Mitochondrial O 2 ⋅ ¯ and H2O2 Mediate Glucose Deprivation-induced …
2004; Elsevier BV; Volume: 280; Issue: 6 Linguagem: Inglês
10.1074/jbc.m411662200
ISSN1083-351X
AutoresIman M. Ahmad, Nükhet Aykin‐Burns, Julia Sim, Susan A. Walsh, Ryuji Higashikubo, Garry R. Buettner, Sujatha Venkataraman, Michael A. Mackey, Shawn W. Flanagan, Larry W. Oberley, Douglas R. Spitz,
Tópico(s)Coenzyme Q10 studies and effects
ResumoThe hypothesis that glucose deprivation-induced cytotoxicity in transformed human cells is mediated by mitochondrial O2⋅¯ and H2O2 was first tested by exposing glucose-deprived SV40-transformed human fibroblasts (GM00637G) to electron transport chain blockers (ETCBs) known to increase mitochondrial O2⋅¯ and H2O2 production (antimycin A (AntA), myxothiazol (Myx), or rotenone (Rot)). Glucose deprivation (2–8 h) in the presence of ETCBs enhanced parameters indicative of oxidative stress (i.e. GSSG and steady-state levels of oxygen-centered radicals) as well as cytotoxicity. Glucose deprivation in the presence of AntA also significantly enhanced cytotoxicity and parameters indicative of oxidative stress in several different human cancer cell lines (PC-3, DU145, MDA-MB231, and HT-29). In addition, human osteosarcoma cells lacking functional mitochondrial electron transport chains (rho(0)) were resistant to glucose deprivation-induced cytotoxicity and oxidative stress in the presence of AntA. In the absence of ETCBs, aminotriazole-mediated inactivation of catalase in PC-3 cells demonstrated increases in intracellular steady-state levels of H2O2 during glucose deprivation. Finally, in the absence of ETCBs, overexpression of manganese containing superoxide dismutase and/or mitochondrial targeted catalase using adenoviral vectors significantly protected PC-3 cells from toxicity and oxidative stress induced by glucose deprivation with expression of both enzymes providing greater protection than was seen with either alone. Overall, these findings strongly support the hypothesis that mitochondrial O2⋅¯ and H2O2 significantly contribute to glucose deprivation-induced cytotoxicity and metabolic oxidative stress in human cancer cells. The hypothesis that glucose deprivation-induced cytotoxicity in transformed human cells is mediated by mitochondrial O2⋅¯ and H2O2 was first tested by exposing glucose-deprived SV40-transformed human fibroblasts (GM00637G) to electron transport chain blockers (ETCBs) known to increase mitochondrial O2⋅¯ and H2O2 production (antimycin A (AntA), myxothiazol (Myx), or rotenone (Rot)). Glucose deprivation (2–8 h) in the presence of ETCBs enhanced parameters indicative of oxidative stress (i.e. GSSG and steady-state levels of oxygen-centered radicals) as well as cytotoxicity. Glucose deprivation in the presence of AntA also significantly enhanced cytotoxicity and parameters indicative of oxidative stress in several different human cancer cell lines (PC-3, DU145, MDA-MB231, and HT-29). In addition, human osteosarcoma cells lacking functional mitochondrial electron transport chains (rho(0)) were resistant to glucose deprivation-induced cytotoxicity and oxidative stress in the presence of AntA. In the absence of ETCBs, aminotriazole-mediated inactivation of catalase in PC-3 cells demonstrated increases in intracellular steady-state levels of H2O2 during glucose deprivation. Finally, in the absence of ETCBs, overexpression of manganese containing superoxide dismutase and/or mitochondrial targeted catalase using adenoviral vectors significantly protected PC-3 cells from toxicity and oxidative stress induced by glucose deprivation with expression of both enzymes providing greater protection than was seen with either alone. Overall, these findings strongly support the hypothesis that mitochondrial O2⋅¯ and H2O2 significantly contribute to glucose deprivation-induced cytotoxicity and metabolic oxidative stress in human cancer cells. Oxidative stress results when the balance between steady-state levels of intracellular prooxidants exceeds cellular anti-oxidant capacity. Many cancer cells have low levels of several antioxidant enzymes (i.e. Mn-SOD 1The abbreviations used are: SOD, superoxide