Mitochondrial Respiration Is Uniquely Associated with the Prooxidant and Apoptotic Effects ofN-(4-Hydroxyphenyl)retinamide
2001; Elsevier BV; Volume: 276; Issue: 49 Linguagem: Inglês
10.1074/jbc.m106559200
ISSN1083-351X
Autores Tópico(s)Bioactive Compounds and Antitumor Agents
ResumoThe synthetic retinoidN-(4-hydroxyphenyl)retinamide (4HPR) is being examined in both chemoprevention and therapy clinical trials. Yet, its mechanism(s) of action is still not fully elucidated. In previous studies, an increase in mitochondrial reactive oxygen species has been proposed as one mechanism through which 4HPR could exert its proapoptotic effects. This study explored whether mitochondrial respiration is required for 4HPR action using human cutaneous squamous cell carcinoma cells and respiration-deficient clones. In parental cells, 4HPR rapidly promoted hydroperoxide production followed by mitochondrial permeability transition, caspase activity, and DNA fragmentation. Short term exposure to 4HPR also inhibited oxygen consumption in parental cells. This activity was reversed by the antioxidant vitamin C indicating the prooxidant effect of 4HPR directly impaired mitochondrial function. In respiration-deficient clones, the proapoptotic qualities of 4HPR were conspicuously diminished illustrating a central role for mitochondrial respiration in 4HPR-induced cell death. In parental cells, various mitochondrial inhibitors were examined to determine potential sites associated with the prooxidant activity of 4HPR. Inhibitors of Complex II as well as center i inhibitors of Complex III enhanced 4HPR-induced hydroperoxide production. Complex I inhibitors, centero inhibitors of Complex III, cyanide, oligomycin A, and coenzyme Q analogues decreased 4HPR-induced hydroperoxide production. The coenzyme Q analogues were very effective in this respect, and they also blocked the enhanced hydroperoxide production obtained when centeri inhibitors were combined with 4HPR. These results suggest the prooxidant property of 4HPR is associated with redox metabolism via an enzymatic process occurring at a quinone-binding site in Complex I and/or center o of Complex III. The synthetic retinoidN-(4-hydroxyphenyl)retinamide (4HPR) is being examined in both chemoprevention and therapy clinical trials. Yet, its mechanism(s) of action is still not fully elucidated. In previous studies, an increase in mitochondrial reactive oxygen species has been proposed as one mechanism through which 4HPR could exert its proapoptotic effects. This study explored whether mitochondrial respiration is required for 4HPR action using human cutaneous squamous cell carcinoma cells and respiration-deficient clones. In parental cells, 4HPR rapidly promoted hydroperoxide production followed by mitochondrial permeability transition, caspase activity, and DNA fragmentation. Short term exposure to 4HPR also inhibited oxygen consumption in parental cells. This activity was reversed by the antioxidant vitamin C indicating the prooxidant effect of 4HPR directly impaired mitochondrial function. In respiration-deficient clones, the proapoptotic qualities of 4HPR were conspicuously diminished illustrating a central role for mitochondrial respiration in 4HPR-induced cell death. In parental cells, various mitochondrial inhibitors were examined to determine potential sites associated with the prooxidant activity of 4HPR. Inhibitors of Complex II as well as center i inhibitors of Complex III enhanced 4HPR-induced hydroperoxide production. Complex I inhibitors, centero inhibitors of Complex III, cyanide, oligomycin A, and coenzyme Q analogues decreased 4HPR-induced hydroperoxide production. The coenzyme Q analogues were very effective in this respect, and they also blocked the enhanced