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Keywords:

  • cytotoxin;
  • apoptosis;
  • p38 MAPK;
  • mitochondrial dysfunction;
  • reactive oxygen species

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The aim of the present study is to elucidate the signalling components related to Naja nigricollis toxin-γ-induced apoptosis in human leukaemia U937 cells. It was found that toxin-γ-induced apoptotic cell death was attributed mainly to activation of p38 mitogen-activated protein kinase (MAPK), reactive oxygen species (ROS) generation and loss of mitochondrial membrane potential (ΔΨm). Subsequent modulation of Bcl-2 family member and cytochrome c release accompanied with activation of caspase-9 and -3 were involved in the death of U937 cells. SB202190 (p38 MAPK inhibitor) and N-acetylcysteine (antioxidant) significantly attenuated toxin-γ-induced cell death and loss of ΔΨm, and completely abolished the production of ROS. In contrast to N-acetylcysteine, degradation of Bcl-2/Bcl-XL and mitochondrial localization of Bax were notably decreased by SB202190. Inhibitors of electron transport (rotenone and antimycin A) or inhibitor of mitochondrial permeability transition pore (cyclosporine A) reduced the effect of toxin-γ on ROS generation, loss of ΔΨm and cytochrome c release. Noticeably, pre-treatment with N-acetylcysteine or rotenone eliminated markedly ROS accompanied with reduction in p38 MAPK activation. Taken together, these results suggest that the cytotoxicity of toxin-γ is initiated by p38-MAPK-mediated mitochondrial dysfunction followed by ROS production and activation of caspases, and that ROS further augments p38 MAPK activation and mitochondrial alteration.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

There are two types of leukaemia: one is lymphocytic leukaemia that originated from lymphocytes in the bone marrow and the other is myelogenous leukaemia that originated mainly from granulocytes or monocytes [1]. Both acute myelogenous leukaemia and adult-onset acute lymphocytic leukaemia are aggressive diseases that respond poorly to conventional chemotherapy. The primary cause of treatment failures in acute myeloid leukaemia is usually the emergence of multidrug resistance [2]. Common mechanisms of resistant disease arise from defects in the apoptotic pathway. Apoptosis is ultimately executed by caspases that operate both through the receptor-mediated pathway containing members of the tumour necrosis factor (TNF) family of death receptor, and the mitochondrial-mediated pathway involving cytochrome c release from the mitochondria [3]. Defects in the receptor- or mitochondrial-mediated pathway may give important prognostic information in the risk stratification of acute myeloid leukaemia patients [4]. In trying to overcome drug resistance and improve clinical outcome, efforts have been made in developing therapeutic agents that overcome defects in apoptotic pathways [5].