dismutase; ETCB, electron transport chain blocker; Mn-SOD, manganese superoxide dismutase; CuZn-SOD, copper-zinc superoxide dismutase; AntA, antimycin A; Myx, myxothiazol; Rot, rotenone; DNP, dinitrophenol; AT, 3-amino-1,2,4-triazole; Me2SO, dimethyl sulfoxide; PBS, phosphate-buffered saline; GPx, glutathione peroxidase; MFI, mean fluorescence intensity; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide.1The abbreviations used are: SOD, superoxide dismutase; ETCB, electron transport chain blocker; Mn-SOD, manganese superoxide dismutase; CuZn-SOD, copper-zinc superoxide dismutase; AntA, antimycin A; Myx, myxothiazol; Rot, rotenone; DNP, dinitrophenol; AT, 3-amino-1,2,4-triazole; Me2SO, dimethyl sulfoxide; PBS, phosphate-buffered saline; GPx, glutathione peroxidase; MFI, mean fluorescence intensity; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide. and catalase) (1Oberley L.W. Buettner G.R. Cancer Res. 1979; 39: 1141-1149PubMed Google Scholar, 2Oberley T.D. Oberley L.W. Yu B.P. Free Radicals in Aging. CRC Press, Boca Raton, FL1993Google Scholar). It has also been suggested that cancer cells may exhibit defects in their mitochondrial electron transport chains that could lead to increased steady-state levels of prooxidants (i.e. superoxide and hydroperoxides) resulting from one-electron reduction of O2 leading to a condition of metabolic oxidative stress (3Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (283) Google Scholar). Most cancer cells maintain a high glycolytic rate; a phenomenon first described over 70 years ago and known as the Warburg effect (4Warburg O. Science. 1956; 132: 309-314Crossref Scopus (9143) Google Scholar). In addition to its role in energy production, glucose metabolism also leads to the formation of pyruvate and NADPH, both of which are believed to function in the cellular detoxification of hydroperoxides (5Nath K.A. Ngo E.O. Hebbel R.P. Croatt A.J. Zhau B. Nutter L.M. Am. J. Physiol. 1995; 268: C227-C236Crossref PubMed Google Scholar, 6Averrill-Bates D.A. Przybytkowski E. Arch. Biochem. Biophys. 1994; 312: 52-58Crossref PubMed Scopus (56) Google Scholar). Pyruvate reacts directly with hydrogen peroxide and organic hydroperoxides (H2O2 and ROOH, respectively), resulting in the deacetylation of pyruvate to acetic acid and reduction of the peroxides to H2O or ROH (5Nath K.A. Ngo E.O. Hebbel R.P. Croatt A.J. Zhau B. Nutter L.M. Am. J. Physiol. 1995; 268: C227-C236Crossref PubMed Google Scholar). NADPH provides electrons for the reduction of glutathione disulfide and oxidized thioredoxin, which is required for the efficient detoxification of H2O2 and ROOH by glutathione peroxidases and peroxiredoxins (3Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (283) Google Scholar, 6Averrill-Bates D.A. Przybytkowski E. Arch. Biochem. Biophys. 1994; 312: 52-58Crossref PubMed Scopus (56) Google Scholar, 7Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 8Blackburn R.V. Spitz D.R. Liu X. Galoforo S.S. Sim J.E. Ridnour L.A. Chen J.C. Daris B.H. Corry P.M. Lee Y.J. Free Radic. Biol. Med. 1999; 26: 419-430Crossref PubMed Scopus (139) Google Scholar). Given these observations it is logical to hypothesize that cancer cells might increase glucose metabolism as a compensatory mechanism to protect against intracellular hydroperoxides generated from mitochondrial electron transport chain activity. Several studies have demonstrated glucose deprivation-induced cytotoxicity and oxidative stress in cancer cells, but the role of mitochondrial O2⋅¯ and H2O2 in this phenomenon has not been clearly demonstrated (3Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (283) Google Scholar, 7Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 8Blackburn R.V. Spitz D.R. Liu X. Galoforo S.S. Sim J.E. Ridnour L.A. Chen J.C. Daris B.H. Corry P.M. Lee Y.J. Free Radic. Biol. Med. 1999; 26: 419-430Crossref PubMed Scopus (139) Google Scholar). We hypothesize that glucose deprivation in human cancer cells will result in a compromised ability to detoxify H2O2 derived from mitochondrial metabolism resulting in steady-state increases in hydroperoxides that contribute to glucose deprivation-induced