hydroperoxide production obtained when centeri inhibitors were combined with 4HPR. These results suggest the prooxidant property of 4HPR is associated with redox metabolism via an enzymatic process occurring at a quinone-binding site in Complex I and/or center o of Complex III. N-(4-hydroxyphenyl)retinamide antimycin A carbonyl cyanide m-chlorophenylhydrazone coenzyme Q coenzyme Q0 coenzyme Q1 cyclosporin A decylubiquinone, DCFDA, 2′,7′-dichlorofluorescin diacetate 2′,7′-dichlorofluorescein 3,3′-dihexyloxacarbocyanine iodide dimethyl sulfoxide diphenyleneiodonium chloride fluorescein isothiocyanate 2-heptyl-4-hydroxyquinoline-N-oxide, H2O2, hydrogen peroxide potassium cyanide ΔΨm, mitochondrial inner transmembrane potential mitochondrial permeability transition oligomycin A respiration-deficient cells lacking mitochondrial DNA reactive oxygen species squamous cell carcinoma theonyltrifluoroacetone, TUNEL, terminal deoxynucleotidyl transferase dUTP nick end-labeling l-ascorbic acid polymerase chain reaction N-(4-Hydroxyphenyl)retinamide (4HPR)1 is a synthetic analog of vitamin A belonging to a growing family of compounds known as retinoids. 4HPR has shown efficacy as an antineoplastic agent in experimental models and clinical trials. In animal models, 4HPR can inhibit carcinogenesis in breast, bladder, lung, ovary, and prostate. Various clinical trials involving chemoprevention of cancers of the breast, prostate, cervix, skin, and lung have also been conducted (1Formelli F. Barua A.B. Olson J.A. FASEB J. 1996; 10: 1014-1024Crossref PubMed Scopus (116) Google Scholar,2Kelloff G.J. Chemoprevention Branch and Agent Development Committee, Division of Cancer Prevention and Control, NCI J. Cell. Biochem. Suppl. 1994; 20: 176-196PubMed Google Scholar). 4HPR promotes apoptosis in a variety of tumor cell lines, which implies a common cellular event may be important with respect to both the chemopreventive and therapeutic effects of this compound (3Reed J. J. Natl. Cancer Inst. 1999; 91: 1099-1100Crossref PubMed Scopus (20) Google Scholar). The mechanism through which 4HPR induces apoptosis is not sufficiently understood. 4HPR can function as a prooxidant in many tumor cell lines (4Oridate N. Suzuki S. Higuchi M. Mitchell M.F. Hong W.K. Lotan R. J. Natl. Cancer Inst. 1997; 89: 1191-1198Crossref PubMed Scopus (203) Google Scholar, 5Delia D. Aiello A. Meroni L. Nicolini M. Reed J.C. Pierotti M.A. Carcinogenesis. 1997; 18: 943-948Crossref PubMed Scopus (125) Google Scholar, 6Hail Jr., N. Lotan R. Cancer Epidemiol. Biomarkers Prev. 2000; 9: 1293-1301PubMed Google Scholar, 7Maurer B. Metelitsa L. Seeger R. Cabot M. Reynolds C. J. Natl. Cancer Inst. 1999; 91: 1138-1146Crossref PubMed Scopus (258) Google Scholar, 8Sun S.-Y. Yue P. Lotan R. Mol. Pharmacol. 1999; 55: 403-410PubMed Google Scholar). These observations led to the assumption that ROS generation is the primary mechanism of action as far as apoptosis induction is concerned (5Delia D. Aiello A. Meroni L. Nicolini M. Reed J.C. Pierotti M.A. Carcinogenesis. 1997; 18: 943-948Crossref PubMed Scopus (125) Google Scholar, 6Hail Jr., N. Lotan R. Cancer Epidemiol. Biomarkers Prev. 2000; 9: 1293-1301PubMed Google Scholar, 9Suzuki S. Higuchi M. Proske R.J. Oridate N. Hong W.K. Lotan R. Oncogene. 1999; 18: 6380-6387Crossref PubMed Scopus (136) Google Scholar). In support of this conclusion, recent reports have illustrated the involvement of ROS and MPT in the process of 4HPR-induced apoptosis, as well as demonstrating a mitochondrial role in 4HPR-induced ROS production (6Hail Jr., N. Lotan R. Cancer Epidemiol. Biomarkers Prev. 2000; 9: 1293-1301PubMed Google Scholar, 9Suzuki S. Higuchi M. Proske R.J. Oridate N. Hong W.K. Lotan R. Oncogene. 