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• W1989775722 abstract "In the present study, we find that cyclopentenone prostaglandins (PGs) of the J2 series, naturally occurring derivatives of PGD2, are potential inducers of intracellular oxidative stress that mediates cell degeneration. Based on an extensive screening of diverse chemical agents on induction of intracellular production of reactive oxygen species (ROS), we found that the cyclopentenone PGs, such as PGA2, PGJ2, Δ12-PGJ2, and 15-deoxy-Δ12,14-PGJ2, showed the most potent pro-oxidant effect on SH-SY5Y human neuroblastoma cells. As the intracellular events that mediate the PG cytotoxicity, we observed (i) the cellular redox alteration represented by depletion of antioxidant defenses, such as glutathione and glutathione peroxidase; (ii) a transient decrease in the mitochondrial membrane potential (Δψ); (iii) the production of protein-bound lipid peroxidation products, such as acrolein and 4-hydroxy-2-nonenal; and (iv) the accumulation of ubiquitinated proteins. These events correlated well with the reduction in cell viability. In addition, the thiol compound,N-acetylcysteine, could significantly inhibit the PG-induced ROS production, thereby preventing cytotoxicity, suggesting that the redox alteration is closely related to the pro-oxidant effect of cyclopentenone PGs. More strikingly, the lipid peroxidation end products, acrolein and 4-hydroxy-2-nonenal, detected in the PG-treated cells potently induced the ROS production, which was accompanied by the accumulation of ubiquitinated proteins and cell death, suggesting that the membrane lipid peroxidation products may represent one of the causative factors that potentiate the cytotoxic effect of cyclopentenone PGs by accelerating intracellular oxidative stress. These data suggest that the intracellular oxidative stress, represented by ROS production/lipid peroxidation and redox alteration, may underlie the well documented biological effects, such as antiproliferative and antitumor activities, of cyclopentenone PGs. In the present study, we find that cyclopentenone prostaglandins (PGs) of the J2 series, naturally occurring derivatives of PGD2, are potential inducers of intracellular oxidative stress that mediates cell degeneration. Based on an extensive screening of diverse chemical agents on induction of intracellular production of reactive oxygen species (ROS), we found that the cyclopentenone PGs, such as PGA2, PGJ2, Δ12-PGJ2, and 15-deoxy-Δ12,14-PGJ2, showed the most potent pro-oxidant effect on SH-SY5Y human neuroblastoma cells. As the intracellular events that mediate the PG cytotoxicity, we observed (i) the cellular redox alteration represented by depletion of antioxidant defenses, such as glutathione and glutathione peroxidase; (ii) a transient decrease in the mitochondrial membrane potential (Δψ); (iii) the production of protein-bound lipid peroxidation products, such as acrolein and 4-hydroxy-2-nonenal; and (iv) the accumulation of ubiquitinated proteins. These events correlated well with the reduction in cell viability. In addition, the thiol compound,N-acetylcysteine, could significantly inhibit the PG-induced ROS production, thereby preventing cytotoxicity, suggesting that the redox alteration is closely related to the pro-oxidant effect of cyclopentenone PGs. More strikingly, the lipid peroxidation end products, acrolein and 4-hydroxy-2-nonenal, detected in the PG-treated cells potently induced the ROS production, which was accompanied by the accumulation of ubiquitinated proteins and cell death, suggesting that the membrane lipid peroxidation products may represent one of the causative factors that potentiate the cytotoxic effect of cyclopentenone PGs by accelerating intracellular oxidative stress. These data suggest that the intracellular oxidative stress, represented by ROS production/lipid peroxidation and redox alteration, may underlie the well documented biological effects, such as antiproliferative and antitumor activities, of cyclopentenone PGs. Oxidative stress is increasingly seen as a major upstream component in the signaling cascade involved in many of the cellular functions such as cell proliferation, inflammatory responses, stimulating adhesion molecule, and chemoattractant production (1Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Clarendon Press, Oxford1989Google Scholar). It has been suggested that some level of oxidative stress may be required in response to cytotoxic agents and converted into the redox regulatory system as a downstream signaling pathway (2Sundaresan M., Yu, Z.