Journal of Biophysical Chemistry
Vol.1 No.3(2010), Article ID:3196,8 pages DOI:10.4236/jbpc.2010.13019

Antioxidative action of N-α-tosyl-L-lysine chloromethyl ketone prevents death of glutathione-depletedcardiomyocytes induced by hydrogen peroxide

Kiyoshi Takahashi, Kyohei Oyama, Hironori Motoshige, Koichi Sakurai*

Division of Biochemistry, Department of Life science, Hokkaido Pharmaceutical University School of Pharmacy, Katsuraoka-cho, Otaru, Hokkaido, Japan; *Corresponding Author: ks51@hokuyakudai.ac.jp

Received 20 August 2010; revised 22 September 2010; accepted 25 September 2010.

Keywords: TLCK; Cardiomyocytes; Oxidative Damage; H2O2; p53; ROS; Antioxidant

ABSTRACT

Hydrogen peroxide (H2O2) induces the hypertrophy in cultured H9c2 cardiomyocytes and cell death in glutathione (GSH)-depleted H9c2 cells. In the present study, we observed that pretreatment with a serine protease inhibitor, N--tosyl-L-lysine chloromethyl ketone (TLCK), significantly prevented the H2O2-induced cell damages in GSH-depleted H9c2 cells in a concentration-dependent manner. The phase contrast microscopy revealed that although the exposure of the GSH-depleted H9c2 cells to H2O2 resulted in a globular shape of the cells, TLCK prevented the occurrence of H2O2-induced morphological changes. TLCK also inhibited the generation of reactive oxygen species in the cells after addition of H2O2, suggesting that the antioxidant action of TLCK is involved in the protection against the cell damages by H2O2. Application of TLCK after ~30 min of exposure to H2O2 could significantly protect the cells from cell damages. The other serine protease inhibitors that were tested could not prevent the cell damages in GSHdepleted H9c2 cells. Pretreatment with an inhibitor of nuclear factor- translocation into the nucleus and a proteasome inhibitor did not prevent the cell damages in GSH-depleted H9c2 cells. An inhibitor of p53 significantly prevented the cell damages in GSH-depleted H9c2 cells. These results suggest that antioxidative action of TLCK prevents the death of GSH-depleted H9c2 cardiomyocytes induced by H2O2.

1. INTRODUCTION

Studies on the reactive oxygen species (ROS), such as superoxide, H2O2, and the hydroxyl radical, have indicated their involvement in pathogenesis of several diseases. Although ROS are involved in atherosclerosis, myocardial ischemia/reperfusion injury, and heart failure [1,2], the mechanism by which ROS initiate these diseases is not yet fully understood. In primary cultured neonatal rats cardiomyocytes, H2O2 at higher doses causes necrotic cell death, whereas at lower doses, it induces apoptosis of a fraction of cells; the surviving cells undergo hypertrophy [3]. We had previously suggested that intracellular free thiol levels determine the fate of H2O2-treated H9c2 cardiomyocytes, i.e., cell death or hypertrophy [4]. In GSH-depleted H9c2 cells, hypertrophy did not occur after treatment with H2O2 at any concentrations, but the viability of the cells decreased markedly in comparison with that of normal GSH-containing cells [4]. These findings suggest that the GSH-depleted cells are vulnerable to oxidative stress. It has been reported that GSH functions as an important cellular defense system against oxidative stress and that GSH participates in the detoxification reactions of free radicals and peroxides, either directly or indirectly as a coenzyme of antioxidant enzymes, such as glutathione peroxidases [5,6].

It is well known that the ratio of GSH to GSSG in cells represents the intracellular redox environment. Additionally, the intracellular redox state of GSH is involved in signal transduction through the redox regulation of a number of proteins that have critical thiols, such as receptors, proteins involved in ubiquitinylation, several protein kinases, and some transcription factors [7,8]. The modulation of the activity of several transcription factors, i.e., nuclear factor-kB (NF-kB), activator protein-1, and heat shock factor-1, by thiol redox state has been reviewed [5].

