טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentTsaba Adili
SubjectMechanistic Studies of Oxidant/Antioxidant Modulated
Apoptosis in Lung Epithelial Cells
DepartmentDepartment of Biotechnology
Supervisors Professor Emeritus Shmuel Yannai (Deceased)
Professor Tzipora Goldkorn
Assistant Professor Fuad Fares


Abstract

Oxidative stress, featuring H2O2 and related free radicals, may result in apoptosis. We have recently shown that H2O2 modulates ceramide signal transduction, which results in apoptosis of airway epithelial cells. In this study we investigated the involvement of caspases, Bcl-2 and crmA, as well as the effect of glutathione (GSH) depletion, in the apoptotic pathway(s) induced by oxidative stress (H2O2) and ceramide. We demonstrated that exposure of A549 lung epithelial cells to 150 μM H2O2 for 1 hour is sufficient to trigger caspase-3 activation and apoptotic cell death. We also show that endogenous ceramide accumulation and C6-ceramide, a cell-permeant ceramide analogue, specifically induce caspase-3 activation and apoptosis. A high degree of correlation between caspase-3 activity and apoptosis was observed. These results suggest that irreversible commitment to H2O2-induced apoptosis occurs within one hour of exposure, and that this apoptotic pathway is mediated by ceramide and caspases. It is highly feasible that caspase-3 plays a major role in this pathway.  In order to determine the mechanistic sequence of events in this apoptotic pathway, Bcl-2 and crmA-overexpressing A549 stable cell lines were generated. Cells overexpressing Bcl-2 were resistant to both H2O2 and C6-ceramide induced apoptosis, whereas cells overexpressing crmA were resistant to H2O2 but not to C6-ceramide. These results suggest that H2O2-mediated ceramide accumulation is dependent on the activity of crmA-inhibitable caspases, while ceramide action sites are situated downstream to crmA-inhibitable caspases, but upstream to caspase-3 (and Bcl-2). In addition, we show that GSH depletion is an effect commonly induced by H2O2, as well as by endogenous and exogenous ceramides, in A549 lung epithelial cells. When GSH is depleted by buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, these cells become sensitized to caspase-3 activation and apoptosis induced by H2O2 and C6-ceramide, suggesting that GSH has a protective role and that its depletion lowers the cellular threshold level of oxidative stress that is sufficient to trigger apoptosis. Taken together, our study substantiates ceramide and caspases as mediators of the H2O2/C6-ceramide-induced apoptosis of A549 lung epithelial cells, orders ceramide accumulation downstream to crmA-inhibitable caspases but upstream to caspase-3 activation, and stresses the significance of GSH depletion in the facilitation of apoptosis.