Cathepsin Inhibition-Induced Lysosomal Dysfunction Enhances Pancreatic Beta-Cell Apoptosis in High Glucose
Figure 1
Inhibition of cathepsins B and L triggers apoptosis in INS-1 cells.
(A) INS-1 cells were treated with E64d (E, 20 μg/mL) and pepstatin A (P, 20 μg/mL) in 11 mM glucose medium for 48 hr, and caspase activation was monitored by western blotting using antibodies detecting cleaved, active forms of caspases. (B) INS-1 cells were treated with cathepsin B inhibitor (CBi, 20 μM), cathepsin L inhibitor (CLi, 20 μM), and cathepsin K inhibitor (CKi, 20 μM) for 48 hr in 11 mM glucose medium, and caspase activation was monitored by western blotting. (C) INS-1 cells stained with Annexin-V and Hoechst were observed using a fluorescence microscope for quantification of apoptotic cells. Cells were treated with E, CBi, CLi and staurosporine (STS, 0.5 μM) in 11 mM glucose medium. Lysosomal protease inhibitors were added daily, and STS was added 6 hr prior to staining (three independent counts). STS is a known prototypic inducer of apoptosis. *p <0.01, ** p <0.05 compared with 11 mM glucose. The scale bar represents 20 μm.