-X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2300) Google Scholar). However, excess oxidative stress may be toxic, exerting cytostatic effects, causing membrane damage, and activating pathways of cell death (apoptosis and/or necrosis). Reactive oxygen species (ROS) 1The abbreviations used are:ROSreactive oxygen speciesPGsprostaglandins15d-PGJ215-deoxy-Δ12,14-PGJ2PGD2prostaglandin D2HNE4-hydroxy-2-nonenalMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideELISAenzyme-linked immunosorbent assayDiOC6(3)3,3′-dihexyloxacarbocyanine iodideCCCPcarbonyl cyanidem-chlorophenylhydrazonePBSphosphate-buffered salinemAbmonoclonal antibodyNACN-acetylcysteineDCFH-DA2′,7′-dichlorodihydrofluorescein diacetateΔψmitochondrial membrane potential generated during oxidative stress may be responsible for these effects due to their ability to damage cellular components, such as membrane lipids. Lipid peroxidation mediated by a free radical chain reaction mechanism yields lipid hydroperoxides as primary products, and subsequent decomposition of the lipid hydroperoxides generates a large number of reactive aldehydes, such as ketoaldehydes, 2-alkenals, and 4-hydroxy-2-alkenals (3Esterbauer H. Schauer R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5847) Google Scholar). There is increasing evidence that these aldehydes are causally involved in most of the pathophysiological effects associated with oxidative stress in cells and tissues.The prostaglandins (PGs) are a family of structurally related molecules that are produced by cells in response to a variety of extrinsic stimuli and regulate cellular growth, differentiation, and homeostasis (4Smith W.L. Biochem. J. 1989; 259: 315-324Crossref PubMed Scopus (763) Google Scholar, 5Smith W.L. Am. J. Physiol. 1992; 263: F181-F191PubMed Google Scholar). PGs are derived from fatty acids, primarily arachidonate, that are released from membrane phospholipids by the action of phospholipases. Arachidonate is first converted to an unstable endoperoxide intermediate by cyclooxygenases and subsequently converted to one of several related products, including PGD2, PGE2, PGF2α, prostacyclin (PGI2), and thromboxane A2, through the action of specific PG synthetases. PGD2 is a major cyclooxygenase product in a variety of tissues and cells and has marked effects on a number of biological processes, including platelet aggregation, relaxation of vascular and nonvascular smooth muscles, and nerve cell functions (6Giles H. Leff P. Prostaglandins. 1988; 35: 277-300Crossref PubMed Scopus (159) Google Scholar). PGD2 readily undergoes dehydration in vivo andin vitro to yield additional biologically active PGs of the J2 series (Fig. 1 A) (7Fizpatrik F.A. Wynalda M.A. J. Biol. Chem. 1983; 258: 11713-11718Abstract Full Text PDF PubMed Google Scholar, 8Kikawa Y. Narumiya S. Fukushima M. Wakatsuka H. Hayaishi O. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1317-1321Crossref PubMed Scopus (164) Google Scholar, 9Hirata Y. Hayashi H. Ito S. Kikawa Y. Ishibashi M. Sudo M. Miyazaki H. Fukushima M. Narumiya S. Hayaishi O. J. Biol. Chem. 1988; 263: 16619-16625Abstract Full Text PDF PubMed Google Scholar). Members of J2 series of PGs, characterized by the presence of a reactive α,β-unsaturated ketone in the cyclopentenone ring (cyclopentenone PGs), have their own unique spectrum of biological effects, including antitumor activity, the inhibition of cell cycle progression, the suppression of viral replication, the induction of heat shock protein expression, and the stimulation of osteogenesis (10Fukushima M. Prostaglandins Leukot. Essent. Fatty Acids. 