While caspases, a family of cysteine proteases, have been predominantly studied in cell death research, serine proteases may also play a significant role in cell death process [9]. The roles of these serine proteases, i.e., Omi /HtrA2, AP24, and thrombin, have been reviewed [10]. During the study that was conducted to clarify whether serine proteases might be involved in the H2O2-induced cell death in cardiomyocytes, we had the opportunity to determine the protective effect of N-a-tosyl-L-lysine chloromethyl ketone (TLCK), a well-known serine protease inhibitor, against the H2O2-induced cell damages in GSH-depleted cardiomyocytes. The serine protease inhibitors inhibited apoptosis-associated proteolysis and DNA fragmentation [9]. TLCK is recognized as an inhibitor of translocation of NF-kB [11] as well as an inhibitor of serine proteases. The translocation of NF-kB is involved in lipopolysaccharide-mediated cytotoxicity in macrophages, and the inhibition of this translocation by TLCK abrogated the cell death in macrophages [12]. Moreover, Gong et al. (1999) and Rideout et al. (2001) demonstrated that TLCK might interfere with p53 dependent cell death pathway [13,14]. Thus TLCK can exert its protective effect through various sites of action. The goal of the present study is to clarify the mechanism through which TLCK protects the GSH-depleted H9c2 cardiomyocytes against the H2O2-induced cell damages.

2. MATERIALS AND METHODS

2.1. Chemicals

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), L-buthionine (S, R) sulfoximine (BSO), TLCK, N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK), pifithrin-a, and catalase were obtained from Sigma Chemical Co., St. Louis, MO. SN50 and MG-132 were obtained from BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA. Phenylmethanesulfonyl fluoride (PMSF) and H2O2 were obtained from Wako Pure Chemical Industries, Ltd., Osaka, Japan. 4- (2-aminoethyl)-benzensulfonyl fluoride hydrochloride (AEBSF) was obtained from Roche Diagnostics GmbH, Mannheim, Germany. Leupeptin was obtained from Peptide Institute, Inc., Osaka, Japan. 2’,7’-dichlorofluorescin diacetate (DCFH2-DA) was purchased from CALBIOCHEM, San Diego, CA. The 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay kit was obtained from Promega Co., Madison, WI. All the other chemicals used in this study were of the highest grade available from commercial suppliers.

2.2. Culture and Treatment of Cells

Rat cardiomyocyte H9c2 cells were obtained from the American Type Culture Collection (Rockville, MD) and were grown in DMEM containing 10% (v/v) heatinactivated FBS. The cells were incubated in an atmosphere of 5% CO2 and 95% air at 37°C. The details of the standard treatment of H9c2 cells are as follows: the cells were seeded onto 96-well plates at a concentration of 1 x 104 cells/well for performing cell survival assays. After overnight preincubation with 0.1 mM BSO, the cells were incubated with different concentrations of H2O2 for various time intervals. TLCK and TPCK were usually added to the cell suspension concurrently with BSO unless otherwise noted, and the other inhibitors were added 30 min (leupeptin, PMSF, SN50) [15] or 1 h (AEBSF, MG-132, pifithrin-a) prior to the addition of H2O2 [15-17]. The details regarding the intracellular GSH levels in H9c2 cells pretreated with or without BSO have been described in our previous report [4].

2.3. Measurement of Cytotoxicity

Cell damages were determined using an MTT assay according to the manufacturer’s instructions. Briefly, after the incubation with H2O2, DMEM containing 10% FBS and a dye solution were added to each of the 96 wells and the assay plates were incubated for 4 h at 37°C. A solubilization/stop solution was added for overnight incubation. The absorbances at 540 nm and 690 nm were recorded using a microplate reader (Labsystems Multiskan Bichromatic, Helsinki, Finland). Other culture conditions were same as those mentioned earlier. Cell morphology was investigated using a phase contrast microscope (Nikon Diaphoto-TMD, Tokyo, Japan).