1999; 18: 6380-6387Crossref PubMed Scopus (136) Google Scholar). Several studies have elegantly established the involvement of the mitochondria and mitochondrial-derived factors in the process of apoptosis (10Pastorino J.G. Chen S.-T. Tafani M. Snyder J.W. Farber J.L. J. Biol. Chem. 1998; 273: 7770-7775Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 11Cai J. Yang J. Jones D.P. Biochim. Biophys. Acta. 1998; 1366: 139-149Crossref PubMed Scopus (649) Google Scholar, 12Dallaporta B. Marchetti P. Pablo M.A. Masse C. Huynh-Thien D. Métivier D. Zamzami N. Geuskens M. Kroemer G. J. Immunol. 1999; 162: 6534-6542PubMed Google Scholar, 13Marchetti P. Zamzami N. Joseph B. Schraen-Maschke S. Méreau-Richard C. Costantini P. Métivier D. Susin S.A. Kroemer G. Fromstecher P. Cancer Res. 1999; 59: 6257-6266PubMed Google Scholar). The mitochondria are attractive targets for cancer chemotherapy. There is growing evidence, both in isolated mitochondria and in intact cells, illustrating many cancer chemotherapeutic agents modulate or interfere with mitochondrial functions to promote MPT (14Costantini P. Jocotot E. Decaudin D. Kroemer G. J. Natl. Cancer Inst. 2000; 92: 1042-1053Crossref PubMed Scopus (499) Google Scholar). Mitochondria constitute 15–50% of the total cytoplasmic volume in most cells and participate in more metabolic functions than any other organelles, especially in cellular energy production (15Modica-Napolitano J.S. Steele G.D. Chen L.B. Cancer Res. 1989; 49: 3369-3373PubMed Google Scholar). In addition, the mitochondria consume ∼90% of cellular oxygen and are a significant source of ROS, which if unchecked, are deleterious to mitochondrial and other cellular functions (16Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4851) Google Scholar, 17Turrens J.F. Biosci. Rep. 1997; 17: 3-7Crossref PubMed Scopus (755) Google Scholar). We have reported previously that the prooxidant property of 4HPR is linked to MPT, which is required for apoptosis in human cutaneous SCC cells (6Hail Jr., N. Lotan R. Cancer Epidemiol. Biomarkers Prev. 2000; 9: 1293-1301PubMed Google Scholar). The present study was conducted to determine whether the prooxidant and apoptotic effects of 4HPR could be inhibited by modulating mitochondrial function in these cells. This approach was taken to determine whether the mitochondria were the primary targets associated with the acute cytotoxicity of 4HPR in SCC cells. The results presented here support the involvement of mitochondrial respiration in 4HPR-induced hydroperoxide production and apoptosis. In particular, the data indicate the prooxidant quality of 4HPR can be explained via an enzymatic process occurring at a quinone-binding site in Complex I and/or center o of Complex III. The COLO 16 cell line was derived from a metastatic lesion in a female patient who succumbed to metastatic SCC (18Moore G.E. Merrick S.B. Woods L.K. Arabasz N.M. Cancer Res. 1975; 35: 2684-2688PubMed Google Scholar). The SRB-12 cell line was derived from cells taken from an epidermal lesion on a patient undergoing skin cancer treatment at the University of Texas M. D. Anderson Cancer Center. SCC cells were routinely cultured in keratinocyte growth medium, consisting of keratinocyte basal medium supplemented with 100 ng/ml human recombinant epidermal growth factor and 0.4% bovine pituitary extract (BioWhittaker/Clonetics, San Diego, CA) unless otherwise specified. Cell cultures were incubated at 37 °C in humidified air containing 5% CO2. Treatment with 4HPR and other agents was performed on subconfluent cultures. 