1992; 47: 1-12Abstract Full Text PDF PubMed Scopus (186) Google Scholar).In the present study, as part of an effort to identify endogenous inducer of intracellular oxidative stress and to elucidate the molecular mechanism underlying the oxidative stress-mediated cell degeneration, we examined the oxidized fatty acid metabolites for their ability to induce intracellular ROS production in a human neuroblastoma SH-SY5Y cell and found that the J2 series of the PGs represent the most potent inducers. In addition, the intracellular ROS production was accompanied by the alteration of cellular redox status and the production of lipid peroxidation-derived highly cytotoxic aldehydes, such as acrolein and 4-hydroxy-2-nonenal (HNE), which could also induced the intracellular ROS production. These data suggest that intracellular oxidative stress constitutes a pivotal step in the pathway of cellular dysfunction induced by the PGs.EXPERIMENTAL PROCEDURESMaterialsPGs and several other lipid peroxidation products were purchased from the Cayman Chemical Co. (Ann Arbor, MI). Horseradish peroxidase-linked anti-goat and anti-mouse IgG immunoglobulins and enhanced chemiluminescence (ECL) Western blotting detection reagents were obtained from Amersham Pharmacia Biotech. The protein concentration was measured using the BCA protein assay reagent obtained from Pierce. 3,3′-Dihexyloxacarbocyanine iodide (DiOC6(3)), carbonyl cyanide m-chlorophenylhydrazone (CCCP), andN-acetylcysteine (NAC) were from Sigma. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was from Molecular Probes Inc. (Eugene, OR).Cell CultureSH-SY5Y cells were grown in Cosmedium-001 (Cosmo-Bio, Tokyo, Japan) containing 5% Nakashibetsu precolostrum newborn calf serum, 100 μg/ml penicillin, and 100 units/ml streptomycin. Cells were seeded in plates coated with polylysine and cultured at 37 °C.Cell ViabilityCell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells incubated with PGs or other chemicals were treated with 10 μl of MTT solution (5 mg/ml) for 4 h. The cells were then lysed with 0.04 n HCl in isopropyl alcohol, and the absorbance was read at 570 nm.Flow Cytometry Analysis of ROS and Mitochondrial Membrane Potential (Δψ)DCFH-DA was employed to measure ROS (11Narayanan P.K. Goodwin E.H. Lehnert B.E. Cancer Res. 1997; 57: 3963-3971PubMed Google Scholar, 12Rothe G. Valet G. J. Leukocyte Biol. 1990; 47: 440-448Crossref PubMed Scopus (780) Google Scholar). Cells were incubated with 10 μm 2′,7′-dichlorofluorescein diacetate (dissolved in dimethyl sulfoxide) for 30 min at 37 °C and then treated with different agents for an additional 30 min at 37 °C. After chilling on ice, the cells were washed with ice-cold PBS, scraped from the plate, and resuspended at 1 × 106 cells/ml in PBS containing 10 mm EDTA. For the detection of Δψ, 40 nm DiOC6(3) (13Li P.-F. Dietz R. Von Harsdorf R. EMBO J. 1999; 18: 6027-6036Crossref PubMed Scopus (426) Google Scholar) in the absence or presence of 15-deoxy-Δ12,14-PGJ2(15d-PGJ2) was added and incubated for 15 min at 37 °C. The fluorescence was measured using a flow cytometer (Epics XL, Beckman Coulter).Glutathione AssayMeasurement of GSH in the cells was performed fluorometrically according to the method of Hissin and Hilf (14Hissin P.J. Hilf R. Anal. Biochem. 1976; 74: 214-216Crossref PubMed Scopus (3659) Google Scholar). In brief, the cells incubated with HNE or NAC were washed twice with PBS (pH 7.0) and extracted with the 25% (w/v) metaphosphoric acid solution containing 5 mm EDTA. After ultracentrifugation (105,000 × g, 30 min), 1.8 ml of 0.1 mphosphate solution (pH 8.0) containing 5 mm EDTA and 100 μl of the o-phthalaldehyde solution (1 mg/ml) were added to the resulting supernatant (100 μl), and then the fluorescence intensity at 420 nm was then determined with activation at 350 nm.Glutathione Peroxidase AssayGSH peroxidase activity was determined according to the method of Lawrence and Burk (15Lawrence R.A. Burk R.F. Biochem. Biophys. Res. Commun. 