitochondria. However, after exposure to UV (right panel), mitochondria became swollen and moved away from the cell periphery to form aggregates around the nucleus, and cytochrome c was released from mitochondria to the cytoplasm.

436


FIGURE 5. Protective effect of coenzyme Q10 on UV-induced apoptosis in cybrids harboring 4,366-bp–deleted mtDNA. (A) UV-induced caspase 3 activation was less pronounced in cybrids pretreated with 100 µM coenzyme Q10 than in cybrids not exposed to coenzyme Q10 (*P < 0.05). (B) UV-induced increase of hydrogen peroxide was less pronounced in cybrids pretreated with 100 µM coenzyme Q10 compared to the cells without pretreatment with coenzyme Q10 (*P < 0.05).

in affected tissues. Indeed, apoptotic features have been reported in muscle fibers of patients carrying a high proportion of mtDNA with large-scale deletion.16 However, a question as to what extent the presence of mutated mtDNA renders cells more susceptible to apoptosis has not been fully answered. In this study, we used cybrids carrying two kinds of large-scale deletions in mtDNA, and we established that UV irradiation at 20 J/m2 triggered caspase 3 and cytochrome c–mediated apoptosis. The cells’ sensitivity to the UV exposure closely correlated with the degree of mitochondrial defects—that is, the higher the proportion of mutated mtDNA the more vulnerable the cybrids were to the UV irradiation (TABLE 1). These results are consistent with our previous study on UV and staurosporine-induced apoptosis in cybrids harboring mtDNA with A3243G mutation, A8344G mutation, and 4,977 bp-deletion, respectively.17 The molecular mechanism of UV-induced apoptosis is not yet fully understood, although previously published studies suggest that the direct UV damage to the cellular macromolecules such as nucleic acids, lipids, and proteins plays a significant role.8 Another possible mechanism may involve the UV-elicited production of ROS such as superoxide anions and singlet oxygen that further increase oxidative stress burden and cellular damage, including the damage to mtDNA. Here we showed that the increased vulnerability to UV irradiation might, indeed, be related to the higher oxidative stress elicited by increased production of ROS in the cybrids harboring mutated mtDNA. Accordingly, modulation of the ROS production by coenzyme Q10 resulted in the reduced UV-induced apoptosis. Coenzyme Q10 is a naturally occurring lipid-soluble antioxidant that is capable of quenching free radical production and preventing oxidative damage to cellular constituents. It is also a critical component of the mitochondrial respiratory chain and is absolutely essential for the production of ATP. And, coenzyme Q10 has been investigated as a potential therapeutic agent to treat cardiovascular diseases, neurodegenerative diseases, and mitochondrial disorders.18–20 Although numerous beneficial outcomes, both experimental and clinical, have been reported in response to


437

coenzyme Q10 supplementation, its full therapeutic potential is greatly limited by the lack of solubility in aqueous media. This limits its bioavailability and a scope of clinical applications. In this study, to offset the damaging effect of UV exposure, we used a unique aqueous coenzyme Q10 formulation developed in Dr. Sikorska’s laboratory at the National Research Council, Canada.14,15 The results showed that pretreatment of the cybrids with 100 µM coenzyme Q10 was protective against UV irradiation. This observation is consistent with the previously reported beneficial effects of coenzyme Q10—that is, improved function of pancreatic beta-cells from patients with diabetes mellitus and MELAS syndrome,21 offsetting a paraquat-induced neurotoxicity,22 mitigating endotoxemic and ischemic brain damage in rodents.23 These studies imply that water-soluble coenzyme Q10 could also be an effective therapeutic agent for the treatment of mitochondrial diseases. In addition to scavenging free radicals, coenzyme Q10 may improve mitochondrial function by stabilizing the inner membrane to facilitate mitochondrial respiration and efficiency of ATP synthesis.24 Taken together, we have demonstrated that large-scale deletions of mtDNA render human cells more vulnerable to apoptosis triggered by UV irradiation. The data support the notion that apoptosis might play an important role in the development of clinical symptoms such as muscle weakness and neurological disorders characteristic of the mitochondrial encephalomyopathies. We have also demonstrated that coenzyme Q10 can interfere with the apoptotic mechanisms, and it therefore might be an effective drug to alleviate the symptoms and slow down the progression of mitochondrial diseases.