cytotoxicity and oxidative stress. To test the hypothesis that mitochondrial O2⋅¯ and H2O2 mediate glucose deprivation-induced oxidative stress and cytotoxicity in human cancer cells, three different approaches were utilized. First, the effect of ETCBs (known to increase O2⋅¯ and H2O2 production in isolated mitochondria) was determined in intact cells during glucose deprivation. Antimycin A was tested as a blocker of Complex III, myxothiazol was tested as a blocker of entry into Complex III, and rotenone was tested as a Complex I blocker (see Fig. 12). Second, rho(0) human cancer cells, deficient in functional mitochondrial electron transport chains, were utilized. Finally, adenovirus-mediated transduction of mitochondrially targeted catalase (MitCat) or Mn-SOD was utilized to overexpress enzymatic scavengers of H2O2 and O2⋅¯, respectively, in human tumor cells prior to glucose deprivation in the absence of ETCBs. The results of these experiments demonstrated that ETCBs dramatically enhanced glucose deprivation-induced cytotoxicity and parameters indicative of oxidative stress in all human tumor cell lines tested. Furthermore, rho(0) cells were resistant to increases in cytotoxicity and parameters indicative of oxidative stress during treatment with glucose deprivation in the presence of ETCBs. Finally, steady-state levels of reactive oxygen species (particularly H2O2) were increased during glucose deprivation in the presence and absence of ETCBs and overexpression of MitCat and Mn-SOD protected PC-3 cells from glucose deprivation-induced cytotoxicity and parameters indicative of oxidative stress in the absence of ETCBs. These results provide strong support for the hypothesis that mitochondria represent a primary source of O2⋅¯ and H2O2 that significantly contributes to glucose deprivation-induced cytotoxicity and oxidative stress in human cancer cells. Cells and Culture Conditions—GM00637G SV40 transformed human fibroblasts were obtained and cultured as described (3Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (283) Google Scholar). HT-29 human colon carcinoma cells were obtained from ATCC and maintained in McCoy's 5A media supplemented with 10% fetal bovine serum. PC-3 human prostate cancer cells were obtained from ATCC, and maintained in F-12 media supplemented with 10% fetal bovine serum. DU145 and MDA-MB231 human prostate and breast cancer cells, respectively, were a gift from Dr. Mary Hendrix, University of Iowa, and maintained in RPMI media supplemented with 10% fetal bovine serum (9Hendrix M.J. Seftor E.A. Seftor R.E. Trevor K.T. Am. J. Pathol. 1997; 150: 483-495PubMed Google Scholar). Dr. Michael King (Thomas Jefferson University, Philadelphia, PA) kindly provided the human osteosarcoma 143BTK–rho(+) and rho(0) cells (10King M.P. Attardi G. Methods Enzymol. 1996; 264: 304-313Crossref PubMed Google Scholar). These cells were grown in DMEM, 4.5 g/liter glucose supplemented with 5% fetal bovine serum and 50 μg/ml uridine for the rho(0) cells. All stock cultures were maintained in 5% CO2 and air in a humidified 37 °C incubator in the absence of antibiotics. All cells were routinely tested for mycoplasma and found to be negative. Glucose Deprivation Conditions and Cell Survival Experiments— Cells were plated in 60-mm tissue culture dishes and grown for 3 days in the presence of antibiotics (gentamycin). At the beginning of each experiment, the cells were rinsed with phosphate-buffered saline (PBS) to remove glucose and placed in glucose-free DMEM or RPMI 1640 media supplemented with 10% dialyzed serum as described previously (7Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 8Blackburn R.V. Spitz D.R. Liu X. Galoforo S.S. Sim J.E. Ridnour L.A. Chen J.C. Daris B.H. Corry P.M. Lee Y.J. Free Radic. Biol. Med. 1999; 26: 419-430Crossref PubMed Scopus (139) Google Scholar). Control cultures were treated identically except glucose was added back at the normal concentration found in the media. Then, drug treatment was initiated as appropriate. Cells were then placed in an incubator and at each time point, cells were trypsinized, counted, diluted, and plated for clonogenic cell survival assay as