1976; 71: 952-958Crossref PubMed Scopus (2880) Google Scholar). One unit was defined as the amount of enzyme required to oxidize 0.5 μmol of NADPH (corresponding to 1 μmol of reduced GSH) per min.Measurements of Acrolein and HNE LevelsThe levels of acrolein and HNE were measured, as their protein-bound forms, by competitive enzyme-linked immunosorbent assays (ELISA), using anti-protein-bound HNE (mAbHNEJ2) (16Toyokuni S. Miyake N. Hiai H. Hagiwara M. Kawakishi S. Osawa T. Uchida K. FEBS Lett. 1995; 359: 189-191Crossref PubMed Scopus (180) Google Scholar) and anti-protein-bound acrolein (mAb5F6) (17Uchida K. Kanematsu M. Sakai K. Matsuda T. Hattori N. Mizuno Y. Suzuki D. Miyata T. Noguchi N. Niki E. Osawa T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4882-4887Crossref PubMed Scopus (544) Google Scholar) monoclonal antibodies, as previously reported (18Satoh K. Yamada S. Koike Y. Toyokuni S. Kumano T. Takahata T. Hayakari M. Tuchida S. Uchida K. Anal. Biochem. 1999; 270: 323-328Crossref PubMed Scopus (70) Google Scholar).Immunoblot AnalysisFor detection of the ubiquitinated proteins, whole cell lysates from SH-SY5Y cells treated with 15d-PGJ2 were incubated with SDS sample buffer for 5 min at 100 °C. The samples ware separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One gel was used for staining with Coomassie Brilliant Blue; the other gel was transblotted onto a nitrocellulose membrane, incubated with Blockace for blocking, washed, and incubated with the anti-ubiquitin polyclonal antibody (Biomeda Co., Foster City, CA). This procedure was followed by the addition of horseradish peroxidase conjugated to rabbit anti-mouse IgG and ECL reagents. The bands were visualized by Cool Saver AE-6955 (ATTO, Tokyo, Japan).ImmunocytochemistryFor immunocytochemistry, cells were fixed overnight in PBS containing 2% paraformaldehyde and 0.2% picric acid at 4 °C. Membranes were permeabilized by exposing the fixed cells to PBS containing 0.3% Triton X-100. The cells were then sequentially incubated in PBS solutions containing blocking serum (5% normal goat serum) and immunostained with anti-protein-bound acrolein monoclonal antibody (mAb5F6) (17Uchida K. Kanematsu M. Sakai K. Matsuda T. Hattori N. Mizuno Y. Suzuki D. Miyata T. Noguchi N. Niki E. Osawa T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4882-4887Crossref PubMed Scopus (544) Google Scholar) or polyclonal antibodies that specifically recognize protein-bound HNE (19Uchida K. Szweda L.I. Chae H.Z. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8742-8746Crossref PubMed Scopus (353) Google Scholar). The cells were then incubated for 1 h in the presence of fluorescein isothiocyanate-labeled goat anti-rabbit and CyTM3-labeled goat anti-mouse, rinsed with PBS containing 0.3% Triton X-100, and covered with anti-fade solution. Images of cellular immunofluorescence were acquired using a confocal laser microscope (Bio-Rad) with a 40× objective (488-nm excitation and 518-nm emission).Statistical AnalysisThe paired Student's ttest was used to compare the significance of the differences between data.RESULTSCyclopentenone PGs as Potential Inducers of Intracellular ROS ProductionTo identify endogenous inducer of intracellular oxidative stress, we screened a large number of lipophilic chemicals, including oxidized fatty acid metabolites, on induction of intracellular ROS production and found that some of the PG derivatives showed potent pro-oxidant effects on human neuroblastoma SH-SY5Y cells. As shown in Fig. 1 B, the intracellular ROS production in SH-SY5Y cells was significantly induced by PGA2 and by the PGD2 metabolites, such as PGJ2, Δ12-PGJ2, and 15d-PGJ2. Among the J2 series of PGs, the intracellular ROS production was most potently induced by 15d-PGJ2, which was followed by it precursors, PGJ2 and Δ12-PGJ2. The level of ROS in the cells exposed to 15d-PGJ2 (10 μm) was ∼10-fold higher than that of the control. Other oxidized fatty acids, including (9R)-hydroxy-(10E,12Z)-octadecadienoic acid, (+)-13-hydroxy-(9Z,11E)-octadecadienoic acid, (9S)-hydroperoxy-(10E,12Z)-octadecadienoic acid, (13S)-hydroperoxy-(9IZ,11IE)-octadecadienoic acid, 9-oxo-(10E,12Z)-octadecadienoic acid, 13-oxo-(9Z,11E)-octadecadienoic acid, (+)-13-hydroxy-(9Z,11E)-octadecadienoic acid cholesteryl ester, and (+)-9(10)-epoxy-(12Z)-octadecenoic acid, had no significant effects on the ROS production (data not shown).Correlation between ROS Production and CytotoxicityTo examine the correlation between ROS production and