ACKNOWLEDGMENTS This work was supported by a grant (NSC92-2321-B-010-011-YC) from the National Science Council and a grant (EX93-9120BN) from the National Health Research Institutes, Executive Yuan, Taiwan. REFERENCES 1. WALLACE, D.C. 1999. Mitochondrial diseases in man and mouse. Science 283: 1482– 1488. 2. LIN, F.H., R. LIN, H.M. WISNIEWSKI, et al. 1992. Detection of point mutations in codon 331 of mitochondrial NADH dehydrogenase subunit 2 in Alzheimer’s brains. Biochem. Biophys. Res. Commun. 182: 238–246. 3. IKEBE, S., M. TANAKA & T. OZAWA. 1995. Point mutations of mitochondrial genome in Parkinson’s disease. Brain Res. Mol. Brain Res. 28: 281–295. 4. MITCHELL, P. 1975. Protonmotive redox mechanism of the cytochrome b-c1 complex in the respiratory chain: protonmotive ubiquinone cycle. FEBS Lett. 56: 1–6. 5. FORSMARK-ANDREE, P. & L. ERNSTER. 1994. Evidence for a protective effect of endogenous ubiquinol against oxidative damage to mitochondrial protein and DNA during lipid peroxidation. Mol. Aspects Med. 15: S73–S81. 6. SANDHU, J.K., S. PANDEY, M. RIBECCO-LUTKIEWICZ, et al. 2003. Molecular mechanisms of glutamate neurotoxicity in mixed cultures of NT2-derived neurons and astrocytes: protective effects of coenzyme Q10. J. Neurosci. Res. 72: 691–703. 7. WEI, Y.H., C.F. LEE, H.C. LEE, et al. 2001. Increases of mitochondrial mass and mitochondrial genome in association with enhanced oxidative stress in human cells harboring 4,977 BP-deleted mitochondrial DNA. Ann. N.Y. Acad. Sci. 928: 97–112.

438


8. KULMS, D. & T. SCHWARZ. 2000. Molecular mechanisms of UV-induced apoptosis. Photodermatol. Photoimmunol. Photomed. 16: 195–201. 9. KING, M.P. & G. ATTARDI. 1989. Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science 246: 500–503. 10. HERRMANN, M., H. M. LORENZ, R. VOLL, et al. 1994. A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Res. 22: 5506–5507. 11. SHIAH, S.G., S.E. CHUANG, Y.P. CHAU, et al. 1999. Activation of c-Jun NH2-terminal kinase and subsequent CPP32/Yama during topoisomerase inhibitor beta-lapachoneinduced apoptosis through an oxidation-dependent pathway. Cancer Res. 59: 391– 398. 12. JIANG, S., J. CAI, D.C. WALLACE, et al. 1999. Cytochrome c-mediated apoptosis in cells lacking mitochondrial DNA. Signaling pathway involving release and caspase 3 activation is conserved. J. Biol. Chem. 274: 29905–29911. 13. LEE, H.C., P.H. YIN, C.Y. LU, et al. 2000. Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells. Biochem. J. 348: 425–432. 14. SIKORSKA, M., H. BOROWY-BOROWSKI, B. ZURAKOWSKI, et al. 2003. Derivatised alphatocopherol as a CoQ10 carrier in a novel water-soluble formulation. Biofactors 18: 173–183. 15. BOROWY-BOROWSKI, M., M. SIKORSKA & P.R. WALKER. 2004. Water-soluble composition of bioactive lipophilic compounds. U.S. Patents 6,045,826 and 6,191,172 B1. 16. MIRABELLA, M., S. DI GIOVANNI, G. SILVESTRI, et al. 2000. Apoptosis in mitochondrial encephalomyopathies with mitochondrial DNA mutations: a potential pathogenic mechanism. Brain 123: 93–104. 17. LIU, C.Y., C.F. LEE, C.H. HONG, et al. 2004. Mitochondrial DNA mutation and depletion increase the susceptibility of human cells to apoptosis. Ann. N.Y. Acad. Sci. 1011: 133–145. 18. BEAL, M.F. 2002. Coenzyme Q10 as a possible treatment for neurodegenerative diseases. Free Radical Res. 36: 455–460. 19. LANGSJOEN, P.H. & A.M. LANGSJOEN. 1999. Overview of the use of CoQ10 in cardiovascular disease. Biofactors 9: 273–284. 20. SOBREIRA, C., M. HIRANO, S. SHANSKE, et al. 1997. Mitochondrial encephalomyopathy with coenzyme Q10 deficiency. Neurology 48: 1238–1243. 21. LIOU, C.W., C.C. HUANG, T.K. LIN, et al. 2000. Correction of pancreatic beta-cell dysfunction with coenzyme Q10 in a patient with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome and diabetes mellitus. Eur. Neurol. 43: 54–55. 22. MCCARTHY, S., M. SOMAYAJULU, M. SIKORSKA, et al. 2004. Paraquat induces oxidative stress and neuronal cell death: neuroprotection by water-soluble coenzyme Q10. Toxicol. Appl. Pharmacol. 201: 21–31. 23. CHUANG, Y.C., J.Y. CHAN, A.Y. CHANG, et al. 2003. Neuroprotective effects of coenzyme Q10 at rostral ventrolateral medulla against fatality during experimental endotoxemia in the rat. Shock 19: 427–432. 24. TURUNEN, M., J. OLSSON & G. DALLNER. 2004. Metabolism and function of coenzyme Q. Biochim. Biophys. Acta 1660: 171–199.