Naja atra Cardiotoxin 1 Induces the FasL/Fas Death Pathway in Human Leukemia Cells.

Jing-Ting Chiou,Long-Sen Chang,Liang-Jun Wang,Yuan-Chin Lee

doi:10.3390/cells10082073

Jing-Ting Chiou, Long-Sen Chang + Show 2 more

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https://doi.org/10.3390/cells10082073

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Abstract

This study aimed to investigate the mechanistic pathway of Naja atra (Taiwan cobra) cardiotoxin 1 (CTX1)–induced death of leukemia cell lines U937 and HL-60. CTX1 increased cytoplasmic Ca2+ and reactive oxygen species (ROS) production, leading to the death of U937 cells. It was found that Ca2+-induced NOX4 upregulation promoted ROS-mediated p38 MAPK phosphorylation, which consequently induced c-Jun and ATF-2 phosphorylation. Using siRNA knockdown, activated c-Jun and ATF-2 were demonstrated to regulate the expression of Fas and FasL, respectively. Suppression of Ca2+-mediated NOX4 expression or ROS-mediated p38 MAPK activation increased the survival of U937 cells exposed to CTX1. FADD depletion abolished CTX1-induced cell death, caspase-8 activation, and t-Bid production, supporting the correlation between the Fas death pathway and CTX1-mediated cytotoxicity. Among the tested N. atra CTX isotoxins, only CTX1 induced Fas and FasL expression. Chemical modification studies revealed that intact Met residues were essential for the activity of CTX1 to upregulate Fas and FasL expression. Taken together, the data in this study indicate that CTX1 induces c-Jun-mediated Fas and ATF-2-mediated FasL transcription by the Ca2+/NOX4/ROS/p38 MAPK axis, thereby activating the Fas death pathway in U937 cells. Furthermore, CTX1 activates Fas/FasL death signaling in the leukemia cell line HL-60.

Highlights

The venom of snakes from the family Elapidae contains a large amount of cardiotoxins (CTXs) [1]
Treatment of U937 cells with CTXs ranging from 0 to 1000 nM for 4 h showed that the half maximal inhibitory concentrations (IC50 ) of cardiotoxin 1 (CTX1), CTX2, CTX3, CTX4, and CTX5 were 500, 200, 150, 400, and 250 nM, respectively (Figure 1A)
The IC50 dose of CTXs was used to examine their effects on Fas and FasL expression in U937 cells

Summary

Introduction

The venom of snakes from the family Elapidae contains a large amount of cardiotoxins (CTXs) [1]. Recent studies have revealed that the blockage of negatively charged carboxyl groups in cobrotoxin, a Naja atra α-neurotoxin, renders the toxin molecule to demonstrate CTX-like activities [4]. This indicates that amino acid substitutions drive the divergence in the biological activities of CTXs and α-neurotoxins during the evolution of snake venom proteins. A previous study suggested that the binding of N. atra CTX3 with cell surface receptor(s) activates apoptosis signaling in lymphocytes [14]. Proteomic studies have revealed that the venom of a single N. atra snake contains many CTX isotoxins [15]

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