cytotoxicity in SH-SY5Y cells exposed to PGs, we examined the cytotoxic effects of PGs by MTT assay. As shown in Fig.2 A, among the PGs tested, the J series of PGs resulted in a rapid decrease in MTT reduction levels to <40% of basal levels within 24 h of exposure. A significant cytotoxicity was also observed when the cells were treated with PGA2 and PGD2 at the concentration of 25 μm (data not shown). In contrast to these PGs, the MTT reduction levels were maintained at 80–90% of basal levels in SH-SY5Y cells exposed to other PGs, such as 13,14-dihydro-15-keto-PGA2, PGB2, PGE2, and 15-keto-PGE2. As shown in Fig.2 B, 15-PGJ2 induced cell death in time- and dose-dependent manners. Even 5 μm15d-PGJ2 did cause a 40% decrease in the MTT reduction level after 24 h of incubation, indicating that 15d-PGJ2, the terminal metabolite of PGD2, may represent the most potent cytotoxic metabolite. Similar results were obtained from other cell viability assays, such as crystal violet and trypan blue exclusion assays (data not shown). As shown in Fig.2 C, the PG-induced ROS production was well correlated with the cytotoxicity. The correlation between ROS production and cytotoxicity was also suggested by the observation that α-tocopherol, a lipophilic antioxidant, significantly inhibited the PG cytotoxicity (Fig. 2 D).Figure 2Correlation between intracellular ROS production and cytotoxicity. A, viability of SH-SY5Y cells exposed to PGs. Cells were exposed to 10 (hatched bar) or 25 μm (closed bar) of PGs for 24 h. The cell viability was measured by the MTT assay. Data are expressed as percent of control culture conditions. B, viability of SH-SY5Y cells exposed to 15d-PGJ2. Time- and dose-dependent reduction of cell viability induced by 5 (open circle), 10 (open square), 25 (closed triangle), and 50 μm (closed circle) of 15d-PGJ2. The cell viability was measured by the MTT assay. Data are expressed as percent of control culture conditions.C, correlation between ROS production and cytotoxicity in the cells exposed to PGs. Cells were exposed to 10 μm of PGs for 1 h for measuring ROS production and for 24 h for measuring cell viability. Symbol numbers: 1, 15-keto-PGE2; 2, PGE2; 3, PGB2; 4, PGD2; 5, 15-keto-PGA2; 6, PGA2; 7, PGJ2; 8, Δ12-PGJ2;9, 15d-PGJ2. D, effect of α-tocopherol (α-Toc) on cell death induced by 15d-PGJ2. The cells were treated with α-tocopherol (0–100 μm) for 4 h, washed twice with PBS, and then exposed to 10 μm 15d-PGJ2 or vehicle for 24 h. A, B, and D, the data represent mean ± S.D. of triplicate determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Redox Alteration Induced by Cyclopentenone PGsA potential pathway that might mediate these effects of cyclopentenone PGs involves alteration of cellular redox status. To examine whether cyclopentenone PGs could influence the redox status, we measured the intracellular GSH levels and GSH peroxidase activity. As shown in Fig.3 A, the GSH levels were partially diminished by treatment with 15d-PGJ2. It was also observed that the 15d-PGJ2 treatment of the cells resulted in a significant decrease in the GSH peroxidase activity (Fig.3 B). To confirm whether the PG-induced redox alteration plays a role in mediating the ROS production and cytotoxicity, SH-SY5Y cells were pretreated with the thiol compound NAC prior to the exposure to 15d-PGJ2, and then the ROS production and cell viability were examined. As shown in Fig. 3, C and D, the NAC pretreatment resulted in an increased survival as well as in the inhibition of ROS production in the cells. These data suggest that redox alteration may be closely related to the action of cyclopentenone PGs.Figure 315d-PGJ2-induced redox alteration and effect of NAC pretreatment on ROS production and cell death induced by 15d-PGJ2. A, changes in intracellular GSH level. Cells were exposed to 10 μm of 15d-PGJ2 for different time intervals as indicated in the figure. Intracellular GSH levels were fluorometrically measured as described under “Experimental Procedures.” B, changes in intracellular GSH peroxidase activity. Cells were exposed to 10 μm of 15d-PGJ2 for different time intervals as indicated in the figure. *, statistical difference between