Exp Neurobiol 2010; 19(2): 90-96
Published online September 30, 2010
© The Korean Society for Brain and Neural Sciences
In-Young Choi and Won-Ki Kim*
Department of Neuroscience, Korea University College of Medicine, Seoul 136-705, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-920-6094, FAX: 82-2-953-6095
Previously, we reported that glucose-deprived astrocytes are more vulnerable to the cytotoxicity of peroxynitrite, the reaction product of nitric oxide and superoxide anion. The augmented vulnerability of glucose-deprived astrocytes to peroxynitrite cytotoxicity was dependent on their proliferation rate. Inhibition of cell cycle progression has been shown to inhibit the apoptotic cell death occurring in cerebral ischemia-reperfusion. In the present study, we demonstrate that the increased death of glucose-deprived astrocytes by peroxynitrte was largely blocked by the cell cycle phase G2/M transition blocker etoposide. However, the cytoprotective effect of etoposide was not associated with its inhibition of cell cycle progression. Instead, etoposide effectively scavenged peroxynitrite. However, etoposide did not scavenge individual nitric oxide and superoxide anion and it did not prevent the hydrogen peroxide-induced cytotoxicity. The present results indicate that etoposide prevents the toxicity of peroxynitrite in astrocytes by directly scavenging peroxynitrite, not by inhibiting cell cycle progression.
Keywords: etoposide, peroxynitrite, glucose deprivation, astrocyte, cell death
Immunostimulated glial cells simultaneously produce nitric oxide (NO) and superoxide anion (O2·-) via expression and activation of inducible nitric oxide (iNOS) (Simmons and Murphy, 1992). NO and O2·- rapidly react in a 1:1 stoichiometry to form a strong oxidant ONOO- (Beckman et al., 1990). ONOO- has been found to play a critical role in tissue injury and appears to mediate many of the cytotoxic effects (Beckman et al., 1990). Previously, we showed that immunostimulated astrocytes themselves became highly vulnerable to glucose deprivation (GD) (Choi and Kim, 1998; Choi et al., 2001). This enhanced vulnerability was attributed to the accumulation of ONOO- due to the disruption of intracellular antioxidant system (i.e., decreased level of reduced glutathione) in glucose-deprived immunostimulated astrocytes (Choi and Kim, 1998a; 1998b; Choi et al., 2000; 2001). Expectedly, the ONOO--releasing reagent 3-morpholinosydnonimine (SIN-1) also augmented the death of glucose-deprived astrocytes. Interestingly, we further found that the augmented death of immunostimulated or SIN-1-treated astrocytes under GD was dependent on the proliferative state of the astrocytes (Choi and Kim, 1998b).
Activation of iNOS was reported to induce cytotoxicity in part by disturbing cell cycle progression (Gu et al., 2000; Kotamraju et al., 2007). Apoptotic death of some cells can be blocked by coordinating the progression through the cell cycle and/or by preventing the inappropriate activation of cell cycle signalling pathways (Park et al., 1997; Zhu et al., 2007). Because proliferating immunostimulated astrocytes are much more vulnerable to GD, it is reasonable to think that the augmented death could be prevented by inhibition of cell cycle progression.
In the present study, therefore, we examined the possible cytoprotective effect of the DNA topoisomerase II inhibitor etoposide on peroxynitrite toxicity in proliferating astrocytes. Interestingly, the present results demonstrates for the first time that etoposide markedly blocks the augmented toxicity of ONOO- in SIN-1-treated/glucose-deprived astrocytes by directly scavenging peroxynitrite, not by inhibiting cell cycle progression.
Astrocytes were cultured from the prefrontal cortices of 2- to 5-day-old Sprague-Dawley rat pups as previously described (Choi and Kim, 1998a) In brief, cells were dissociated by mild trypsinization (10 min at 37℃ with Dulbecco's modified Eagle medium (DMEM) containing 0.1% trypsin) and passed through a sterile nylon sieve (130µm pore size) into DMEM containing 10% heat-inactivated fetal bovine serum (FBS). Cells were then plated (about 50,000 cells/ml) onto poly-L-lysine (20µg/ml)-coated 75-cm2 culture bottles and maintained for one week in DMEM/F12 supplemented with 10% FBS. Astrocytes were then trypsinized, washed and re-plated in the growth medium onto poly-L-lysine (20µg/ml)-coated 48-well plates. Cells were used for the experiments 5 to 7 days later.
Astrocytic injury and death was assessed by morphological examination of cells using phase-contrast microscopy and quantified by measuring the amount of lactate dehydrogenase (LDH) released into the bathing medium. Activity of LDH was measured using a diagnostic kit (Sigma Chemical Co., St. Louis, MO). In brief, NADH and pyruvate (0.1 w/v %) were added and the samples incubated at 37℃ for 30 min. The samples then were incubated with the coloring reagent for 20 min. The reaction was stopped by adding 0.4 N NaOH, and the activity (unit/ml) of LDH in each sample was calculated from the standard curve. Total LDH, which corresponds to complete astrocyte death, was measured in sister cultures frozen and thawed after the experiments. Cell viability was expressed as percentage of total LDH.