described previously (11Spitz D.R. Malcolm R.R. Roberts R.J. Biochem. J. 1990; 267: 453-459Crossref PubMed Scopus (110) Google Scholar). Surviving colonies were fixed and stained after 14 days and counted under a dissecting microscope. Drug Treatment—AntA, Myx, Rot, dinitrophenol (DNP), and 3-amino-1,2,4-triazole (AT) were obtained from Sigma and used without further purification. Drugs were added to cells at final concentrations of 10 μm AntA and Myx, 50 μm Rot, 2 μm DNP, and 50 mm AT. Stock solutions of 10 mm AntA and Myx, 50 mm rotenone, and 2 mm DNP were dissolved in Me2SO, whereas 5 m AT was dissolved in PBS, and the required volume was added directly to the cells to achieve the desired final concentrations. All cells were incubated in glucose-free medium containing dialyzed serum with different drugs for the specified times. Measurement of Glutathione Levels—Following treatment, cells were scrape-harvested in PBS at 4 °C, centrifuged, the PBS discarded, and the cell pellets frozen at–80 °C. Samples were thawed and whole homogenates were prepared as described (7Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 8Blackburn R.V. Spitz D.R. Liu X. Galoforo S.S. Sim J.E. Ridnour L.A. Chen J.C. Daris B.H. Corry P.M. Lee Y.J. Free Radic. Biol. Med. 1999; 26: 419-430Crossref PubMed Scopus (139) Google Scholar). Total glutathione (GSH + GSSG) and glutathione disulfide (GSSG) were determined using a spectrophotometeric recycling assay (7Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 8Blackburn R.V. Spitz D.R. Liu X. Galoforo S.S. Sim J.E. Ridnour L.A. Chen J.C. Daris B.H. Corry P.M. Lee Y.J. Free Radic. Biol. Med. 1999; 26: 419-430Crossref PubMed Scopus (139) Google Scholar). All biochemical determinations were normalized to protein content using the method of Lowry et al. (12Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). EPR Spin Trapping of Oxygen Centered Radicals—DMPO was purchased from Sigma (D-5766). The stock solution of DMPO was prepared with nanopure water. The solution was purified by multiple centrifugations through activated charcoal (Sigma, C-5385) and filtered. The concentration of DMPO was determined with a HP 8453 UV-visible spectrometer at ϵ227 = 7800 m–1 cm–1. Purity was confirmed using EPR. EPR spectra were obtained using a Bruker X-band EMX spectrometer at room temperature. Instrument settings were: 9.77 GHz, microwave frequency; 100 kHz, modulation frequency; 40 milliwatt, nominal microwave power (13Buettner G.R. Kiminyo K.P. J. Biochem. Biophys. Meth. 1992; 24: 147-151Crossref PubMed Scopus (44) Google Scholar); 1.0 G, modulation amplitude; 3480 G center field for DMPO/·OH; 80 G/84 s scan rate; and 164-ms time constant. Each sample for EPR contained 7 × 106 freshly trypsinized cells in PBS and each spectrum was signal averaged, n = 15, to increase the signal-to-noise ratio. Transduction of Antioxidant Enzymes—Replication incompetent adenoviral vectors, AdCMV BglII (AdBglII), AdCMV catalase (AdCAT), AdCMV mitochondrial catalase (AdMitCat), and AdCMV Mn-SOD (AdMn-SOD) were manufactured at The University of Iowa Gene Transfer Vector Core by inserting the gene of interest into the E1 region of an Ad5 E1/particle E3 deleted replication-deficient adenoviral vector. The cDNAs were all under the control of a CMV promotor. Except for AdMitCat, the adenovirus constructs were originally prepared by John Engelhardt, University of Iowa (14Zwacka R.M. Dudus L. Epperly M.W. Greenburger J.S. Engelhardt J.F. Hum. Gene Ther. 1998; 9: 1381-1386Crossref PubMed Scopus (109) Google Scholar). The full-length catalase cDNA with the Mn-SOD mitochondrial leader sequence added to the construct were originally prepared by Dr. Andre Melendez (15Bai J. Rodriquez A.M. Melendez J.A. Cederbaum A.I. J. Biol. Chem. 1999; 274: 26217-26224Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Cells were seeded until attached (overnight), and then the desired amount of viral particles was added with 1.8 ml of complete media per 60-mm dish for 24 h, then the media was changed and replaced by 4 ml of complete media and left for another 24 h prior to each experiment. Catalase Activity Assay—Catalase activity was determined on whole homogenates by measuring the disappearance of 10 mm hydrogen peroxide (Δϵ240 = 39.4 m–1 cm–1) in 50 mm potassium phosphate, pH 7.0, monitored at 240 nm and the units were expressed as milli-k units/mg of protein as described (16Spitz D.R. Elwell J.H. Sun Y. Oberley L.W. Oberley T.D. Sullivan S.J. Roberts R.J. Arch. Biochem. Biophys. 