4HPR was obtained from Dr. Ronald Lubet (Division of Cancer Prevention and Control, NCI, National Institutes of Health, Bethesda, MD). AA, CoQ0, CoQ1, capsaicin, carboxin, CCCP, DB, Me2SO, H2O2 (30% solution), HQNO, KCN, myxothiazol, rotenone, stigmatellin, TTFA, and Vit-C were purchased from Sigma Chemical Co. CsA, DPI, and OA were purchased from Biomol (Plymouth Meeting, PA). Dihydroethidium, DCFDA, and DiOC6 (3Reed J. J. Natl. Cancer Inst. 1999; 91: 1099-1100Crossref PubMed Scopus (20) Google Scholar) were purchased from Molecular Probes, Inc. (Eugene, OR). Respiration-deficient SCC cells (ρ0 clones) were isolated according to a previously described method (19King M.P. Attardi G. Methods Enzymol. 1996; 264: 304-313Crossref PubMed Google Scholar) with limited modifications. Briefly, COLO 16 and SRB-12 cells were cultured for 8 weeks in enriched medium consisting of Dulbecco's modified Eagle's medium containing 4.5 mg/ml glucose (Sigma); 110 mg/ml pyruvate (Sigma); 50 mg/ml uridine (Sigma); 100 ng/ml ethidium bromide (Sigma); and 2% dialyzed (3,500 molecular weight cutoff) fetal bovine serum (Life Technologies, Inc. Grand Island, NY). Clones were isolated by limiting dilution culture in enriched medium. The effects of chronic exposure to ethidium bromide on mitochondrial DNA and respiration in isolated clones were assessed by three methods. First, when cultured in enriched medium without uridine the clones exhibited greatly diminished cell survival (not shown). ρ0 cells have been reported to become pyrimidine auxotrophs, suggesting respiration is defective in these cells (19King M.P. Attardi G. Methods Enzymol. 1996; 264: 304-313Crossref PubMed Google Scholar). Second, a PCR procedure was used to detect mitochondrial DNA sequences in DNA samples from parental cells and their respective clones. The PCR reaction was conducted as specified for 40 cycles in a thermal cycler using a primer set (L1, 5′-AACATACCCATGGCCAACCT-3′ and H1, 5′-GGCAGGAGTAATCAGAGGTG-3′) designated for the detection of total mitochondrial DNA (20Wei Y.-H. Kao S.-H. Lee H.-C. Ann. N. Y. Acad. Sci. 1996; 786: 24-42Crossref PubMed Scopus (84) Google Scholar). Using this method, PCR products derived from mitochondrial DNA were detected in parental cells, but were absent in their ρ0 clones (not shown). Finally, oxygen consumption rates in the isolated clones were ∼15% of those observed in parental cells (see Fig. 3 A), illustrating that the ρ0clones were functionally deficient in respiration. Treatment of ρ0 clones was conducted 24 h after culture in enriched medium without ethidium bromide. Oxygen consumption was measured polarographically using a Clark-type oxygen electrode and YSI Model 5300 Biological Oxygen Monitor (Yellow Spring Instrument Co., Yellow Springs, OH). Approximately 6 × 106 cells suspended in 3 ml of their respective culture medium were added to a 3-ml respiration chamber in a circulating water bath at 37 °C. Oxygen consumption was measured over a 10-min period after equilibration of the electrode in the respiration chamber. Respiration rates for cells were normalized (nmol of O2/min/106 cells) assuming an O2concentration of 220 μm in air-saturated media at 37 °C (21Estabrook R.W. Methods Enzymol. 1967; 10: 41-47Crossref Scopus (1897) Google Scholar). In certain determinations, 4HPR was added directly to cell suspensions (final concentration 10 μm) in the respiration chamber for a 10-min exposure. Control cells received an equal volume of the vehicle Me2SO. For longer exposures, 4HPR and/or other agents were added directly to the media of cells cultured in 10-cm plastic tissue culture plates. The cells were harvested by trypsinization, pelleted by centrifugation, resuspended at a density of ∼2 × 106 cells/ml in fresh media at 37 °C, and 3 ml of the cell suspension was placed in the respiration chamber. Oxygen consumption rates were obtained during the final 10 min of exposure. Cell viability was routinely checked via Trypan