Abundant cardiotoxins (CTXs), a group of major polypeptides of around 60 amino acid residues, are present in the elapid family of snakes [6]. According to their structure, CTXs can be classified into groups I and II [7]. Recent studies show that Naja naja atra (Taiwan cobra) CTX3, a group II CTX, induces apoptotic cell death on human leukaemia cells and human prostate cancer cells [8, 9], indicating that CTX may be a potential agent for leukaemia treatment. Alternatively, the cytotoxic mechanism of group I CTX had not been elucidated. To clarify the cytotoxic events associated with group I CTXs, toxin-γ isolated from the venom of N. nigricollis was employed in the present study. In this report, toxin-γ was found to induce p38-MAPK-mediated mitochondrial dysfunctions in human leukaemia U937 cells. These included a decrease in mitochondrial membrane potential (ΔΨm), release of cytochrome c and finally activation of caspase-9 and -3. Our data also showed that toxin-γ enhanced the generation of reactive oxygen species (ROS), which further augmented p38 MAPK activation and dissipation of ΔΨm. Noticeably, the cytotoxic effect of toxin-γ on peripheral blood mononuclear cells (PBMCs) was weaker than on that on U937 cells. This reveals that toxin-γ may effectively discriminate between normal and malignant cells, implying a new paradigm for multidrug-resistant leukaemia therapy.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Naja nigricollis toxin-γ (Sigma, St Louis, MO, USA) was purified according to the procedure previously described [10]. N-acetylcysteine, digitonin, MTT, propidium iodide, SB202190, SP600126, U0126, rotenone, antimycin A and cyclosporin A were obtained from Sigma. Anti-p38 MAPK, anti-phospho-p38 mitogen-activated protein kinase (MAPK), anti-extracellular signal-regulated kinase (ERK) and anti-phospho-ERK, anti-c-Jun N-terminal kinases (JNK), anti-phospho-JNK, anti-caspase-9, anti-poly(ADP-ribose) polymerase (PARP), anti-Bcl-2 and anti-Bax antibodies were products of Cell Signaling (Danvers, MA, USA). Anti-caspase-3 antibodies, anti-caspase-8 antibodies and Z-VAD-fmk were purchased from Calbiochem (San Diego, CA, USA), and anti-β-actin antibodies were obtained from Chemicon (Billerica, MA, USA). Anti-cytochrome c, anti-Bak, anti-Bcl-XL and anti-Bid antibodies were products of BD Pharmingen (San Jose, CA, USA), and horseradish peroxidase-conjugated secondary antibodies were obtained from Pierce (Rockford, IL, USA). H2DCFDA and rhodamine-123 were products of Molecular Probes (Carlsbad, CA, USA). Cell culture supplies were purchased from GIBCO/Life Technologies Inc (Carlsbad, CA, USA). Unless otherwise specified, all other reagents were of analytical grade.

Cell viability assay

Human lymophoma cancer cell line U937 obtained from ATCC (Rockville, MD, USA) was grown in Roswell Park Memorial Institute medium (RPMI), 1640 medium supplemented with 10% foetal calf serum (Gibco BRL), 2mM L-glutamine, 100 U/ml penicillin/streptomycin and 1% sodium pyruvate incubating at 37°C in a humidified air containing 5% CO2. Exponentially growing cells (1 × 105) were plated in 96-well plates and treated with a series of concentrations of toxin-γ in serum-free medium after 24 hrs of growth. For pharmacological experiments, culture cells were pre-treated with 2 mM N-acetylcysteine, 10 μM SB202190, 10 μM SP600126, 10 μM U0126, 1 μM cyclosporin A, 10 μM antimycin A or 1 μM rotenone before toxin was added. At suitable time intervals, MTT solution was added to each well at a final concentration of 0.5 mg/ml and incubated for 4 hrs. Formazan crystals resulting from MTT reduction were dissolved by addition of 100 μl dimethyl sulfoxide (DMSO) per well. The absorbance was detected at 595 nm using a plate reader.

Sub-G1 analysis

Sub-G1 distribution was determined by staining DNA with propidium iodide. Briefly, 1 × 106 cells were incubated with toxin-γ for 4 hrs. Cells were then washed in phosphate buffer saline (PBS) and fixed in 70% ethanol. Cells were again washed with PBS and then incubated with propidium iodide (10μg) with simultaneous treatment of RNase at 37°C for 30 min. The percentages of cells having the sub-G1 DNA content were measured with a Beckman Coulter Epics XL flow cytometer (Beckman Coulter, Fullerton, CA, USA) and analysed using EXPO32 ADC software (Beckman Coulter).

Measurement of intracellular ROS production

H2DCFDA was employed to detect the intracellular generation of ROS. Toxin-γ-treated cells were collected and incubated with 10 μM H2DCFDA (dissolved in DMSO) for 20 min. prior to harvesting, and then washed with PBS. Then the fluorescence intensity was measured by Beckman Coulter Paradigm™ Detection Platform with excitation at 485 nm and emission at 530 nm. Protein concentration was measured using Bradford method (BIO-RAD) with bovine serum albumin as a standard and results were shown as fold increase in fluorescence intensity per microgram of proteins compared with the control group. The levels of fluorescence were also analysed by flow cytometry (Coulter Epics XL, Beckman Coulter Inc.).