2.4. Measurement of Antioxidant Effect

Intracellular ROS levels were assessed using DCFH2- DA [18,19]. Briefly, DCFH2-DA was preincubated with cells in culture medium at a final concentration of 10 μM for 15 min. Following incubation with H2O2 for 15 min, the cells were trypsinized, collected and washed with PBS by centrifugation at 300 ´ g for 5 min. The cell pellets were resuspended with PBS (1 ml) and analyzed using a flow cytometer (Beckman Coulter, Fullerton, CA). DCF fluorescence was measured by fluorescence channel 1 of the flow cytometer [20,21]. Antioxidant effect of TLCK on lipid peroxidation was examined in rat liver microsomes incubated with ascorbic acid and FeCl3 [22]. Briefly, microsomes (0.5 mg protein/mL) prepared from male Wistar strain rats were incubated with ascorbic acid (0.5 mM) and FeCl3 (10 μM) in 50 mM phosphate buffer, pH 7.4 at 37°C for 1 h. The production of lipid peroxidation was assessed using thiobarbituric acid reactive substance (TBARS). TLCK and TPCK were added to reaction system at the beginning of incubation. The TBARS values represented in the data was corrected by subtracting the value obtained in the reaction system without ascorbic acid and FeCl3 from total value.

2.5. Data Analysis

The data are expressed as mean ± SD of at least three experiments. Student’s t-test was used to analyze statistical difference. P values less than 0.05 were considered significant.

3. RESULTS

3.1. Protective Effects of TLCK against CellOxidative Damages by H2O2

We had previously reported that H2O2 induces hypertrophy in H9c2 cardiomyocytes and cell death in GSHdepleted H9c2 cells. An exposure to 100 µM H2O2 for more than 3 h induced an increase in the damages of the GSH-depleted H9c2 cells (Figure 1(a)), as assessed by the function of MTT reduction. The damages were inhibited by the addition of antioxidants such as Trolox and butylated hydroxyanisol (data not shown). H2O2 (100 µM) alone did not induce the damages in the cells pretreated without BSO. These results suggest that H2O2 induces the oxidative damages in the GSH-depleted H9c2 cells. Pretreatment with TLCK significantly inhibited the cell damages induced by H2O2 up to 6 h of test (Figure 1(a)). Although data not shown, the loss of MTT reductive activity was observed in the cells treated with H2O2 for 24 h in the presence of TLCK, suggesting that TLCK prevented oxidative damages 6 h but dit not the cell death 24 h after the addition of H2O2. Therefore, we selected 6 h as the duration of H2O2 treatment for the GSH-depleted cells in further experiments unless otherwise stated. Regarding the dose-related cytotoxic effect of H2O2, an increase in the concentration of H2O2 resulted in an increase in the damages of the cells; however, TLCK significantly prevented H2O2-induced cell damages caused by ~250 µM H2O2 (Figure 1(b)). TLCK at a concentration of more than 30 µM, particularly a concentration of 100 µM, significantly prevented the cell damages induced by H2O2 (Figure 1(c)). On the other hand, TLCK at a concentration of 300 µM exerted a deleterious effect in a preliminary experiment (data not shown). Therefore, we selected 100 µM TLCK as the dose in further experiments. An analysis using a phase contrast microscope was performed to observe the effect of TLCK on morphological changes in the H2O2-treated cells. Exposure to 100 µM H2O2 for 6 h resulted in a globular shape of the cells and detachment of the cells from the well in the GSH-depleted H9c2 cells (Figure

(a)(b)(c)

(a) GSH-depleted H9c2 cells pretreated with (solid circles) or without (open circles) TLCK were incubated with 100 µM H2O2. *p < 0.01 vs. corresponding TLCK-untreated cells. (b) GSH-depleted H9c2 cells pretreated with (shaded bars) or without (open bars) TLCK were incubated with various concentrations of H2O2 for 6 h. (c) GSH-depleted H9c2 cells pretreated with various concentrations of TLCK were incubated with (solid bars) or without (open bars) 100 µM H2O2 for 6 h.

