EPC-K1 ATTENUATES PEROXYNITRITE-INDUCED APOPTOSIS IN CEREBELLAR GRANULE CELLS

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1 Vol. 46, No. 1, September 1998 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Poges EPC-K1 ATTENUATES PEROXYNITRITE-INDUCED APOPTOSIS IN CEREBELLAR GRANULE CELLS Taotao Wei, Chang Chen, Baolu Zhao, and Wenjuan Xin* Institute of Biophysics, Academia Sinica, Beijing , China Akitane Moil Department of Neuroscience, Institute of Molecular and Cellular Medicine Okayama University Medical School, Okayama, Japan Received May 10, 1998 SUMMARY Apoptosis induced by peroxynitrite in cultured cerebellar granule cells was confirmed morphologically by chromatin condensation and biochemically by DNA laddering. A 30 min exposure to peroxynitrite (10 pm) initiated oxidative stress, which caused the formation of thiobarbituric acid-reactive substances (TBARS) and the alteration of cell membrane fluidity. Peroxynitrite treatment also caused ATP decrease and thus activated the apoptotic program. Pre-treating cells with antioxidant EPC-K1 (L-ascorbic acid 2-[3,4-dihydro-2,5,7,8- tetramethyl-2-(4,8,12-trimethyltridecyl)-2h-l-benzopyran-6-yl-hydrogen phosphate] potassium salt), a new water-soluble derivative of vitamin C and vitamin E, attenuated oxidative injury and prevents cells from apoptosis. The results suggest that EPC-K1 might be used as a potential therapeutic agent for diseases associated with NO/ONOO--mediated neuronal injury. Keywords: cerebellar granule cells, apoptosis, peroxynitrite, antioxidants, EPC-K1 INTRODUCTION Apoptosis, also termed as programmed cell death, is a fundamentally important biological process that is required to maintain the integrity and homeostasis of multicellular organisms. (1). However, inappropriate apoptosis plays important roles in the pathogenesis of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and various forms of cerebellar degeneration (2,3), while more and more evidence has suggested that reactive oxygen species (ROS) might play important roles in the cascade of events leading to neuronal apoptosis (4). Nitric oxide (NO), a short-life free radical generated endogenously, exerts a number of important functions including neurotransmission, synaptic plasticity and long-term memory in central nervous system (CNS) (5,6). When * Corresponding author /98/ ) /0 Copyright by Academic Press Australia. All rights of reproduction in any form reserved

2 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL produced with superoxide concurrently, NO converts to peroxynitrite (ONOO-) at high rates (k _> 6.7 x 10 9 M-I-s-I), which acts as a pathological mediator in some neurodegenerative diseases (7), but the mechanisms are still not clear. Accordingly, antioxidants might be useful drugs for these neuronal diseases (8,9). EPC-K1 is a novel phosphate ester derivative of vitamin C and vitamin E, which acts as an inhibitor on lipid peroxidation (10) and protects brain from ischemia-reperfusion injury (11,12), while the effect on peroxynitrite-mediated neurotoxicity has not been reported yet. In the present work, the protective effect of EPC-K 1 on peroxynitrite-induced neuronal cell apoptosis was studied, and the mechanisms were also discussed. MATERIALS AND METHODS Materials. Wistar rats were purchased from Beijing Medical University, China. Cell culture plastic ware was purchased from Costar (Cambridge, MA, USA). Cell culture reagents, culture medium, fetal bovine serum, proteinase K, and RNase A were products of Gibco BRL (Grand Island, NY, USA). Agarose, ethidium bromide, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), and poly-l-lysine were purchased from Sigma (St. Louis, MO, USA). 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was purchased from Aldrich (Milwaukee, WI, USA). L-ascorbic acid 2-(3,4-dihydro-2,5,7,8-tetramethyl-2- (4,8,12-trimethyltridecyl)-2H-l-benzopyran-6-yl-hydrogen phosphate) potassium salt (EPC- K1) was a generous gift of Senju Pharmaceutical Co. Ltd., Japan. Peroxynitrite was synthesized in a quenched flow reactor as described previously (13) and was diluted with 10 mm NaOH just before use. Other reagents made in China were of analytical grade. Cell culture. Primary cultures of rat cerebellar granule cells were prepared following procedures we described previously (14). Cells were plated on 6-well multidishes (2 x 10 6 cell/ml, 2 ml/well) or 24-well multidishes (2.5 x 10 6 cell/ml, 0.4 ml/well) previously coated with poly-l-lysine and maintained at 37 ~ in a humidified 5 % CO2-95 % air atmosphere. Treatment with peroxynitrite, Culture media were removed, cells were washed twice with HBSS and incubated in HBSS supplemented with 25 mm HEPES (ph 7.4) at 37 ~ for 15 min. Antioxidants, when employed, were added during this period. Aliquots of peroxynitrite stock solution were rapidly added to wells followed by gently mixing to contribute the peroxynitrite. Control cells were treated with the same volumes of 10 mm NaOH solution. After incubation with peroxynitrite at 37 ~ for 30 min, cells were washed twice with 1-IBSS, the original culture media were restored, and cells were cultured for indicated time. Under these experiment conditions, the addition ofperoxynitrite only caused slight ph shifts. Morphological studies. Ultrastructure of cells was observed by electron microscopy. Samples were prepared following standard protocols (15). Briefly, cells were fixed with 2.5 % glutaraldehyde at 4 ~ for 1 h and post-fixed with 1% OsO4 at 4 ~ for 1 h, dehydrated through a series of graded ethanol solutions, and embedded in resin. Ultrathin sections of samples were stained with uranyl acetate and lead citrate and observed with an electron microscope. Detection of DNA fragmentation, The laddering pattern of DNA fragmentation was detected by agarose gel electrophoresis according to previous report (16). Briefly, a quantity of cells 90