Measurement of mitochondrial membrane potential

After incubating in serum-free medium with or without the addition of toxin-γ at the indicated time periods, the cells were incubated with 20 nM rhodamine-123 at 37°C for 20 min. The cells were then washed twice with PBS, and rhodamine-123 intensity was determined by flow cytometry. Cells with reduced fluorescence (less rhodamine-123) were counted as having lost some of their ΔΨm.

Subcellular fractionation

Following induction of apoptosis, cytosolic and pellet (mitochondrial) fractions were generated using a digitonin-based subcellular fractionation technique. Briefly, 1 × 107 cells were harvested by centrifugation at 800 ×g, washed in PBS and repelleted. Cells were digitonin-permeablilized for 5 min. on ice at a density of 3 × 107/ml in cytosolic extraction buffer (75 mM NaCl, 1 mM NaH2PO4, 8 mM Na2HPO4, 250 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin and 0.05% digitonin). Following centrifugation step 800 ×g at 4°C for 10 min., the supernatant was separated from the pellet comprising mitochondria and cellular debris. The supernatant containing cytoplasmic protein was further purified by centrifugation at 13,000 ×g at 4°C for 10 min. The pellets were solubilized in the same volume of mitochondrial lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM ethylene glycol tetraacetic acid (EGTA), 0.2% Triton X-100, 0.3% NP-40, 1 mM phenylmethylsulfonyl fluoride,5 μg/ml leupeptin, 5 μg/ml aprotinin). After centrifugation at 12,000 ×g at 4°C for 10 min., the supernatant were collected and used as the mitochondrial fraction. Cytochrome c and proteins of Bcl-2 family were detected by Western blotting analysis.

Separation of human peripheral blood mononuclear cells

Blood was obtained from three healthy adult volunteers (10 ml plus 0.1 ml of heparin, 1000 U/ml). The blood was centrifuged at 2000 rpm with a vasculant rotor for 10 min. at room temperature. The layer of white cells plus some red blood cells was taken and transferred to tubes with PBS and centrifuged at 1000 rpm for 10 min. The white layer was taken, completed to 10 ml PBS, and placed on 5 ml Ficoll-Hypaque, and after centrifugation at 2000 rpm for 30 min., the monocyte layer was taken and cultured in RPMI 1640 containing 10% foetal calf serum.

Western blotting

After specific treatments, cells were incubated in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 1 mM Na3VO4, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor mixtures for 20 min. on ice. After insoluble debris was precipitated by centrifugation at 13,000 ×g at 4°C for 15 min., the supernatants were collected and assayed for protein concentration using the Bradford method. An equal amount of protein per sample (15 μg) was resolved on 10% SDS-PAGE and transferred onto a polyvinyl fluoride (PVDF) membrane. The transferred membranes were blocked for 1 hr in 5% nonfat milk in PBST (PBS containing 0.05% Tween 20) and incubated with appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies. The immune complexes were detected by SuperSignal West Pico Chemiluminescent substrate kit (Pierce) and quantified by imaging densitometry. Mean densitometry data from independent experiments were normalized to the control.

Statistical analysis

All data are presented as mean ± S.D. Significant differences among the groups were determined using the unpaired Student’s t-test. A value of P < 0.05 was taken as an indication of statistical significance. All the figures shown in this article were obtained from at least three independent experiments with similar results.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Toxin-γ induces apoptosis in human leukaemia U937 cells

As shown in Fig. 1A, upon exposure to toxin-γ, U937 cells showed a concentration- and time-dependent decrease in cell viability. Because the dose required for half-maximum inhibition of viability was around 0.5 μM after toxin-γ treatment for 4 hrs, this single treatment was used for further assessment of cytotoxicity. As shown in Fig. 1B, flow cytometric analyses of hypodiploid cell populations revealed that toxin-γ induced an increased accumulation of cells in the sub-G1 phase (15.87%) as compared with untreated control cells (0.92%). Immunoblotting analyses revealed a decrease in the level of procaspase-3 and procaspase-9 after toxin-γ treatment (Fig. 1C). Moreover, the production of active caspase-3 and -9 and the degradation of PARP (a caspase-3 substrate) were also noted. These supported the argument that toxin-γ induced apoptotic death of U937 cells. However, there was no significant degradation of procaspase-8 upon toxin-γ treatment.