Cells were washed with phosphate-buffered saline (PBS), trypsinized and pooled with the cells collected from corresponding suspension at the end of treatment. After washing once with PBS, cells were resuspended in citrate buffer (250 mM sucrose, 40 mM sodium citrate, pH 7.6). Cells were stained for 30 min in ice-cold DNA staining/lysis buffer (Mg2+ and Ca2+-free PBS containing 10µg/ml RNase, 20µg/ml propidium iodide, 0.5% NP-40, 0.5 mM EDTA, pH 7.2). Flow cytometric analysis was performed on a FACS Caliber (Becton Dickinson, Mountain View, CA) equipped with aircooled 488 nm argon-ion laser. Using a CELLQuest software (Becton Dickinson, Mountain View, CA), cell cycle distribution was analysed with a minimum of 10,000 cells.
The ONOO--dependent oxidation of dihydrorhodamine 123 (DHR) to rhodamine 123 (RH) was measured, as described before (Choi et al., 2002). Briefly, SIN-1 (200µM) was added into phosphatebuffered saline solution (PBS) containing DHR (10 µM) in the absence and presence of etoposide. After a 10 min incubation at room temperature, the fluorescence of RH was measured at an excitation wavelength of 485 nm and emission wavelength of 530 nm (slit widths 20 and 25 nm, respectively) using a fluorescence microplate reader (FL600, Bio-tek Instruments, Inc., Winooski, VT).
As previously described by us (Choi et al., 2002), the amount of NO was measured with a commercially available Clark-type electrode (ISO-NO, Mark II, World Precision Instruments, Sarasota, FL). NO is oxidized on the surface of a prepolarized working electrode, permeable only for gases with low molecular weights, resulting in electrical current. This current (pA) was recorded at 0.5 Hz and converted to a nM concentration of NO using a calibration curve that was generated daily. NO was released from 200µM S-nitroso-N-acetylpenicillamine (SNAP) by incubation in PBS containing 4 mM cysteine as a reducing agent and various concentrations of etoposide. All reagents were freshly prepared before use. The formation of NO was quantified from the initial rate of release using the data acquisition system Duo18 (World Precision Instruments, Sarasota, FL).
As previously described by us (Choi et al., 2002), the amount of superoxide anion was monitored with a Berthold LB9505 luminometer (Bad Wildbad, Germany). The reaction mixtures contained 40 mU/ml xanthine oxidase, 5µM lucigenin, and various concentrations of etoposide in 1 ml of air-saturated PBS (pH 10). The superoxide-producing reaction was initiated by adding xanthine to yield a final concentration of 40µM. All reagents were freshly prepared in PBS. Chemiluminesence was measured at 1-min intervals for 10 min, and data are expressed as the integrated area under the 10-min curve. Mean blank values obtained by adding 300 U/ml SOD were subtracted from all chemiluminescent measurements.
For the measurement of glutathione (GSH), astrocytes were washed with PBS, and then incubated at 37℃ for 10 min with 50µM monochlorobimane. Cells were washed with PBS and then lysed with PBS containing 0.2% Triton X-100. The fluorescence of the monochlorobimane-GSH complex was measured with a fluorescence microplate reader (FL600, Bio-tek Instruments, Inc., Winooski, VT) (excitation wavelength of 400 nm and emission wavelength of 480 nm). The GSH content of the samples was calculated from the standard curve prepared from GSH standards incubated in the presence of monochlorobimane and glutathione transferase (Sigma Chemical Co., St. Louis, MO). The concentration of GSH was expressed as nmol/mg protein; protein content was determined using the bicinchoninic acid (Smith et al., 1985).
Hydrogen peroxide (H2O2) was measured spectrophotometrically with some modifications at 550 nm at 37℃ by the peroxydase-catalysed conversion of 4-aminophenazone to a pink-colored substance. Measurement was started after adding reagent to solution of various concentrations of etoposide and H2O2. This reagent consisted of 10 parts of solution A [4.2 mM 3,5-dichloro-2-hydroxybenzenesulphonic acid, dissolved in Mops buffer (50 mM Mops/50 mM KCl, pH 7.4)], 1 part of solution B (33 mM 4-aminophenazone) and 2.3 units/ml peroxydase (final activity).
Data are expressed as the mean±standard error of mean (S.E.M.) and analysed for statistical significance using ANOVA followed by Scheffe's test for multiple comparison. A p value <0.05 was considered significant.
As we previously reported (Choi and Kim, 1998a; Choi et al., 2002a; 2002b), SIN-1 potentiated the LDH release (i.e., a cell death marker) in glucose-deprived astrocytes (Fig. 1). Presence of etoposide (10µM) 16 h before (i.e., pre-treatment) and during GD (i.e., co-treatment) completely blocked the potentiated cell death (Fig. 1). Similarly, cotreatment of etoposide alone also blocked the increased cell death. However, FACS analysis using propidium iodide showed that co-treatment of etoposide alone did not arrest the cell cycle progression (Table 1). It implies that the cytoprotective effect of etoposide may not caused by its inhibition of cell cycle progression.