3 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL (1.2 x 107) were pelletted, rinsed with cold PBS, and lysed in 10 mm Tris, 10 mm EDTA, 0.2 % Triton X-100, ph 7.5. After 15 min on ice, the lysate was centrifugated at x g for 10 min. The supernatant (which contained RNA and fragmented DNA but not intact chromatin) was treated with 0.5 mg/ml proteinase K and 0.5 mg/ml RNase A at 50 ~ for 3 h. After phenol extraction and chloroform extraction, DNA was perticipated with 0.1 volume of 10 M ammonium acetate and 2 volumes of ethanol at -20 ~ overnight, and harvested by centrifugation at x g for 15 rain. After washed with 70 % ethanol, DNA was dissolved in TE (10 mm Tris, 1 mm EDTA, ph 8.0) and analyzed by 1.5 % agarose gel electrophoresis. Assessment of cell viability. Cell viability was assessed by MTT assay (17). MTT was added to cells cultured in 24-well multidishes (0.5 mg/ml, final concentration). After incubation at 37 ~ for 30 min, 1 ml oflysis solution (10 % SDS, 25 % DMF, ph 3.5) was added and optical density at 570 nm was measured. Measurement of lipid peroxidation. Lipid peroxidation in cerebellar granule cells was measured by thiobarbituric acid (TBA) assay according to the method discribed previously (18). Spin labelling. Cell membrane fluidity was determined by ESR spin labelling technique. A quantity of cells (1.2 x 107) cells were incubated with 100 IxM 5-doxyl-stearic acid in PBS at 37 ~ for 30 min, washed 3 times with PBS, and transfered into quartz capillaries for ESR measurement. ESR spectrum was recorded by a Varian E-109 spectrometer with measurement conditions as: X-band, 100 khz modulation with 0.1 mt modulation amplitudes, 10 mw microwave power, 25 ~ The order parameter (S) was calculated from the ESR spectrum as discribed (19). Measurement of ATP. Cellular ATP content was measured by HPLC (20). Briefly, 1.2 x 107 cells were peuetted and treated with 0.5 M perchloric acid for 30 min on ice. After centrifugation at x g for 10 min, the supernatant was neutralized and then analyzed by HPLC (Zorbax 5 Bm ODS column, 250 x 4.6 mm). The eluent was 0.05 M phosphate buffer (ph 6.0) containing 3.75 % methanol. Statistical analysis. Each experiment was performed at least three times and results are presented as mean +_ SD. Statistical analyses were performed using one-way A_NOVA or student's t-test. A probability of< 0.05 was considered significant. RESULTS Characteristics of apoptosis. After exposure to 10 BM peroxynitrite for 30 min, cerebellar granule cells underwent apoptosis, which was morphologically characterized by neurite breakdown, cell shrinkage, chromatin condensation, and the formation of apoptotic bodies (Figure 1). Cells pre-treated with antioxidants EPC-K1, vitamin C or Trolox (100 BM) were protected from death. Agarose gel electrophoresis of DNA extracted from peroxynitritetreated cells showed a ladder pattern, which is a well-accepted characteristic of apoptosis (Figure 2). Pre-treating cells with 50 pm EPC-K1 effectively prevented DNA from fragmentation. Vitamin C or Trolox also showed protective effects at higher concentrations (100 gm). 91

