Abstract
The purpose of this study is to investigate the effects of eosinophil cationic protein (ECP) on human bronchial epithelial cells (Beas-2B cells). Eosinophilic granulocytes are important for immune system in human body. Many derived cationic proteins with cytotoxic activities such as ECP and eosinophil derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, has been widely used to be a biomarker for asthma. ECP is reported to inhibit cell viability, but it is never fully verified its mechanism of cell death. In this study, it was the novel discovery that rECP could cause cell apoptosis and involve in caspase-8 pathway in Beas-2B cells. First, we found that rECP could inhibit the cell viability in Beas-2B cells with IC50 of 21.03 uM. The cell death undergoing apoptotic pathway was investigated by chromatin condensation, cleavage of PARP, sub-G1 of cell cycle and Annexin V. To elucidate the specific apoptotic pathway, the general caspase inhibitor (Z-VAD-FMK) and caspase-8 inhibitor (Z-IETD-FMK) were used to prove the caspase and caspase-8 dependent apoptosis. Further, the cleavage of caspase 8 was also detected after rECP treatment. Although apoptosis occurred, both mitochondrial membrane potential (MMP) and endoplasmic reticulum (ER) response were not obviously involved in the rECP-induced apoptosis. We also found that TNF-□ was released from Beas-2B cells into medium during rECP-induced apoptosis. Therefore, we hypothesized that caspase-8 mediated the specific apoptosis pathway which was trigger by TNF-a functioned as autocrine. We also assumed that the cytotoxicites of different RNases such as ECP, EDN and RNase A depended on their isoelectric pH (pI). Finally, we found that rECP also induced apoptosis in lung carcinoma cells (A549 cells).