1990; 279: 249-260Crossref PubMed Scopus (100) Google Scholar). Mn-SOD Activity Assay—SOD activity of whole homogenates prepared on ice following 2 freeze thaw cycles in 50 mm potassium phosphate buffer (pH 7.8, with 1.34 mm diethylenetriaminepentaacetic acid) was determined using an indirect competitive inhibition assay (17Spitz D.R. Oberley L.W. Anal. Biochem. 1989; 179: 8-18Crossref PubMed Scopus (582) Google Scholar). This assay is based on the competition between SOD and an indicator molecule (nitro blue tetrazolium) for superoxide production from xanthine and xanthine oxidase, according to the method of Spitz and Oberley (17Spitz D.R. Oberley L.W. Anal. Biochem. 1989; 179: 8-18Crossref PubMed Scopus (582) Google Scholar). Incubation for at least 45 min with 5 mm sodium cyanide was used to inhibit CuZn-SOD activity to measure Mn-SOD activity. Preparation of Mitochondria—Twelve 15-cm culture dishes of PC-3 cells (transduced with AdMitCat or vector control) grown to 80% confluence were harvested and mitochondria were prepared as described previously (18Dobson A.W. Xu Y. Kelley M.R. LeDoux S.P. Wilson G.L. J. Biol. Chem. 2000; 278: 37518-37523Abstract Full Text Full Text PDF Scopus (118) Google Scholar). Western Blot Analysis—To assay for catalase immunoreactive protein levels whole homogenates, intact mitochondria, and cytosolic fractions prepared as described above were sonicated at a duty cycle of 30% (50% for whole homogenates) (Sonics Vibracell, VC750) and an output control of 3 (4 for whole homogenates) for 10 s (20 s for whole homogenates), and the protein concentration was measured (Bradford method). Five to 40 μg of denatured protein was resolved on 12% SDS-PAGE and electroblotted onto nitrocellulose membranes (Bio-Rad). The membrane to measure catalase was incubated with rabbit anti-human catalase polyclonal antibody as the primary antibody (1:1,000) (Athens, Inc., Athens, GA), whereas for cytochrome c, the blots were incubated with rabbit anti-human cytochrome c polyclonal antibody as the primary antibody (1:500) (Santa Cruz), followed by incubation with horseradish peroxidase conjugated to goat anti-rabbit IgG (Sigma) as the second antibody (1:10,000) for both. Detection by the chemiluminescence reaction was carried out for 5 min using the ECL kit (Amersham Biosciences), followed by exposure to Kodak X-Omat x-ray film (Eastman Kodak). Intracellular Prooxidant Production—Prooxidant production was determined using the oxidation-sensitive 5- (and-6)-carboxy-2′, 7′-dichlorodihydrofluorescein diacetate (C-400, 10 μg/ml) and oxidation-insensitive 5- (and-6)-carboxy-2′, 7′-dichlorofluorescein diacetate (C-369, 10 μg/ml) fluorescent dyes (dissolved in Me2SO) obtained from Molecular Probes. The oxidized form of the dye acts as a control for changes in uptake, ester cleavage, and efflux, so that any changes in fluorescence seen between groups with the oxidation-sensitive dye can be directly attributed to changes in dye oxidation. Cells were treated for the indicated period and then harvested at 37 °C using trypsin, re-suspended in 37 °C glucose-free medium with or without drugs, labeled with the fluorescent dyes for 15 min at 37 °C, placed on ice, and analyzed using a FACScan flow cytometer (BD Biosciences, Mountain View, CA) (excitation 488 nm, emission 535 nm). Normal glucose levels were added to the +glucose controls (5 mm). The mean fluorescence intensity of 20,000 cells was analyzed in each sample and corrected for autofluorescence from unlabeled cells. Hydrogen Peroxide Production—A method for estimating H2O2 production by monitoring the irreversible inactivation of catalase in the presence of AT was developed based on previous reports (19Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4764) Google Scholar). PC-3 cells were infected with 