4 Vol. 46, No. ], ]998 BIOCHEMISTRYondMOLECULAR BIOLOGY INTERNATIONAL B ~i 9 84 i ~ 9!i Figure 1. Ultrastructure of normal (A, x 12,000) and peroxynitrite-treated 03, x 10,000) cerebellar granule cells. Cell viability. Dead cells increased gradually after exposure to peroxynitrite as assessed by MTT assay. EPC-K1 effectively prevented cells from death (Figure 3). Vitamin C or Trolox showed similar protective effects at higher concentrations (100 IxM). Lipid peroxidation in cells. Exposure of cultures to peroxynitrite induced lipid peroxidation in cells as indicated by the significant increase in TBARS levels. In cultures pre-treated with 50 IxM EPC-K1, the formation of TBARS was markedly suppressed, which suggested that EPC- K1 inhibited membrane lipid peroxidation effectively (Figure 4). Pre-treated cells with 100 lam vitamin C or Trolox also markedly inhibited the formation of TBARS. Cell membrane fluidity alteration. Order parameter calculated from 5-doxyl spin label ESR spectrum indicates the membrane fluidity. After exposure to peroxynitrite, cell membrane fluidity decreased significantly, and antioxidants EPC-K1 (50 ~ or vitamin C (100 ~tm) 92

5 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL "'! Figure 2. Peroxynitrite-induced DNA fragmentation in cultures of cerebellar granule cells. Cells were pretreated with 50 IxM EPC-K1, 100 I.tM vitamin C or 100 I.tM Trolox and then exposed to 10 ktm peroxynitrite for 30 min and cultured for 24 h. DNA was extracted and analyzed by 1.5 % agarose gel electrophoresis as described in Materials and Methods. Lane 1: DNA marker (~, DNA/EcoR I + Hind III); lane 2: peroxynitrite-treated cells; lane 3: normal cells; lane 4, lane 5, and lane 6: cells pretreated with EPC-K1, vitamin C, and Trolox, respectively, and then exposed to peroxynitrite ' ' ' ; ' ' ' ' ' 90-9,-~ =--- Normal Cells " ~ Y ~> 9 -~ t~-- 10 ~M ONOO (,) A 50 p.m EPC-K ~.M ONOO 60 r~ --T lam vitamin C + 10 p.m ONOO -----o ~tm Trolox + 10 p.m ONOO 0 t 0 ;0 1'5 2'0 25 Culture Time (h) Figure 3. Time-course of peroxynitrite-induced cell death in cultures of cerebellar granule cells and the protective effects of EPC-K1, vitamin C and Trolox. Cerebellar granule cells were pretreated with 50 lam EPC-K1, 100 ktm vitamin C or I00 IxM Trolox, and then exposed to 10 p.m peroxynitrite for 30 rain and cultured for indicated time. Cell viability was assessed by MTT assay as described in Materials and Methods. Data were means + SD of six different experiments. 93

6 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL 3OO 250 "~ 200 o 2; q., O I Normal Cells Colatrol EpC-K1 TrotOX Vitarv~C25 Ixmol/L ONOO 10 ~.mofl ONOO Figure 4. Peroxynitrite-induced lipid peroxidation in cultures of cerebellar granule cells. Cells were pretreated with 50 I.tM EPC-K1, 100 I.tM vitamin C or 100 lam Trolox, and then exposed to 10 t.tm peroxynitrite for 30 min and cultured for 24 h. Lipid peroxidation was measured by TBA assay. Data were means + SD of four different experiments. * : p < 0.01 in comparison with normal cells; ** : p < 0.01 in comparison with peroxynitrite-treated cells prevented cell membrane fluidity from alteration, but Trolox did not show significant protective effect at a concentration of 100 IxM (Figure 5). ATP content. The cellular ATP content decreased time-dependantly after exposure to peroxynitrite for 30 min (Figure 6). However, the decrease of ATP was suppressed by pretreating cells with antioxidants EPC-KI, vitamin C or Trolox. DISCUSSION Reactive oxygen species are associated with many neuronal diseases (21). Central nervous system is especially prone to oxidative damage for several reasons. First, the brain has a very high oxygen consumption ratio, which may cause the formation of endogenous ROS; second, neuronal cell membrane is rich in polyunsaturated fatty acid side chains, which are sensitive to ROS attack; third, the antioxidant defense systems in CNS are relatively poor. Nitric oxide can be generated endogenously by several kinds of neuronal cells including cortical neurons, cerebellar granule cells, and astrocytes. Thus, peroxynitrite might be formed in CNS under some pathological conditions. Peroxynitrite (and its breakdown product, hydroxyl radicals) can 94