image

Figure 1. Toxin-γ induces apoptotic death of U937 cells. (A) Concentration- and dose-dependent induction of cell death by toxin-γ. U937 cells were treated with varying concentrations of toxin-γ for indicated time periods. Cell viability was determined using MTT assay. The values represent averages of three independent experiments with triplicate measurement (error bars, mean ± S.D.). (B) Cell cycle analysis of U937 cells treated with toxin-γ. Flow cytometry analyses showed an increase in the sub-G1 DNA content of U937 cells after treatment with 0.5 μM toxin-γ for 4 hrs. (C) Degradation of procaspase-9, procaspase-3 and PARP in toxin-γ-treated U937 cells. In contrast to procaspases-9 and -3, procaspase-8 was not degraded in toxin-γ-treated cells. Cells were treated with 0.5 μM toxin-γ for indicated time periods.

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Production of ROS and mitochondrial depolarization in toxin-γ-treated U937 cells

Increasing evidence suggests that altered mitochondrial function is linked to apoptosis and a decreasing mitochondrial transmembrane potential is associated with mitochondrial dysfunction [11]. Rhodamine fluorescence was employed to determine the ΔΨm in U937 cells following treatment with 0.5-μM toxin-γ. As shown in Fig. 2A, flow cytometry analysis showed that, in untreated control U937 cells, more than 97% of cells were functionally active with high rhodamine-123 signals. Increasing population of U937 cells exhibited a time-dependent loss of ΔΨm after toxin-γ treatment (Fig. 2C). As shown in Fig. 2D, a time-dependent release of cytochrome c into cytosol was detected relative to gradual decrease in mitochondrial cytochrome c. These suggested that toxin-γ-induced apoptosis was mediated via the mitochondrial pathway.

image

Figure 2. Toxin-γ induces loss of ΔΨm, production of ROS and release of cytochrom c in U937 cells. (A) The loss of mitochondrial membrane potential (ΔΨm) in toxin-γ-treated cells. Cells were treated with 0.5 μM toxin-γ for 4 hrs and incubated with 5 nM Rhodamine-123 for 15 min. at 37°C. (B) The production of ROS in toxin-γ-treated U937 cells. U937 cells were treated with 0.5 μM toxin-γ for 4 hrs, and then incubated with ROS indicator H2DCFDA for 20 min. at room temperature. The cells were harvested and subjected to analysis by flow cytometry. The black peak represents the cell treated with toxin-γ, and the white peak denotes the control. (C) Time-dependent ROS generation and collapse of ΔΨm in toxin-γ-treated cells. ROS was measured by fluorescence ELISA reader, and the dissipation of ΔΨm was estimated from cytometry analyses. (D) Western blotting analyses of cytochrome c release from mitochondria in toxin-γ-treated cells. U937 cells were treated with 0.5 μM toxin-γ for indicated time periods.

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ROS has been demonstrated to be an early signal that mediates apoptosis [12]. Flow cytometry analyses showed an increased ROS generation in toxin-γ-treated cells (Fig. 2B). As shown in Fig. 2C, following treatment with 0.5 μM toxin-γ, a significant generation of ROS appeared as early as 1 hr. A time-dependent increase in ROS levels was observed in U937 cells following toxin-γ treatment, and maximal ROS generation determined by fluorescent ELISA reader was achieved after toxin-γ treatment for 6 hrs.

Expression of Bcl-2 family members in toxin-γ-treated U937 cells

As shown in Fig. 3, the expression of antiapoptotic proteins, Bcl-2 and Bcl-XL, was down-regulated by treatment with toxin-γ. Alternatively, up-regulation of apoptotic proteins Bax/Bak was noted among toxin-γ-treated cells. Moreover, marked translocation of Bax to mitochondria was observed after toxin-γ treatment for 2 hrs, while the cleaved product of Bid was not observed. These results suggested that toxin-γ-induced mitochondrial alteration should be partly mediated through proteins of Bcl-2 family.