pre- and post-15d-PGJ2 treatment (p < 0.05).C, effect of NAC pretreatment on ROS production induced by 15d-PGJ2. The cells pretreated with NAC (0–400 μm) for 4 h were incubated with 10 μmDCFH-DA for 30 min, washed twice with PBS, and then treated with 10 μm 15d-PGJ2 for 1 h. After washing with PBS, the cells were resuspended in PBS containing 10 μmEDTA, and then the fluorescence intensity of more than 10,000 cells was analyzed using a flow cytometer. D, effect of NAC pretreatment on cell death induced by 15d-PGJ2. The cells were treated with NAC (0–500 μm) for 4 h, washed twice with PBS, and then exposed to 20 μm15d-PGJ2 or vehicle for 24 h. Cell viability was then measured by the MTT assay. Data are expressed as percent of control culture conditions. A, B, and D, the data represent means ± S.D. of triplicate determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mitochondria as the Source of ROSIt is believed that mitochondrial oxidative phosphorylation is the major endogenous source of the ROS and is involved in a wide variety of disorders (20Wallace D.G. Science. 1999; 283: 1482-1488Crossref PubMed Scopus (2585) Google Scholar). Therefore, it was anticipated that the ROS detected in the cells exposed to PGs may originate from the mitochondria, one of the major ROS-producing organella. In this context, we measured the alteration of the mitochondrial membrane potential (Δψ), which is a component of the overall proton motive force that drives the ATP production in the mitochondria. As shown in Fig.4 A, 15d-PGJ2induced a significant decrease in mitochondrial Δψ, suggesting that the PG acted on the process of oxidative phosphorylation. We then examined the effect of CCCP treatment on the 15d-PGJ2-induced ROS production. CCCP has a dissociable proton and acts by carrying protons across the inner mitochondrial membrane, resulting in depletion of mitochondrial electrochemical gradient (Δψ) by dissipating the proton gradient. As shown in Fig.4 B, CCCP alone did not cause ROS production, whereas the pretreatment of CCCP led to a dose-dependent inhibition of intracellular ROS production induced by 15d-PGJ2. These results suggest that mitochondrial electron transport chain is involved in the 15d-PGJ2-induced ROS production.Figure 4Mitochondria as the source of intracellular ROS. A, time-dependent alterations of Δψ induced by 15d-PGJ2. The cells were incubated with 40 nm DiOC6(3) in the absence or presence of 15d-PGJ2 for 15 min at 37 °C. B, effect of CCCP pretreatment on the 15d-PGJ2-induced ROS production. The cells incubated with 10 μm DCFH-DA for 30 min were pretreated with CCCP (0–100 μm) for 30 min and then treated with 10 μm 15d-PGJ2 for 30 min.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Accumulation of Protein-bound Aldehydes and Ubiquitinated Proteins in SH-SY5Y Cells Exposed to Cyclopentenone PGsIn addition to the ROS production, we found that the PG cytotoxicity was accompanied by the production of lipid peroxidation-derived highly cytotoxic aldehydes, such as acrolein and HNE (Fig.5 A), in the cells. The levels of acrolein and HNE were measured, as the protein-bound forms, by competitive ELISA and immunocytochemical assays. As shown in the Fig.5 B, 15d-PGJ2 enhanced the productions of the protein-bound acrolein and HNE in a time-dependent manner. Maximum 3- and 8-fold increases in the production of acrolein and HNE, respectively, was observed, and the production of both modified proteins persisted for at least 24 h. Noncytotoxic PGs, such as PGA2, PGB2, and PGE2, did not produce the protein-bound aldehydes (Fig. 5, C andD). Immunocytochemical analyses showed that exposure of the cells to 15d-PGJ2 resulted in the appearance of acrolein and HNE reactivity in essentially all cells (Fig.6). Since these aldehydes are known to be the most reactive electrophiles, it is likely that the cytotoxic effect of 15d-PGJ2 was potentiated by these aldehydes.Figure 5Detection of protein-bound reactive aldehydes in SH-SY5Y cells exposed to 15d-PGJ2. A,chemical structures of lipid peroxidation products, acrolein, and HNE.B, competitive ELISA analysis of protein-bound acrolein (open square) and protein-bound HNE (closed