Figure 1. Effects of TLCK on the cytotoxicity of GSH-depleted H9c2 cells exposed to H2O2.

2(b)) in comparison with the H2O2-unexposed control cells (Figure 2(a)). Moreover, the cells remained attached had shrunken nuclei and cytoplasm (Figure 2(b)). When the cells were pretreated with TLCK, none of these morphological changes was observed in the cells even after incubation with H2O2 for 6 h (Figure 2(c)), however, marked decrease of cell numbers was observed for 24 h. These results show that TLCK protects the GSH-depleted H9c2 cells against the H2O2-induced cell oxidative damages and morphological changes, but not the cell death.

(a)(b)(c)

H9c2 cells pretreated with BSO (A, B) or BSO and TLCK (C) were incubated without (A) or with (B, C) 100 µM H2O2 for 6 h. The scale bar represents 50 µm.

Figure 2. TLCK prevents H2O2-induced morphological changes in GSH-depleted H9c2 cells.

3.2. Effect of Addition Time of TLCK andCatalase on the Cell Damages Induced by H2O2

TLCK was added to the GSH-depleted H9c2 cells at different time intervals before or after the addition of H2O2. TLCK could prevent the H2O2-induced cell damages when it was added to the cells at the same time as H2O2, 15 h before the addition of H2O2, or 0.5 h after the addition of H2O2. However, it could not prevent the cell damages when added 1 h after the addition of H2O2 (Figure 3(a)). On the other hand, catalase completely prevented the cell damages when it was added at the same time as H2O2. However, it did not prevent cell damages when it was added 15 min after addition of H2O2 (Figure 3(b)). These results suggest that H2O2 induces the cell damages within 15 min after its addition and that TLCK may prevent the H2O2-induced cell damages by interfering with some steps in the signaling pathway that results in H2O2-induced cell damages, but not by quenching H2O2.

(a)(b)

GSH-depleted H9c2 cells were treated with 100 µM TLCK (a) or 100 units of catalase (b) at different time intervals before or after the addition of 100 µM H2O2. The cytotoxicity was measured 6 h after the addition of H2O2. *, p < 0.05 vs. H2O2 alone (shaded bars).

Figure 3. Effects of addition time of TLCK and catalase on the cytotoxicity of GSH-depleted H9c2 cells exposed to H2O2.

3.3. Effects of Various Inhibitors on H2O2induced Cell Damages

To clarify the mechanism through which TLCK protects the GSH-depleted H9c2 cells against the H2O2- induced cell damages, we first tested whether inhibitors of serine protease other than TLCK could prevent the cell damages as with TLCK. The inhibitors tested in the present study included TPCK, leupeptin, PMSF, and AEBSF; and their test concentrations are shown in the Figure 4(a). None of these four inhibitors could protect the cells against the H2O2-induced cell damages; in fact, higher concentrations of TPCK and AEBSF exerted by themselves a deleterious effect on the ability of the cells for MTT reduction (Figure 4(a)). These results suggest that the protection of cells by TLCK against the H2O2-induced cell damages could not be due to its inhibitory effect on serine protease.

TLCK blocks the activation of NF-kB by inhibiting

(a)(b)(c)

GSH-depleted H9c2 cells were pretreated with inhibitors of serine protease (A), NF-kB (B), or p53 (C) before the addition of H2O2. Then, the cells were incubated with (solid bars) or without (open bars) 100 µM H2O2 for 6 h.