blue exclusion after oxygen consumption determinations. Hydroperoxide production was determined using DCFDA. Cells in 10-cm plastic tissue culture plates were treated for 30 min with 20 μm DCFDA and 4HPR or Me2SO. The cells were harvested by trypsinization, washed in 5 ml of phosphate-buffered saline at 37 °C, pelleted by centrifugation, resuspended in 1 ml of phosphate-buffered saline at 37 °C and analyzed immediately by flow cytometry. Evaluations of ΔΨm dissipation, superoxide production, and DEVDase activity were adapted from previously published methods (13Marchetti P. Zamzami N. Joseph B. Schraen-Maschke S. Méreau-Richard C. Costantini P. Métivier D. Susin S.A. Kroemer G. Fromstecher P. Cancer Res. 1999; 59: 6257-6266PubMed Google Scholar) with limited modifications. For concurrent determination of ΔΨm dissipation and superoxide production, cells in 10-cm plastic tissue culture plates were treated with agents alone or in combination for various periods. Twenty minutes before the cells were harvested, DiOC6 (3Reed J. J. Natl. Cancer Inst. 1999; 91: 1099-1100Crossref PubMed Scopus (20) Google Scholar) and dihydroethidium were added directly to the culture medium to a final concentration of 30 nm and 5 μm respectively. The cells were harvested as described above, resuspended in 1 ml of phosphate-buffered saline at 37 °C, and analyzed immediately by flow cytometry. DEVDase-like caspase activity was determined with PhiPhiLux-G1D2 (Oncoimmunin Inc., Gaithersburg, MD) a cell permeant fluorogenic substrate (DEVD-rhodamine), which is cleaved in a DEVD-dependent manner (13Marchetti P. Zamzami N. Joseph B. Schraen-Maschke S. Méreau-Richard C. Costantini P. Métivier D. Susin S.A. Kroemer G. Fromstecher P. Cancer Res. 1999; 59: 6257-6266PubMed Google Scholar). Cells were treated in 10-cm plastic tissue culture plates as described above. Following treatment, the cells were harvested by trypsinization, incubated in 10 μm PhiPhiLux-G1D2, and washed according to the manufacturer's recommendations. The resulting cell suspension was analyzed immediately by flow cytometry. Intranucleosomal DNA fragmentation was evaluated using an apoptosis detection kit (Phoenix Flow Systems, Inc., San Diego, CA) that is based on the TUNEL technique. The reaction labels the 3′-hydroxyl termini of DNA fragmented during apoptosis with fluorescein isothiocyanate-conjugated dUTP (22Li X. Traganos F. Melamed M.R. Darzynkiewicz Z. Cytometry. 1995; 20: 172-182Crossref PubMed Scopus (154) Google Scholar). Cells were also stained with propidium iodide as a relative indicator of DNA content. After treatment, the cells were detached with trypsin. The cells were then combined with their respective culture medium that was removed prior to trypsinization. The cells were pelleted by centrifugation, fixed, and stained using the protocol provided in the apoptosis detection kit with limited modifications (6Hail Jr., N. Lotan R. Cancer Epidemiol. Biomarkers Prev. 2000; 9: 1293-1301PubMed Google Scholar). All flow cytometric procedures were performed on a Coulter XL flow cytometer, and data analysis was accomplished using System II XL software (Coulter Corp., Miami, FL). Approximately 10,000 events (cells) were evaluated for each sample. In all cytofluorometric determinations, cell samples were initially gated to exclude cell debris and clumps. Rates of cellular hydroperoxide generation were determined using DCFDA as described previously (6Hail Jr., N. Lotan R. Cancer Epidemiol. Biomarkers Prev. 2000; 9: 1293-1301PubMed Google Scholar). Briefly, cells were seeded in their respective culture medium in 6-well tissue culture plates and allowed to attach and proliferate for 24 h. The wells were washed twice with 2 ml of buffer A (Krebs-Ringer buffer