50 m.o.i. AdCAT for 24 h, allowed to recover for 24 h in the absence of virus, and then placed in media in the presence and absence of glucose for 15 h. At the end of the 15-h incubation, 50 mm AT was added to the cultures and cells were harvested for catalase activity assay at 0 to 360 min of incubation. Inhibition of catalase activity was fit to a steady-state model as described previously (20Mackey M.A. Roti Roti J.L. J. Theor. Biol. 1992; 156: 133-146Crossref PubMed Scopus (17) Google Scholar). It was assumed that a transition to a new, final steady-state value of catalase activity was achieved over time according to the differential equation, dA/dt=−k(A−Af) where A is the catalase activity, k is a constant, and Af is the final steady-state catalase activity detected by the assay. Solving the above differential equation, we arrive at the following expression for the catalase activity as a function of time, A(t)=Af(1−e−kt)+A0e−kt where A0 is the catalase activity at time t0. Nonlinear least-squares regression analysis of the experimental data yielded estimates for the parameters Af, and k. Experimental data were fit to the above equation using the Levenberg-Marquardt nonlinear least-squares method (21Press W.H. Flannery B.P. Teukolsky S.A. Vetterling W.T. Numerical Recipes in C. Cambridge University Press, New York1988Google Scholar). This method yields the estimated parameter values, along with standard errors of these estimates. One-tailed Student's t tests were used to determine statistical significance of differences in fit parameters (p < 0.05). Measurement of Intracellular ATP Levels—Intracellular ATP was measured with the Bioluminescent Somatic Cell Assay Kit (Sigma) based on a described previously luciferin-luciferase assay (22Stanley P.E. Methods Enzymol. 1986; 133: 14-22Crossref PubMed Scopus (74) Google Scholar, 23Singh G. Lakkis C.L. Laucirica R. Epner D.E. J. Cell. Physiol. 1999; 180: 431-438Crossref PubMed Scopus (66) Google Scholar). Briefly, 25,000 GM00637G SV40-transformed human fibroblasts were plated per well in 24-well plates. Two days later cells were washed twice with PBS and incubated for 2 or 8 h with (+Glu) or without (–Glu) glucose in the presence of DNP (2 μm) and AntA (10 μm) in triplicate. At the time of the assay, experimental medium was aspirated off and cells received 250 μl of a 2:1:1 mixture of somatic cell releasing agent (provided in the kit), water, and culture medium (glucose and serum-free). Cells were then incubated at 37 °C for 10 min. Plates were swirled twice and 100 μl of the mixture from each well transferred into a 96-well plate, which already contained 100 μl of ATP assay mixture. Readings were obtained with a model 392 Luminoskan Ascent luminometer (ThermoLabsystems, Finland) with a 10-s delay time, a 30-s integrate time, and no pre-delay time. Results were obtained by comparison with a standard curve and normalized per mg of cellular protein as determined by the method of Bradford (24Bradford M.A. Anal. Biochem. 1976; 72: 248-256Crossref PubMed Scopus (211983) Google Scholar). NADP+/NADPH Measurement—Following the 24-h treatment with (+Glu) or without (–Glu) glucose, PC-3 cells were washed with PBS twice and scrape-harvested in PBS at 4 °C. After centrifugation at 320 × g for 5 min, cell pellets were resuspended in 150 μl of extraction buffer containing 0.1 m Tris-HCl, pH 8.0, 0.01 m EDTA, and 0.05% (v/v) Triton X-100. The cell suspension was sonicated at a duty cycle of 34% (Sonics Vibracell, VC750) for 2 min at 30-s intervals in a cup horn filled with ice water. The solution was centrifuged at 5500 × g for 5 min. The supernatants were collected and analyzed immediately for NADP+ and NADPH using a described previously method (25Zhang Z. Yu J. Stanton R.C. Anal. Biochem. 