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Figure 3. Western blotting analyses of Bcl-2 family members in toxin-γ-treated U937 cells. U937 cells were treated with 0.5 μM toxin-γ for indicated time periods.

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Inhibition of p38 MAPK activation and ROS generation rescue cell death induced by toxin-γ

Since activated MAPKs are common components of the apoptotic program [13], the activated MAPK family members including p-JNK, p-p38 MAPK and p-ERK were examined in toxin-γ-treated U937 cells. As shown in Fig. 4A, phosphorylation of ERK increased significantly after toxin-γ treatment for 5 min. and then declined to basal level within 30 min., while JNK was not significantly activated in either short- or long- time treatment with toxin-γ. Alternatively, phosphorylation of p38 MAPK was notably increased after toxin-γ treatment for 2 hrs, and sustained at least till 12 hrs. In contrast to JNK inhibitor (SP600125) and ERK inhibitor (U0126), pre-treatment with p38 MAPK inhibitor (SB202190) restored significantly the cell viability of toxin-γ-treated cells (Fig. 3A). Moreover, N-acetylcysteine (an antioxidant) rescued significantly the cell viability of toxin-γ-treated cells. These results indicated that toxin-γ-induced apoptosis is partly associated with p38 MAPK activation and ROS generation.

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Figure 4. p38 MAPK activation associated with toxin-γ-induced death of U937 cells. (A) Western blotting analyses of phosphorylated MAPK. U937 cells were treated with 0.5 μM toxin-γ for indicated time periods. (B) Effect of MAPK inhibitors and N-acetylcysteine (antioxidant) on cell viability of toxin-γ-treated cells. U937 cells were pre-treated with 2 mM N-acetylcysteine (NAC) for 1 hr or 10 μM MAPK inhibitors (SB202190, p38 MAPK inhibitor; SP600125, JNK inhibitor; U0126, ERK inhibitor) for 2 hrs before toxin-γ treatment for 4 hrs. Cell viability was analysed by MTT assay (*P < 0.05).

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Figure 5A and 5B show that pre-treatment with either N-acetylcysteine or SB202190 resulted in attenuating ROS generation and the loss of ΔΨm in toxin-γ-treated cells. In contrast to the almost abrogated ROS generation (Fig. 5A), the loss of ΔΨm was partly restored by N-acetylcysteine and SB202190 (Fig. 5B). Nevertheless, co-incubation with N-acetylcysteine and SB202190 could not further restore ΔΨm. Pre-treatment with

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Figure 5. N-Acetylcysteine and SB202190 attenuated ROS generation, loss of ΔΨm and cytochrome c release in toxin-γ-treated U937 cells. U937 cells were pre-treated with 2 mM N-acetylcysteine (NAC) for 1 hr or 10 μM SB202190 for 2 hrs before toxin-γ (0.5 μM) treatment for 4 hrs. ROS generation (A) and dissipation of ΔΨm (B) in toxin-γ-treated cells were attenuated by pre-treatment with NAC and SB202190 (*P < 0.05). (C) Pre-treatment with NAC and SB202190 attenuated cytochrome c release in toxin-γ-treated cells.

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N-acetylcysteine and SB202190 prevented significantly toxin-γ-induced release of cytochrome c from the mitochondria (Fig. 5C). Taken together, these likely suggested that p38 MAPK activation and ROS generation were upstream events related to mitochondrial alterations. However, it should be noted that the disruption of ΔΨm could not be attributed entirely to p38 MAPK activation and ROS generation.

p38 MAPK activation and ROS generation are involved in regulation of the expression of Bcl-2/Bcl-XL and translocation of Bax on mitochondria

