Mechanism of cytotoxicity of human embryonic kidney cells induced by gliotoxin

AIM: To study the mechanism underlying gliotoxin-induced cytotoxicity of human embryonic kidney (HEK) cells. METHODS: Crystal violet assay was used to determine cell viability. DNA fragmentation of HEK cells was measured based on Burton's method. The activity of caspase-3-like proteases was measured as increases in hydrolysis of fluorogenic tetrapeptide substrate, Ac-DEVD-7-amino-4-methylcoumarin and caspase-3 protein abundance was observed by Western blot. Based on fluorescence probe label method, DNA content and reactive oxygen species (ROS) of HEK cells were detected by flow cytometry. RESULTS: Gliotoxin induced HEK cell death in a concentration-dependent manner within 0.4-1.0 mg·L^-1. Under gliotoxin treatment at 1.0 mg·L^-1, cell membrane of HEK cells kept intact associated with hypodiploid nuclei and DNA fragmentation which suggested gliotoxin killed HEK cells via apoptosis. Boc-aspartyl (OMe)-fluoromethylketone (BAF) and z-DEVD. fmk, commonly used as caspase-3-like proteases inhibitor, significantly abolished gliotoxin-induced cell death at 100 and 200 μmol·L ^-1, respectively, suggesting the cytotoxicity induced by gliotoxin was mediated by caspases. N-acetylcysteine concentration-dependently attenuated the HEK cells death induced by gliotoxin, significantly inhibited the generation of ROS of HEK cells upon exposure with gliotoxin, which indicated that ROS was involved in the cytotoxicity of HEK cells induced by gliotoxin. CONCLUSION: Gliotoxin-induced cytotoxicity of HEK cells proceeded via apoptosis, which was mediated by caspases and ROS.