Development of cell death-based method for the selectivity screening of caspase-1 inhibitors

TY - JOUR

T1 - Development of cell death-based method for the selectivity screening of caspase-1 inhibitors

AU - Chopra, Puneet

AU - Gupta, Shashank

AU - Dastidar, Sunanda G

AU - Ray, Abhijit

PY - 2009

Y1 - 2009

N2 - Caspase-1 selective inhibitors are novel therapeutic agents for inflammatory diseases. Selectivity assays for caspases can be initiated with purified enzyme, making these assays very costly and time consuming. Therefore, there is a need to develop a fast and reliable cell-based assay, which can be used for the selectivity screening of multiple caspases in a biologically relevant context in a single assay. In this study, we have developed an assay in which DNA fragmentation, a hallmark of apoptosis, of Jurkat cell line was examined post induction with etoposide in the presence or absence of inhibitors of caspases 1, 3, 8, 9 and pan-caspase inhibitors. We observed that caspases-3, -8, -9 and pan caspase inhibitors resulted in significant inhibition of etoposide-induced DNA fragmentation. However, caspase-1 specific inhibitor failed to prevent DNA fragmentation, suggesting that either caspases belonging to caspase-1 family (1, 4 and 5) are not present in the Jurkat cells or might not be involved in the etoposide-induced DNA fragmentation. Since the inhibition of caspases 3, 8 and 9 is accompanied by the down regulation of the activity of a cascade of caspases (caspases 2, 6, 7, 9 and 10), selectivity of caspase-I inhibitors can be ascertained for the above panel (caspases 2, 6, 7, 8, 9 and 10) of caspases from this single assay.

AB - Caspase-1 selective inhibitors are novel therapeutic agents for inflammatory diseases. Selectivity assays for caspases can be initiated with purified enzyme, making these assays very costly and time consuming. Therefore, there is a need to develop a fast and reliable cell-based assay, which can be used for the selectivity screening of multiple caspases in a biologically relevant context in a single assay. In this study, we have developed an assay in which DNA fragmentation, a hallmark of apoptosis, of Jurkat cell line was examined post induction with etoposide in the presence or absence of inhibitors of caspases 1, 3, 8, 9 and pan-caspase inhibitors. We observed that caspases-3, -8, -9 and pan caspase inhibitors resulted in significant inhibition of etoposide-induced DNA fragmentation. However, caspase-1 specific inhibitor failed to prevent DNA fragmentation, suggesting that either caspases belonging to caspase-1 family (1, 4 and 5) are not present in the Jurkat cells or might not be involved in the etoposide-induced DNA fragmentation. Since the inhibition of caspases 3, 8 and 9 is accompanied by the down regulation of the activity of a cascade of caspases (caspases 2, 6, 7, 9 and 10), selectivity of caspase-I inhibitors can be ascertained for the above panel (caspases 2, 6, 7, 8, 9 and 10) of caspases from this single assay.

U2 - 10.1007/s10616-009-9217-9

DO - 10.1007/s10616-009-9217-9

M3 - Journal article

C2 - 19655264

SN - 0920-9069

JO - Cytotechnology

JF - Cytotechnology

ER -