Figure 4. Effects of various inhibitors on H2O2-induced cytotoxicity.

the proteolytic cleavage of IkB, a cytosolic inhibitor of NF-kB. Accordingly, we examined the effects of SN50, an inhibitor of NF-kB translocation into the nucleus, and MG-132, a proteasome inhibitor that inhibits NF-kB activation by preventing IkB degradation, on the H2O2- induced cell damages. Neither SN50 nor MG-132 could protect the cells against the H2O2-induced cell damages; however, at higher concentrations, both the inhibitors had a little tendency to increase the damages of the control cells (Figure 4(b)). These results indicate that the protection of cells by TLCK against the H2O2-induced cell damages may not be mediated by the inhibition of NF-kB activation.

TLCK prevents neuronal cell death, which is induced by UV irradiation or by treatment with camptothecin, through the inhibition of the induction and nuclear accumulation of p53 [14]. H2O2 induces phosphorylation of p53, leading to accumulation of p53 and transcription of its target gene p21Cip1/WAF1 in endothelial cells [21]. If p53 participates in the H2O2-induced cell damages in our model, suppression of p53 should prevent the cell damages. Therefore, we examined the effect of pifithrin-a, an inhibitor of p53 [22], on the H2O2-induced cell damages. Figure 4(c) shows that pifithrin-a at a concentration of more than 5 µM protected the GSH-depleted H9c2 cells against H2O2-induced cell damages in a concentration-dependent manner, and 20 µM pifithrin-a almost completely blocked the cell damages. Pifithrin-a itself did not exert any deleterious effects on the ability of the cells for MTT reduction in the range of concentrations that were tested.

3.4. Antioxidant Effects of TLCK

To evaluate the effect of TLCK on oxidative stress in the GSH-depleted H9c2 cells after the addition of H2O2, the generation of intracellular ROS was measured using a fluorescent probe DCFH2-DA. DCFH2 reacts with ROS to generate a highly fluorescent derivative DCF [23]. As shown in Figure 5(a), an increase in the intensity of DCF fluorescence was observed after the addition of H2O2 in the GSH-depleted H9c2 cells, while TLCK significantly inhibited it after addition of H2O2. In the cells preincubated with BSO, TLCK does not affect the intensity of DCF fluorescence. TLCK did not induce the increase of GSH levels in the cells pretreated with BSO, or rather TLCK slightly decreased the level (16 ± 8.0%, p > 0.05). To characterize the antioxidative action of TLCK, we evaluated the effect of TLCK on lipid peroxidation of rat liver microsomes in the reaction system of ascorbic acid and FeCl3. As shown in Figure 5(b), TLCK significantly inhibited the production of TBARS in a dose-  dependent manner. However, TPCK did not inhibit the

(a)(b)

A, H9c2 cells were pretreated with BSO or BSO and TLCK overnight. Then, the cells were incubated with (solid bars) or without (open bars) 100 µM H2O2. B, Antioxidant effect of TLCK on lipid peroxidation.

Figure 5. TLCK inhibits generation of ROS in GSH-depleted H9c2 cells.

increase in production of TBARS, suggesting that TLCK may have some antioxidant action in the cells.

4. DISCUSSION

In our experiments, TLCK, which is known as an inhibitor of serine proteases, significantly protected GSHdepleted H9c2 cardiomyocytes from oxidative damages (Figure 1) and morphological changes (Figure 2) induced by H2O2 up to 6 h of test, but did not from cell death for 24 h, suggesting that TLCK prevented the oxidative damages and delayed the induction of cell death. The other serine protease inhibitors, i.e., TPCK, leupeptin, PMSF, and AEBSF, did not exert any protective effect on the damages (Figure 4(a)); however, pharmacological inhibitors of serine proteases have been shown to prevent cell death under several other conditions [14]. These results suggest that serine proteases may not be involved in H2O2-induced cytotoxicity of GSH-depleted cardiomyocytes. Therefore, we also investigated the other effects of TLCK on GSH-depleted H9c2 cardiomyocytes apart from the effect of serine protease inhibition.