In brain, one of many well-known actions of astrocytes is to produce antioxidants, which scavenge reactive oxygen species and buffer oxidative stress. Astrocytes are known to be the main source of GSH which acts as a free radical scavenger and is important in recycling other antioxidants (Makar et al., 1994; Ju et al., 2000). Thus, astrocytes appear to be resistant to the actions of the strong oxidant ONOO- (Barker et al., 1996). In the present study, the level of GSH in astrocytes was synergistically reduced by SIN-1 and GD (Fig. 2A). Co-treatment of etoposide prevented the synergistic decrease of GSH (Fig. 2A). Pretreatment of Lbuthionine-S,R-sulfoximine (BSO, a relatively specific inhibitor of GSH biosynthesis) for 16 h depleted the intracellular GSH (Fig. 2B). Depletion of GSH itself did not induce the cell death, but increased the release of LDH in astrocytes exposed to SIN-1 or SIN-1/GD (Fig. 2C). Although etoposide did not block the GSH depletion by BSO, it inhibited the increased peroxynitrite toxicity by BSO (Fig. 2C).
The ONOO- plays a critical role in the enhanced vulnerability of astrocytes to GD (Ju et al., 2000; Choi et al., 2002a; 2002b). To quantify the ONOO- scavenging effect of etoposide, we used the dihydrorhodamine 123. We found that etoposide effectively inhibited ONOO- (SIN-1)-induced oxidation of dihydrorhodamine 123 to rhodamine 123 in the presence of catalase (Fig. 3A). However, etoposide was little or less efficacious in scavenging NO (Fig. 3B) and O2·- (Fig. 3C).
Previously, we reported that GD increased the intracellular concentration of H2O2 in immunostimulated astrocytes, leading to astroglial cell death (Choi et al., 2004). Our present study showed that both GSH depletion and death in astrocytes simultaneously exposed to GD and H2O2 were not reversed by etoposide (Fig. 4A, B, respecively). We further found that etoposide did not effectively scavenge H2O2 (Fig. 4C).
Many researchers have used etoposide for experimentally inducing apoptosis in tumor cells. Studies on etoposide-induced apoptotic cell death include the processing of stabilized cleavage complexes into frank DNA strand breaks, sensing of the DNA damage, leading to activation of stress-asociated signaling pathways and cell cycle arrest (Kubota et al., 1990; Ryan et al., 1991; Tsao et al., 1992). Etoposide can also activate a pre-existing group of enzymes and enzyme precursors, typified by the cysteine-dependent aspartate-directed protease (caspases) (Henderson et al., 2005). The present study, however, demonstrates that the anti-tumoric DNA topoisomerase II inhibitor etoposide can reduce the peroxynitrite toxicity in glucose-deprived astrocytes by directly scavenging peroxynitrite, not by inhibiting cell cycle progression. Furtherore, etoposide interacts with ONOO- most likely due to the high reactivity of ONOO-, but not with other products NO, O2·- and H2O2.
The mammalian cell cycle maintains internal checkpoints at which cells are arrested when previous events have not been completed in response to external signals (Hartwell and Weinert, 1989). The cell cycle checkpoints are thought to exist at the G1/S and G2/M transitions (Agarwal et al., 1995). Etoposide has been used as a potent and reversible blocker of the cell cycle that appears to act at the G2/M transition. Previously, we found that the G1/S transition blockers mimosine and ciclopirox also blocked the peroxynitrite toxicity. Like etoposide, mimosine reduced the augmented death of glucose-deprived immunostimulated astrocytes by directly scavenging peroxynitrite rather than suppressing the cell cycle progression (Choi et al., 2002a). Unlike mimosine, however, ciclopirox blocked the peroxynitrite toxicity in glucose-deprived SIN-1-treated astrocytes by attenuating peroxynitrite-induced mitochondrial dysfunction, not by scavenging peroxynitrite (Choi et al., 2002b; Lee et al., 2005). Ciclopirox neither prevented the depletion of reduced glutathione in glucose-deprived SIN-1-treated astrocytes, nor scavenged oxidants such as peroxynitrite, nitric oxide, superoxide anion, hydrogen peroxide and hydroxyl radical (Choi et al., 2002b). Because of those various mechanisms of action, therefore, such cell cycle inhibitors should be carefully selected for the studies on oxidative stress-related cell deaths.
Table 1. Changes of cell cycle distribution by etoposide. Astrocytes were exposed to 200 mM SIN-1 and/or glucose deprivation with or without pre-(16 h) and co-treatment, or just co-treatment of etoposide
N=7. *p<0.05, **p<0.001, compared with the group simultaneously treated with SIN-1 and GD in the absence of etoposide.