Abstract
Background
Epsilon toxin, synthesized by Clostridium perfringens, is a toxin from the class of pore-forming proteins that are associated with the development of enterotoxaemia in ruminants. As an agent of bioterrorism, exposure of toxin aerosol form causes endothelial cell damage and cytotoxicity in human lung cells. However, little information is available regarding the cytotoxicity and the mechanisms associated with lung cancer cell lines. The aim of the present study was to explore the cytotoxic effects of epsilon toxin on the human lung cell line A549 and its involvement in the PI3K/AKT/mTOR signaling pathway to clarify the underlying molecular mechanism.
Methods and Results
Following treatment of A549 cells with epsilon toxin, the cytotoxicity by the MTT and LDH assays and the levels of reactive oxygen species (ROS), cell cycle, and cell apoptosis using flow cytometry assay as well as detection of cell apoptosis with Hoechst 33258 staining were conducted. The expression levels of apoptosis-, autophagy- and PI3K/Akt/mTOR signaling-related mRNA and proteins were measured by qRT-PCR and Western blot analysis. Epsilon toxin concentration-dependently induced a reduction in cell viability and an increase in membrane leakage along with ROS generation. Epsilon toxin upregulated the levels of beclin-1, LC3 II/I, and p62 expression while downregulated the PI3K/Akt/mTOR expression levels. Epsilon toxin significantly arrested cell-cycle at the Sub-G1 phase and could further promote apoptosis in A549 cells via increasing the expression level of P53, Bax and caspase-3 while reducing the expression of Bcl-2, confirmed with annexin V/PI and Hoechst 33258 staining.
Conclusions
These findings demonstrated that the epsilon toxin-induced cytotoxicity associated with apoptosis and autophagy processes via the PI3K/AKT/mTOR signaling pathway.