The present study showed that pifithrin-a, an inhibitor of p53 function [23], significantly prevented the H2O2- induced cell damages (Figure 4(c)). The transcription factor p53, originally identified as a tumor suppressor protein, is activated in response to DNA damage, resulting in the induction of the transcription of the regulatory genes involved in cell cycle and DNA repair [24]. When DNA damage is serious and irreparable, p53 causes the cell death by inducing an apoptosis program in order to prevent formation of neoplasia in mammals [25]. It has also been demonstrated that oxidative stress is involved in the activation of p53 [26]. Gong et al. reported that TLCK prevented p53 induction and protected leukemic cells against ionizing radiation-induced cell death [13]. Rideout et al. also reported that TLCK prevented the induction and accumulation of p53 that was induced by DNA damage in cortical neurons, leading to the increase in the survival of the cells [14]. In the present study, pifithrin-a exerted a protective effect on the damages, suggesting the involvement of the p53-dependent pathway in the cytotoxicity. It has also been reported that TLCK prevents the activation of NF-kB through the inhibition of IkBa-protease [11,27]. We demonstrated that SN50, an inhibitor of translocation of NF-kB into nucleus, and MG-132, an inhibitor of proteasome that is responsible for the proteolysis of IkBa, did not protect the cells against the cytotoxicity. This result suggests that activation of NF-kB is not involved in the cell damage process.

Pretreatment with BSO presumably decreases the ability of cells to scavenge ROS, because BSO inhibits de novo synthesis of intracellular GSH [4,28]. The present data showed that an increase in the intensity of DCF fluorescence was observed after the addition of H2O2 on BSOpretreated cells, suggesting that ROS are concerned with the cell damages. TLCK markedly inhibited the generation of ROS in the cells after addition of H2O2, and the production of TBARS in rat liver microsomes incubated with ascorbic acid and FeCl3. However, the levels of GSH in the cells pretreated with BSO were not significantly affected by the addition of TLCK. These results suggest that TLCK have the ability to scavenge ROS.

We had previously demonstrated that H2O2 induces the hypertrophy in the normal (BSO-nontreated) cells and cell death in the GSH-depleted cells, suggesting that the intracellular free thiol levels determine the signal pathway resulting in the cell death or hypertrophy in H2O2-treated cardiomyocytes [4]. We demonstrated that TLCK did not induce the increase of GSH levels in H9c2 cells pretreated with BSO. These results suggest that neither the increase of GSH levels in the cells nor the interfering with the depletion of GSH by the pretreatment with BSO are involved in the protective action of TLCK on the oxidative damages. In addition, TLCK significantly inhibited the microsomal lipid peroxidation in the reaction system of ascorbic acid with FeCl3. We guess that rich electrons on 3- and 5-position of benzene ring and/or the primary amino group can participate in the protective action of TLCK against the production of TBARS and the oxidative damage(s) in the cells.

The H2O2-induced cell damages were abolished when catalase was added along with H2O2 (Figure 3(b)), indicating that the cell damages are caused by the oxidative damages initiated by H2O2. After its addition, H2O2 initiates the process of the cell damages within 15 min, because no cytoprotection was observed when catalase was added 15 min after the addition of H2O2 (Figure 3(b)). In contrast, TLCK prevented the cell damages even when it was added 30 min after the addition of H2O2; however, it did not prevent cell damages when added 1 h after the addition of H2O2 (Figure 3(a)). These results suggest a possibility that TLCK also block the some signaling pathway involving p53, that is activated within 1 h after stimulation. Based on these results, we inferred that TLCK abrogated the H2O2-induced cell damages via the antioxidant effect accompanied by the inhibitory effect on p53 function under defined experimental conditions. Further investigations are required to clear the target molecules and the protective mechanism of TLCK in cardiomyocyte.

5. ACKNOWLEDGEMENTS

We thank Mr. Ryota Fujiki for helping with the measurement of GSH amounts.

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