Reactive oxygen species and p38 mitogen-activated protein kinase induce apoptotic death of U937 cells in response to Naja nigricollis toxin-gamma

Abstract

The aim of the present study is to elucidate the signalling components related to Naja nigricollis toxin--induced apoptosis in human leukaemia U937 cells. It was found that toxin--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 (deltapsim). 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--induced cell death and loss of deltapsim, 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- on ROS generation, loss of deltapsim 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- 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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.