As shown in Fig. 6, unlike N-acetylcysteine, pre-treatment with SB202190 significantly abolished down-regulation of antiapoptotic proteins, Bcl-2/Bcl-XL, in response to toxin-γ treatment. Compared with cells treated with toxin-γ alone (Fig. 3), translocation of Bax to mitochondria was also markedly affected by pre-treatment with N-acetylcysteine and SB202190. Nevertheless, toxin-γ-induced up-regulation of Bak was not affected by the two inhibitors. Figure 6 shows that N-acetylcysteine attenuated significantly p38 MAPK activation and SB202190 abolished p38 MAPK activation. Moreover, compared with cells treated with toxin-γ alone (Fig. 1), the level of active caspase-3 and cleaved PARP were reduced by N-acetylcysteine and SB202190. As illustrated in Fig. 6C, N-acetylcysteine, SB202190 and Z-VAD-fmk notably inhibited the cytotoxicity of toxin-γ even after toxin-γ treatment for 24 hrs. Taken together, this emphasized the notion that p38MAPK- and ROS-mediated caspase activation was involved in toxin-γ-induced cell death. Given that ROS generation was eliminated by pre-treatment with SB202190 (Fig. 5A), p38 MAPK activation should be an upstream event responsible for generating ROS and regulating the expression of Bcl-2 family members. Moreover, the production of ROS could further .augment p38 MAPK activation.

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Figure 6. Effect of N-acetylcysteine and SB202190 on expression of Bcl-2 family members, activation of p38MAPK and degradation of procaspase-3 and PARP. (A) U937 cells were pre-treated with 2 mM N-acetylcysteine (NAC) for 1 hr before toxin-γ (0.5 μM) treatment for 4 hrs. (B) U937 cells were pre-treated with 10 μM SB202190 for 2 hrs before toxin-γ (0.5 μM) treatment for 4 hrs. (C) N-Acetylcysteine, SB202190 and Z-VAD-fmk (caspase inhibitor) effectively rescued viability of U937 cells after treatment with toxin-γ for 24 hrs.

Cyclosporin A, rotenone and antimycin A attenuate the cytotoxicity of toxin-γ

To assess whether toxin-γ-induced cell death was associated with opening the permeability transition pore, the effect of cyclosporin A, an inhibitor of mitochondrial permeability transition pore, on CTX-induced cell death was examined. As shown in Fig. 7A and B, cyclosporin A attenuated the loss of ΔΨm and the production of ROS in toxin-γ-treated cells. Rotenone and antimycin A, inhibitors of mitochondrial electron transport chain complexes I and III, were employed to evaluate whether ROS generation in toxin-γ-treated cells arose exclusively from mitochondrial alterations. As shown in Fig. 7A and B, rotenone and antimycin A prevent significantly ROS generation and the loss of ΔΨm in toxin-γ-treated cells. The ability of rotenone to reduce ROS generation was higher than that of antimycin A (Fig. 7B). This reveals that toxin-γ affected most mitochondrial electron transport chain complexes I in causing ROS generation. Moreover, the cytotoxicity of toxin-γ was reduced by pre-treatment with rotenone, antimycin A and cyclosporine A (Fig. 7C). Nevertheless, rotenone reduced significantly p38 MAPK activation compared with cyclosporin A and antimycin A (Fig. 7D). These again implied that ROS could augment p38 MAPK activation.

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Figure 7. Effect of rotenone, antimycin A and cyclosporin A on toxin-γ-induced ROS generation, loss of ΔΨm, cell death and p38 MAPK activation. (A) U937 cells were pretreated with 1 μM rotenone (Rot), 10 μM antimycin A (Atm), or 1 μM cyclosporin A (CsA) for 3 hrs, and then co-incubated with 0.5 μM toxin-γ for 4 hrs. (B) ROS generation in toxin-γ-treated cells was attenuated by pre-treatment with rotenone, antimycin A and cyclosporine. (C) Pre-treatment with rotenone, antimycin A and cyclosporine A led to an increase in cell viability of toxin-γ-treated cells. (D) Effect of rotenone, antimycin A and cyclosporine A on p38 MAPK activation in toxin-γ-treated cells.

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Toxin-γ shows less cytotoxicity towards PBMCs

In addition, we examined whether toxin-γ showed cytotoxicity in normal PBMCs. PBMCs isolated from three volunteers were treated with 0.5 μM toxin-γ for 24 hrs. As show in Fig. 8A, compared with untreated PBMC, the viability of PBMC was reduced by approximately 40%. Moreover, ROS generation and loss of ΔΨm were notably lower in PBMCs than in U37 cells.