containing 10 mm d-glucose, 120 mm NaCl, 4.5 mm KCl, 0.15 mm CaCl2, 0.7 mmNa2HPO4, 1.5 mmNaH2PO4, and 0.5 mmMgCl2 (pH 7.4 at 37 °C)). The wells were then covered with 2 ml of buffer A containing 20 μm DCFDA with Me2SO as a control, or with the indicated concentrations of other reagents and/or 4HPR. The plates were rocked for 2 min to ensure adequate mixing. Fluorescence was measured at time 0 (immediately following mixing) and subsequently at 30-min intervals over a 120-min period using a CytoFluor 4000 spectrofluorimeter (Perseptive Biosystems, Inc. Framingham, MA). The spectrofluorimeter preformed 13 fluorescence measurements per well and provided an average value. Hydroperoxide generation rates were derived from the slopes of lines obtained between 30 and 120 min from triplicate wells. Cultures were incubated at 37 °C between the 30-min intervals for fluorescence determination. Cell viability was routinely checked via Trypan blue exclusion at the conclusion of fluorescence determinations. Previous studies have used the oxidation of 2′,7′-dichlorofluorescin to monitor 4HPR-induced hydroperoxide production (4Oridate N. Suzuki S. Higuchi M. Mitchell M.F. Hong W.K. Lotan R. J. Natl. Cancer Inst. 1997; 89: 1191-1198Crossref PubMed Scopus (203) Google Scholar, 5Delia D. Aiello A. Meroni L. Nicolini M. Reed J.C. Pierotti M.A. Carcinogenesis. 1997; 18: 943-948Crossref PubMed Scopus (125) Google Scholar, 6Hail Jr., N. Lotan R. Cancer Epidemiol. Biomarkers Prev. 2000; 9: 1293-1301PubMed Google Scholar, 7Maurer B. Metelitsa L. Seeger R. Cabot M. Reynolds C. J. Natl. Cancer Inst. 1999; 91: 1138-1146Crossref PubMed Scopus (258) Google Scholar, 8Sun S.-Y. Yue P. Lotan R. Mol. Pharmacol. 1999; 55: 403-410PubMed Google Scholar, 9Suzuki S. Higuchi M. Proske R.J. Oridate N. Hong W.K. Lotan R. Oncogene. 1999; 18: 6380-6387Crossref PubMed Scopus (136) Google Scholar), as well as the retention of cationic probes to monitor changes in ΔΨm promoted by 4HPR treatment (6Hail Jr., N. Lotan R. Cancer Epidemiol. Biomarkers Prev. 2000; 9: 1293-1301PubMed Google Scholar,9Suzuki S. Higuchi M. Proske R.J. Oridate N. Hong W.K. Lotan R. Oncogene. 1999; 18: 6380-6387Crossref PubMed Scopus (136) Google Scholar). Thus, to gain a perspective on the importance of hydroperoxide production in 4HPR-induced cell death, a kinetic analysis of the salient features associated with this process was conducted using COLO 16 cells. The experiment depicted in Fig.1 A shows that a 30-min exposure to 4HPR was sufficient to promote a shift in the mean DCF fluorescence intensity that was approximately 5-fold higher than Me2SO-treated control cells. This experiment was repeated substituting DCFDA, which specifically reacts with hydroperoxides, with dihydroethidium (5 μm), which is oxidized to ethidium via superoxide (23Buxser S.E. Sawada G. Raub T.J. Methods Enzymol. 1999; 300: 256-274Crossref PubMed Scopus (49) Google Scholar). However, cells treated with 4HPR under these conditions for 30, 60, or 120 min exhibited ethidium fluorescence intensities similar to Me2SO-treated cells (not shown), indicating hydroperoxides are the primary ROS generated after 4HPR treatment. Whether 4HPR-induced hydroperoxide production is also contingent on superoxide dismutase activity remains to be determined. Several hallmark events occur in the mitochondria and cytoplasm of apoptotic cells that precede DNA fragmentation. Three such events are dissipation of ΔΨm, enhanced superoxide production, and caspase activation that are associated with MPT (24Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1384) Google Scholar, 25Kroemer G. Petit P. Zamzami N. Vayssière J.