Inhibition of noncaspase proteases, calpain and proteasome, via ALLN and Bortezomib contributes to cell death through low degradation of pro-/anti-apoptotic proteins and apoptosis induction.

Dysfunction at any regulatory point along the apoptotic signaling pathway is closely related to many diseases including cancers. The apoptotic protein expression level is an important cause of cancer-related death, and the correct degradation of apoptotic proteins is involved in tumor development. Therefore, understanding of a regulatory point that underlying cancer-related death may help the development of new strategies to overcome the clinical challenges. Here, proteasome inhibitor Bortezomib and calpain inhibitor ALLN were examined on protein levels of caspase-3, caspase-9, XIAP, and E3-ligase PARC in HEK293T cells overexpressing XIAP and caspase-9. ATP depletion and caspase-3 activation were as a consequence of Bortezomib and ALLN function. Higher numbers of PI-stained cells provided evidence of cell death by both inhibitors. Western blotting analysis showed that both ALLN and Bortezomib equally inhibited degradation of XIAP, but only ALLN was effective at inhibiting caspase proteolytic degradation. Moreover, treatment of cells with both types of inhibitors significantly increased the level of E3-ligase PARC. Our findings showed that inhibition of proteasome and calpains enhanced the level of anti-apoptotic, XIAP and PARC, and pro-apoptotic, caspase-9 and 3 proteins, which totally promote cell death significantly.