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Figure 8. Effect of toxin-γ on viability of human normal PBMCs. PBMCs 1–3 represent the cells from three volunteers. (A) Human normal PBMCs were treated with 0.5 μM toxin-γ for indicated time periods. Cell viability was determined using MTT assay. (B) ROS generation and loss of ΔΨm in PBMCs after treatment with 0.5 μM toxin for 24 hrs.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Apoptosis can be initiated through the extrinsic pathway involving activation of death receptors or via the cell-intrinsic pathway triggered by various forms of cellular stress [3]. In the cell-intrinsic pathway, apoptotic signals converge on the mitochondria to trigger the release of cytochrome c into the cytosol, causing caspase-9 and -3 activation and cell death. Activation of death receptor leads to cleavage of caspase-8, which may subsequently activate caspase-3 or initial apoptosis through the mitochondrial pathway [14]. In the mitochondrial pathway, caspase-8 converts the Bid from the inactivate form (22 kD) to the active form (15 kD) which is called truncated Bid (tBid). tBid is associates with the mitochondrial outer membrane, disrupts ΔΨm, and releases cytochrome c into the cytoplasm. In the present study, degradation of procaspase-8 and Bid was not found in toxin-γ-treated cells. Obviously, the ΔΨm in toxin-γ-treated cells should not dissipate through caspase-8-mediated pathway. This ruled out the possible participation of death receptors and their downstream cascades in toxin-γ-induced apoptosis.

Several studies have reported how increased mitochondrial permeability and ΔΨm loss may be involved in the release of cytochrome c, and the crucial role of Bax/Bcl-2 is well established [15–18]. It is known that antiapoptotic Bcl-2/Bcl-XL proteins protect cells against apoptosis via interaction with proapoptotic Bax/Bak proteins. An excess of Bax/Bak proteins produced caused them to be inserted into the outer mitochondrial membrane where they oligomerize and form protein-permeable channels which promote cell death by release of cytochrome c and other lethal factors from the mitochondria [16, 17, 19–23]. Our results showed that inhibition of p38 MAPK activation by SB202190 suppressed toxin-γ-induced accumulation of Bax to mitochondria, down-regulation of Bcl-2/Bcl-XL, the loss of ΔΨm and cell death. Phosphorylation of Bcl-2 or Bax by p38 MAPK has been identified to play a key event in inducing of apoptosis in embryonic fibroblasts or human hepatoma HepG2 cells [24, 25]. Moreover, several lines of evidence show that phosphorylation of Bcl-2 family members enhances their degradation or mitochondrial translocation [24, 26–28]. Thus, this study concludes that activation of p38 MAPK regulates the expression and mitochondrial translocation of Bcl-2 family members in toxin-γ-treated cells.

Mitochondria are known to be a significant source of ROS as the impairment of the electron transfer chain results in active generation of ROS. Moreover, growing evidence suggests that mitochondrial dysfunction plays a key role in oxidative stress [29, 30]. Once generated, ROS further impairs mitochondrial electron transport and enhances ROS production [12, 31]. In some contexts, intracellular ROS is a downstream consequence of the opening of the permeability transition pore [32]. However, increased mitochondrial formation of ROS has been shown to trigger the intrinsic apoptotic pathway by (1) increasing the permeability of the outer mitochondrial membrane through the opening of the permeability transition pore [32–35] and (2) inducing dissociation of cytochrome-c from cardiolipin on the inner mitochondrial membrane [36, 37]. Moreover, several studies reveal the involvement of ROS in Bax/Bak-dependent release of cytochrome c [30, 38].