-L. FASEB J. 1995; 9: 1277-1287Crossref PubMed Scopus (967) Google Scholar). Given ROS production can be both a cause and consequence of MPT (26Zoratti M. Szabò I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2199) Google Scholar, 27Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar), mitochondrial parameters associated with MPT were examined in 4HPR-treated cells. The dissipation of ΔΨm represented by decreased retention of the cationic probe DiOC6 (3Reed J. J. Natl. Cancer Inst. 1999; 91: 1099-1100Crossref PubMed Scopus (20) Google Scholar), and enhanced superoxide production represented by the oxidation of dihydroethidium can be used concurrently to monitor MPT in intact cells (13Marchetti P. Zamzami N. Joseph B. Schraen-Maschke S. Méreau-Richard C. Costantini P. Métivier D. Susin S.A. Kroemer G. Fromstecher P. Cancer Res. 1999; 59: 6257-6266PubMed Google Scholar). As shown in Fig. 1 B, control cells were gated assuming they exhibited high DiOC6 (3Reed J. J. Natl. Cancer Inst. 1999; 91: 1099-1100Crossref PubMed Scopus (20) Google Scholar) fluorescence and low ethidium fluorescence. A 2-h exposure to 4HPR did not markedly influence the fluorescence intensities of either probe (not shown). However, after 4 h, ∼64% of the cell population had shifted to low DiOC6 (3Reed J. J. Natl. Cancer Inst. 1999; 91: 1099-1100Crossref PubMed Scopus (20) Google Scholar) fluorescence. 8 h after treatment ∼95% of the cell population had shifted to low DiOC6 (3Reed J. J. Natl. Cancer Inst. 1999; 91: 1099-1100Crossref PubMed Scopus (20) Google Scholar) fluorescence, and ∼36% of this population also exhibited high ethidium fluorescence indicating both a substantial dissipation of ΔΨm and enhanced superoxide production resulting from the disintegration of mitochondrial electron transport (13Marchetti P. Zamzami N. Joseph B. Schraen-Maschke S. Méreau-Richard C. Costantini P. Métivier D. Susin S.A. Kroemer G. Fromstecher P. Cancer Res. 1999; 59: 6257-6266PubMed Google Scholar). After 12 h, ∼59% of the cells displayed low DiOC6 (3Reed J. J. Natl. Cancer Inst. 1999; 91: 1099-1100Crossref PubMed Scopus (20) Google Scholar) fluorescence and high ethidium fluorescence with the remaining cells low for both DiOC6 (3Reed J. J. Natl. Cancer Inst. 1999; 91: 1099-1100Crossref PubMed Scopus (20) Google Scholar) and ethidium fluorescence. These results imply 4HPR-induced hydroperoxide production precedes MPT in COLO 16 cells. The process of MPT did indeed enhance superoxide production apparently as a peripheral event because this phenomenon was not observed until most of the cell population displayed reduced ΔΨm. MPT induction results in the release of cytochromec and other soluble mitochondrial factors capable of inducing caspase activity (13Marchetti P. Zamzami N. Joseph B. Schraen-Maschke S. Méreau-Richard C. Costantini P. Métivier D. Susin S.A. Kroemer G. Fromstecher P. Cancer Res. 1999; 59: 6257-6266PubMed Google Scholar, 27Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). 4HPR promoted DEVDase-like caspase activity in COLO 16 cells. As with enhanced superoxide production, this was not profoundly evident until 8 h after treatment (Fig.1 C). Interestingly, this activity decreased slightly after 12 h. These results suggest 4HPR-induced hydroperoxide production triggered MPT, and MPT was involved with caspase activity. Examination of DNA fragmentation promoted by 4HPR treatment revealed at least 10 h of exposure were required before >50% of the treatment population exhibited positive TUNEL staining (Fig.1 D). Me2SO-treated cells exhibited the same degree of TUNEL staining as 4HPR-treated cells after 6 h (∼9%) and remained basically unchanged at subsequent time-points. Between 8 and 10 h after 4HPR treatment, DNA fragmentation appeared to be most prominent with only a slight increase detectable after the following 