2000; 285: 163-167Crossref PubMed Scopus (98) Google Scholar). Briefly, an aliquot (50 μl) of the extract was incubated with 950 μl of extraction buffer at 37 °C for 5 min and an absorbance measurement was taken at 340 nm. This reading measures the total amount of NADPH and NADH in the sample (A1). Another 50-μl aliquot of the extract was pre-incubated at 37 °C for 5 min in a reaction mixture containing 5.0 IU of glucose-6-phosphate dehydrogenase, 0.1 m Tris-HCl, pH 8.0, 0.01 m MgCl2, and 0.05% (v/v) Triton X-100. The reaction was initiated by the addition of 5 mm glucose 6-phosphate. After incubation of the mixture at 37 °C for 5 min, absorbance measurement was taken at 340 nm. This reaction converted NADP+ to NADPH (A2). Finally, a 50-μl aliquot of the extract was preincubated at 25 °C for 5 min in a reaction mixture containing 5.0 IU of glutathione reductase, 0.1 m phosphate buffer, pH 7.6, 0.05 mm EDTA, and 0.05% (v/v) Triton X-100. The reaction was initiated by the addition of glutathione disulfide (GSSG, 5 mm) to convert NADPH to NADP+. The tubes were incubated for an additional 5 min at 25 °C, and absorbance at 340 nm was determined (A3). Subtraction of A3 from A1 represents the total amount of NADPH in the sample. The total amount of NADP+ was calculated by subtracting the A1 from A2 (25Zhang Z. Yu J. Stanton R.C. Anal. Biochem. 2000; 285: 163-167Crossref PubMed Scopus (98) Google Scholar). Results were obtained by comparison with a standard curve and normalized per mg of cellular protein as determined by the method of Bradford (24Bradford M.A. Anal. Biochem. 1976; 72: 248-256Crossref PubMed Scopus (211983) Google Scholar). Glutathione Reductase Measurement—Following 24 h treatment with (+Glu) or without (–Glu) glucose, PC-3 cells were washed with PBS twice and scrape-harvested in PBS at 4 °C. After centrifugation at 320 × g for 5 min, cell pellets were resuspended in potassium phosphate (100 mm), EDTA (3.4 mm) buffer. For each sample, a mixture containing 650 μl of ddH2O, 1500 μl of potassium/EDTA buffer, 350 μl of NADPH (0.8 mm), 100 μl of 30 mm GSSG, and 1% bovine serum albumin was incubated at 37 °C for 5 min. An aliquot (100 μl) of the cell homogenate was added to this mixture and absorbance measurement was taken at 340 nm for 5 min. Maximum linear rates for both samples and blank were used to calculate units of glutathione reductase activity as described previously based on the extinction coefficient of β-NADPH at 340 nm (26Mavis R.D. Stellwagen E. J. Biol. Chem. 1968; 243: 809-814Abstract Full Text PDF PubMed Google Scholar). Results were normalized per milligram of cellular protein as determined by the method of Bradford (24Bradford M.A. Anal. Biochem. 1976; 72: 248-256Crossref PubMed Scopus (211983) Google Scholar). Statistical Analysis—All results are expressed as mean ± 1 S.D. For analysis limited to two groups, Student's t test was employed (p < 0.05). Statistical comparisons among more than 2 treatment groups were accomplished using analysis of variance and the least significant difference test (p < 0.05) to determine differences between individual means. Mitochondrial Electron Transport Chain Blockers Enhance Glucose Deprivation-induced Cytotoxicity and Parameters Indicative of Oxidative Stress—The effect of ETCBs on glucose deprivation-induced cytotoxicity and parameters indicative of oxidative stress was determined in GM00637G SV40-transformed human fibroblasts. GM00637G cell survival decreased dramatically after 8 h of glucose deprivation in the presence of AntA, Myx, and Rot (relative to Me2SO control), but these drugs were not toxic in the presence of glucose during this time frame (Fig. 1A). No cytotoxicity was seen in cells treated with DNP in the presence or absence of glucose. Glucose deprivation (8 h) in the presence of ETCBs also increased a parameter indicative of oxidative stress (GSSG) in GM00637G-transformed human fibroblasts (Fig. 1B). As was noted in the survival assay, the presence of DNP caused no changes in GSSG, relative to the Me2SO control. In addition, no significant changes in ATP levels could be detected during 8 h in the presence or absence of glucose in GM00637G cells treated with AntA or DNP (Table I), supporting the hypothesis that ATP depletion was unlikely to contribute to the observed effects.Table IATP levels in GM00637G cells treated with +/–Glu for 2 or 8 h (n = 3)GroupGlucoseATP levels2 h8 hnmol/mg proteinControl+0.253 ± 0.0150.249 ± 0.037-0.249 ± 0.0240.270 ± 0.001Ant A (10 μm)+0.260 ± 0.0260.306 ± 0.040-0.
Referência(s)