Pre-treatment with N-acetylcysteine attenuated the loss of ΔΨm and cytochrome c release, suggesting that the production of ROS indeed aggravates mitochondrial alteration. In contrast to N-acetylcysteine, which could not completely abrogate activation of p38 MAPK, inhibition of p38 MAPK abolishes entirely the production of ROS. It reflects that p38-MAPK-mediated mitochondrial permeability should facilitate ROS generation. In the meantime, elimination of ROS by N-acetylcysteine and rotenone reduced p38 MAPK activation, implying that ROS can further trigger phosphorylation of p38 MAPK. Thus, it is not surprising to find that N-acetylcysteine suppresses significantly the effect of toxin-γ on the expression of Bcl-2 family members. In addition to mitochondrial respiration, ROS are formed as by-products of oxidases including nicotine adenine diphosphate oxidase, xanthine oxidase and certain arachidonic acid oxygenases [31]. Co-incubation with rotenone results in a notable inhibition of ROS generation, suggesting that toxin-γ-induced oxidative stress arises mostly from mitochondrial dysfunction. Noticeably, pre-treatment with SB202190 and N-acetylcysteine could not completely abolish the loss of ΔΨm and cell death induced by toxin-γ. This implies that another route in parallel to p38 MAPK activation governs toxin-γ-induced cell death. Previous studies reveal that CTXs could internalize into cells for exerting their cytotoxicity [39–41], and CTXs disrupt directly the integrity of intracellular organelles that vitally induces cell death. Thus, the possibility that penetration of toxin-γ into U937 cells leads to cell death is plausibly considered.

In sum, toxin-γ activates selectively p38 MAPK, which alters the expression of Bcl-2 family members (Fig. 9). Subsequently, increase in the mitochondrial permeability induces ΔΨm loss and ROS generation. Moreover, toxin-γ-induced ROS generation further activates p38 MAPK and aggravates the collapse of ΔΨm. Finally, cytochrome c release activates caspase-9 and -3, thus resulting in apoptotic death of human leukaemia U937 cells. Nevertheless, compared with U937 cells, toxin-γ shows less cytotoxicity towards PBMCs. Moreover, as listed in Table 1, EC50 of toxin-γ on leukaemia cells is at least 80-fold lower than that on other cancer cells, reflecting that toxin-γ selectively induces death of leukaemia cells. To overcome drug resistance and improve clinical outcome, identification and evaluation of novel therapeutic agents that have less toxicity in normal cells are important for treatment of multiple myeloma and acute myelogenous leukaemia are important works. Thus, in addition to an elucidation of the mechanism involved in toxin-γ-induced apoptosis, our data suggest that toxin-γ may be a promising molecule that merits extensive investigation for leukaemia therapy.

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Figure 9. Schematic drawing showing p38 MAPK activation-loss of ΔΨm-ROS generation signalling pathway in toxin-γ-induced cell death. p38 MAPK activation in toxin-γ-treated cells leads to down-regulation of Bcl-2 and translocation of Bax to the mitochondria, which alters subsequently mitochondrial membrane permeability in causing ROS generation. Moreover, toxin-γ-induced ROS generation further augments p38 MAPK activation and aggravates loss of mitochondrial membrane potential. Finally, cytochrome c release activates caspase-9 and -3, thus resulting in apoptotic death of U937 cells.

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Table 1.  Lethal dose of toxin-γ towards leukaemia cells, liver cancer cells, neuroblastoma, colon cancer cells, breast cancer cells, lung cancer cells and cervical cancer cells
Cell type EC50 (μM)*
  1. *Cells were incubated with various concentrations of toxin-γ for 24 hrs, and cell viability was determined by MTT assay. Compared with that of untreated control cells, viability of toxin-γ-treated cells was reduced by 50% at indicated concentration.

Human leukaemic monocyte lymphoma U9370.03 ± 0.02
Human chronic myelogenous leukaemia K5620.05 ± 0.03
Human hepatoma HepG26.73 ± 0.57
Human hepatoma SK-Hep16.61 ± 0.77
Human hepatoma Hep3B>10
Human neuroblastoma SK-N-SH7.37 ± 0.22
Human colon adenocarcinoma HT296.26 ± 0.86
Human breast adenocarcinoma MCF-73.75 ± 0.46
Human lung adenocarcinoma A5492.34 ± 0.56
Human cervical carcinoma HeLa4.97 ± 0.48

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by grant NSC95-2320-B110-007-MY3 from the National Science Council, ROC (to L.S.C.), and grant of National Sun Yat-Sen University – Kaohsiung Medical University Joint Center.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References