Cigarette Smoke Extract Induces Bronchial Epithelial Cell Death by Caspase 1-dependent Mechanism

Abstract

Introduction: Cigarette smoke, which contains over 4,000 chemical compounds, could affect cell death in various manners. Although several mechanisms including apoptosis and necrosis were proven in cell deaths caused by cigarette smoke, pyroptosis which is caspase 1-dependent cell death was not demonstrated yet. The aim of this study is to prove pyroptosis of bronchial epithelial cells triggered by cigarette smoke. Methods and Results: BEAS-2B cells were treated with different concentration of cigarette smoke extract (CSE). After CSE treatment for 12, 24 and 48 hours, MTT assay was performed to measure cell viability according to CSE concentration. When cells were treated for 24 hours with 7.5% CSE, cell viability began to decline significantly. However, cell survival rate was increased when 2.5% CSE was treated. As pyroptosis is initiated by caspase 1 activation, caspase 1 expression and activity were assessed. Increased caspase 1 expression by CSE was confirmed by western blotting. Caspase 1 activity measured by calorimetric kit was also increased with CSE treatment. Because the key feature of pyroptosis is pore formation, ethidium bromide (EtBr) uptake was measured. Cell death following pore formation was evaluated with MTT assay and LDH release assay. EtBr uptake and cell deaths were increased when CSE was treated. However, when caspase 1 inhibitor (Z-YVAD-FMK 10μM) was treated together, EtBr uptake and cell deaths (MTT assay, LDH release assay) were significantly decreased. Moreover, flow cytometry result showed increased size of CSE treated cells suggesting pore formation and cell swelling. However, as cell death could not be wholly explained by pyroptosis, the hallmarks of apoptosis and autophagy were measured. CSE activated caspase 3 and this mediated PARP cleavage. Moreover, Bcl-xL level was decreased by CSE treatment. Flow cytometry using fluorescein-conjugated Annexin V also showed the increased percentage of apoptotic cells by CSE. Finally, cell viability using MTT assay and LDH release assay under CSE treatment was decreased when caspase 3 inhibitor (Z-DEVD-FMK 10μM) was added. The hallmarks of autophagy (Beclin 1 and LC-3B) were also activated by CSE. In addition to the hallmarks of apoptosis and autophagy, senescence markers were also evaluated. p21 and p27 were activated by CSE and these activation could explain the reason why cell survival rather than cell death occurred in a certain concentration of CSE. Conclusions: Cigarette smoke could induce caspase 1-dependent cell death (pyroptosis) of bronchial epithelial cells. Moreover, other cell death mechanisms such as apoptosis or autophagy as well as pyroptosis could contribute to the cell death by cigarette smoke.