The Mechanism of Nereis Active Protease-induced Apoptosis in Lung Cancer SPC-A-1 Cells
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Abstract:
This study aims to investigate the mechanism of Nereis active protease (NAP)-induced SPC-A-1 cell apoptosis. The effect of NAP on the activity of SPC-A-1 cells was examined by MTT assay. Typical morphologic changes were observed in the SPC-A-1 cells by inverted microscopy and AO/EB staining. The early-stage apoptotic rate and membrane potential were detected using flow cytometry, and the protein expressions of apoptosis-related genes were examined via western blotting. The results showed that NAP significantly inhibited the activity of SPC-A-1 cells in a time- and dose-dependent manner. The flow cytometry (FCM) studies revealed that the percentage of the early-stage apoptotic SPC-A-1 cells that were treated with increasing concentrations of NAP for 24 h increased from 13.50% to 22.98%, and the percentage of cells with decreased mitochondrial membrane potential increased from 12.59% to 25.28%. After treatment with 50 μg/mL NAP for 24 h, the Bcl-2/Bax expression ratio relative to the control group increased by a factor of 6.05. The expression levels of Cyt-C, Cleaved-caspase 9, Cleaved-caspase 3, and Cleaved-PARP relative to the control group significantly increased by a factor of 2.32, 3.07, 3.68, and 1.36, respectively. In conclusion, NAP inducing apoptosis of SPC-A-1 cells by decreasing the expression of the anti-apoptotic protein Bcl-2 and upregulating the expression of Bax, thereby inducing the decrease of mitochondrial membrane potential which then promoting the transfer of Cyt-C and activate the cascade of caspase family.