7 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL t~ O 0.74 k NNNN r///// (((((~ :));;A Normal Control EpC-l~l "l?~olox Vi*ta-~ C 25 pmol/l Cells 10 rtmol/l ONO0 ONOO Figure 5. Peroxynitrite-induced cell membrane fluidity alteration in cultures of cerebellar granule cells. Cells were pretreated with 50 lam EPC-K1, 100 p.m vitamin C or 100 lam Trolox, and then exposed to I0 p.m peroxynitrite for 30 min and cultured for 24 h. Cell membrane was spin labeled with 5-doxyl and ESR spectrum was measured. Order parameter, an indicator of cell membrane fluidity, was calculated from the ESR spectrum. Data were means + SD of four different experiments. * : p < 0.01 in comparison with normal cells; ** : p < 0.05 in comparison with peroxynitrite-treated cells. # : p > 0.05 in comparison with peroxynitrite-treated cells. oxidize biomacromolecules including membrane (22), protein (23) and DNA (18), and cause oxidative injury. In the present work, ONOO--induced neuronal cell injury was employed as the model ofno/onoo--initiated neurotoxicity. It was found that treating cerebellar granule cells with peroxynitrite triggered apoptosis, and antioxidants EPC-K1 could inhibit this process to some extent. Peroxynitrite-induced lipid peroxidation in cerebellar granule cells damaged the normal structure and function of the neuronal cell membrane. Oxidative stress may alter the permeability of cell membrane and trigger a series of signals such as Ca 2 influx (24), which activates NOS to produce more NO (25). Furthermore, peroxynitrite may oxidize the cysteine and tyrosine residues of enzymes including complex I, complex II-III, and complex IV of the mitochondrial electron transport chain and influence their activity (26), which was confirmed by experiment result that exposure to peroxynitrite led to the the decrerase of ATP content. The inactivation of mitochondrial enzymes and the decrease of ATP might trigger a cascade of signals leading to apoptosis (27). 95

8 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL 120 fj 100 ~X~t T 80 Z o ~ 6o g 40 ~ 20 < I I Normal Cells 10 rtm ONOO" ~1 50 ~tm EPC-K ~tm ONOO" 100 rtm Trolox + 10 )t/vl ONOO" I~ 100)tMVitaminC+ 10)tMONOO- I ~////&..\\~ I I I I I I~ I 3 Culture Time (h) 24 Figure 6. Peroxynitrite-induced cellular A'lt' content decrease in cultures of cerebellar granule cells. Cells were pretreated with 50 ~M EPC-K1, 100 I-tM vitamin C or 100 I.tM Trolox, and then exposed to 10 ~M peroxynitrite for 30 min and cultured for indicated time. Cellular ATP content was analyzed by HPLC. Data were means + SD of three different experiments. * : p < 0.0l in comparison with normal cells; ** : p < 0.01 in comparison with peroxynitrite-treated cells. EPC-K1 is a kind of diester derivative of vitamin C and vitamin E. Its special structure endowed it with potent antioxidative abilities on scavenging both hydrophilic and hydrophobic radicals. In comparison with vitamin C and Trolox, EPC-K1 shows better protective effect on ONOO--induced cell apoptosis. For application, EPC-K1 is soluble in water and can be injected into body and then spreaded all around the body by circulation. It also has a large hydrophobic substituent, which makes it possible to go through the blood vessel wall and thus enter the brain. This suggests that EPC-K1 may be used as a potential therapeutic agent on neurodegenerative diseases associated with oxidative damage. REFERENCES 1 Jacobson, M. D., Weil, M., and Raft, M. C. (1997) Cell 88, Bredesen, D. E. (1995) Ann. Neurol. 38, Thompson, C. B. (1995) Science 267, Greenlund, L. J. S., Deckwerth, T. L., and Johnson, E. M., Jr. (1995) Neuron 14,

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