Gliotoxin-induced cytotoxicity proceeds via apoptosis and is mediated by caspases and reactive oxygen species in LLC-PK1 cells.
Renal failure associated with aspergillosis is caused by pathogenic fungi. Gliotoxin is a toxic epipolythiodioxopiperazine metabolite produced by the pathogens. The present study investigated the cytotoxicity and underlying mechanisms induced by gliotoxin in LLC-PK1 cells, a porcine renal proximal tubular cell line. Gliotoxin at 100 ng/ml did not show a cytotoxic effect, but unmasked a dose-dependent cell death induced by TNF-alpha. TNF-alpha-induced cell death in the presence of gliotoxin was associated with hypodiploid nuclei and activation of caspase-3-like proteases. Blockade of caspases by boc-aspartyl (OMe)-fluoromethylketone and z-DEVD.fmk inhibited TNF-alpha-induced cell death. As the concentrations of gliotoxin were increased, gliotoxin killed the cells directly in a dose-dependent manner. Further analyses of DNA fragmentation, hypodiploid nuclei, mitochondrial membrane potential, and plasma membrane integrity revealed that cell death proceeded via apoptosis. Gliotoxin-induced apoptosis was associated with dose-dependent and time-dependent activation of caspase-3-like proteases. Boc-aspartyl (OMe)-fluoromethylketone attenuated the killing effect. Gliotoxin also increased the intracellular levels of reactive oxygen species as measured by flow cytometry. N-acetylcysteine, a well-known antioxidant, completely abolished the gliotoxin-induced caspase-3-like activity, cytotoxicity, and reactive oxygen species. In conclusion, (1) gliotoxin at 100 ng/ml unmasks the ability of TNF-alpha-induced apoptosis, and the effect of TNF-alpha is mediated by caspase-3-like proteases; and (2) at higher concentrations gliotoxin itself induces cell death, which is via apoptosis and dependent on caspase-3-like activity and reactive oxygen species.
Zhou
X
X
Department of Medicine, Uniformed Services University, Bethesda, Maryland 20814, USA. xiazhou@usuhs.mil
Zhao
A
A
Goping
G
G
Hirszel
P
P
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000096:biosynthesis*; D053938:DNA Fragmentation / Q000187:drug effects; D005840:Gentian Violet; D005912:Gliotoxin / Q000633:toxicity*; D007166:Immunosuppressive Agents / Q000633:toxicity*; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects; D018374:LLC-PK1 Cells; D008564:Membrane Potentials / Q000187:drug effects; D011419:Propidium; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D007166:Immunosuppressive Agents; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D011419:Propidium; D005912:Gliotoxin; D020169:Caspases; D005840:Gentian Violet