square) in the cells exposed to 10 μm15d-PGJ2. C, competitive ELISA analysis of protein-bound HNE in the cells exposed to 10 μm PGs.D, competitive ELISA analysis of protein-bound acrolein in the cells exposed to 10 μm PGs. B, C, andD, the data represent means ± S.D. of triplicate determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Immunocytochemical detection of protein-bound HNE and protein-bound acrolein in SH-SY5Y cells exposed to 15d-PGJ2. The cells were incubated with 10 μm 15d-PGJ2 at 37 °C. The digitized images were colorized and combined using Adobe Photoshop, 3.0. Fluorescein isothiocyanate fluorescence (protein-bound HNE, green) is shown in the left column of panels (a–d); CyTM3 fluorescence (protein-bound acrolein, red) is shown in the center column of panels (e–h), and the corresponding combined (superimposed) images are shown in theright column of panels (i–l) (yellowrepresents colocalization).View Large Image Figure ViewerDownload Hi-res image Download (PPT)On the other hand, it was anticipated that the PG-induced oxidative stress leading to the formation of oxidatively modified proteins provokes the misfolding of proteins, which may then be targeted for degradation by the ubiquitin-dependent proteolytic pathway (21Stadtman E.R. Biochemistry. 1990; 29: 6323-6331Crossref PubMed Scopus (185) Google Scholar). To examine whether the ubiquitin pathway is activated by the PG-induced oxidative stress, ubiquitin-protein conjugates generated in the cells were analyzed by an immunoblot analysis. As shown in Fig.7 A, the J series of PGs most significantly induced the generation of ubiquitin-protein conjugates with high molecular weights (>100 kDa); other PGs, including PGA2, 15-keto-PGA2, PGB2, PGD2, PGE2, and 15-keto-PGE2, were less effective or ineffective. Upon incubation with 15d-PGJ2, the ubiquitinated proteins were detected from 30 min to 24 h and returned to the level of the control at 48 h (Fig. 7 B). These data suggest that the PG-induced oxidative stress may lead to the increased ubiquitination of aberrant proteins, including oxidatively modified proteins.Figure 7Immunoblot analysis of ubiquitin-protein conjugates. A, generation of ubiquitin-protein conjugates in the cells exposed to 10 μm PGs for 8 h. B, time-dependent generation of ubiquitin-protein conjugates in the cells exposed to 15d-PGJ2. The cells were incubated with 10 μm15d-PGJ2 at 37 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Intracellular ROS Production and Cell Death Induced by Reactive AldehydesMore strikingly, we found that the lipid peroxidation-derived reactive aldehydes could be a second source of ROS in the cells. As shown in Fig.8 A, when SH-SY5Y cells were treated with a variety of reactive aldehydes, both acrolein and HNE caused a significant increase in the ROS levels. Interestingly, the α,β-unsaturated aldehydes, such as crotonaldehyde and 2-nonenal, possessing an analogous functionality to acrolein and HNE, were all inactive. We also found that both acrolein and HNE showed the most potent cytotoxicity (Fig. 8 B). In addition, generation of ubiquitinated protein was observed in the cells exposed to the aldehyde (HNE) (Fig. 8 C). These results are consistent with the observation (Fig. 2) that the ROS production in the cells exposed to PGs was closely associated with the cytotoxicity. These data suggest that the reactive aldehydes, such as acrolein and HNE, may potentiate the effect of 15d-PGJ2 by accelerating the ROS production and redox alteration in the cells (Fig.9).Figure 8Cell death, intracellular ROS production, and accumulation of ubiquitinated proteins induced by lipid peroxidation" @default.
• W1989775722 created "2016-06-24" @default.
• W1989775722 creator A5004162735 @default.
• W1989775722 creator A5035336165 @default.
• W1989775722 creator A5055822025 @default.
• W1989775722 creator A5057254057 @default.
• W1989775722 creator A5087932160 @default.
• W1989775722 date "2001-04-01" @default.
• W1989775722 modified "2023-10-16" @default.
• W1989775722 title "Cyclopentenone Prostaglandins as Potential Inducers of Intracellular Oxidative Stress" @default.
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