2 h. Previous examination of COLO16 cells revealed a gradual increase in the number of TUNEL-positive cells from ∼60% to 90% of the treatment population 12–48 h after 4HPR treatment (6Hail Jr., N. Lotan R. Cancer Epidemiol. Biomarkers Prev. 2000; 9: 1293-1301PubMed Google Scholar). Therefore, it can be concluded the culmination (DNA fragmentation) of 4HPR-induced cell death is a protracted process in COLO 16 cells evidently set in motion by hydroperoxide production and MPT. Hydroperoxide production appears to be the first detectable event associated with 4HPR-induced apoptosis. Hydrogen peroxide and other ROS can interfere with electron transport and disrupt mitochondrial function (17Turrens J.F. Biosci. Rep. 1997; 17: 3-7Crossref PubMed Scopus (755) Google Scholar, 28Zhang Y. Marcillat O. Giulivi C. Ernster L. Davies K. J. Biol. Chem. 1990; 265: 16330-16336Abstract Full Text PDF PubMed Google Scholar). If hydroperoxide production is involved with the acute cytotoxic effects of 4HPR, we would expect to observe a decline in cellular oxygen consumption since mitochondria consume ∼90% of cellular oxygen (17Turrens J.F. Biosci. Rep. 1997; 17: 3-7Crossref PubMed Scopus (755) Google Scholar). To test this hypothesis, COLO 16 cells were treated with 4HPR first by adding the drug directly to cells suspended in the respiration chamber. However, a 10-min exposure neither enhanced (denoting an uncoupling effect) nor inhibited respiration relative to Me2SO-treated controls (not shown). As shown in Fig. 2 A, a 30-min exposure to 4HPR promoted a slight decrease in respiration, but this was negligible compared with an equivalent exposure to the Complex IV inhibitor cyanide (17Turrens J.F. Biosci. Rep. 1997; 17: 3-7Crossref PubMed Scopus (755) Google Scholar). In addition, the effect of cyanide would indicate cellular oxygen consumption was predominantly of mitochondrial origin in COLO 16 cells. Sustained exposures to 4HPR decreased oxygen consumption by ∼40% after 2 h and ∼60% after 4 h (Fig.2 A). These results illustrate 4HPR can modulate mitochondrial function in intact cells. The antioxidant Vit-C can inhibit the prooxidant and apoptotic effects of 4HPR in COLO 16 cells (6Hail Jr., N. Lotan R. Cancer Epidemiol. Biomarkers Prev. 2000; 9: 1293-1301PubMed Google Scholar). This prompted examination of this agent to determine whether it could also inhibit the decrease in respiration promoted by 4HPR. Exposure to Vit-C, while having no considerable impact on oxygen consumption at any of the time points examined, was able to block the effects of 4HPR. In addition, the activity of 4HPR appears to be distinctly different from cyanide because the decrease in respiration promoted by cyanide was not markedly influenced when combined with Vit-C (Fig. 2 A). These results suggest the prooxidant property of 4HPR is associated with the decrease in mitochondrial oxygen consumption. We have reported previously that CsA can reduce 4HPR-induced hydroperoxide production in SCC cells. This led to the conclusion some of the ROS production was associated with MPT (6Hail Jr., N. Lotan R. Cancer Epidemiol. Biomarkers Prev. 2000; 9: 1293-1301PubMed Google Scholar). If this were the case, CsA, like Vit-C, should also inhibit the decrease in oxygen consumption promoted by 4HPR. However, a 2-h exposure to CsA alone also inhibited respiration in COLO 16 cells and only slightly suppressed the effect of 4HPR (Fig. 2 A). The decrease in respiration promoted by CsA can potentially be explained as follows. CsA binds to cyclophilin D to stabilize the closed matrix conformation of the adenine nucleotide translocator (26Zoratti M. Szabò I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2199) Google Scholar, 27Green D.R. Reed J.
Referência(s)