PS-341 (Bortezomib) Induces Lysosomal Cathepsin B Release and a Caspase-2-dependent Mitochondrial Permeabilization and Apoptosis in Human Pancreatic Cancer Cells

Abstract

PS-341 (bortezomib) is a potent and reversible proteosome inhibitor that functions to degrade intracellular polyubiquitinated proteins. PS-341 induces apoptosis and has shown broad antitumor activity with selectivity for transformed cells. We studied the effect of PS-341 on lysosomal and mitochondrial permeabilization, including the role of caspase-2 activation in apoptosis induction in the BxPC-3 human pancreatic carcinoma cell line. PS-341 induced a dose-dependent apoptosis in association with reactive oxygen species generation and cleavage of caspase-2 to its 33-and 14-kDa fragments. PS-341 disrupted lysosomes with redistribution of cathepsin B to the cytosol, as shown using fluorescence confocal microscopy, that was blocked by the free radical scavenger tiron but not by a caspase-2 inhibitor (benzyloxycarbonyl (Z)-VDVAD-fluoromethyl ketone (FMK)). PS-341-induced caspase-2 activation was attenuated by a selective pharmacological inhibitor of cathepsin B (R-3032), suggesting that cathepsin B release occurs upstream of caspase-2. PS-341-induced mitochondrial depolarization was attenuated by Z-VDVAD-FMK, tiron, and an inhibitor of the mitochondrial permeability transition pore (bongkrekic acid). Regulation of mitochondrial permeability by caspase-2 was confirmed using caspase-2 small interfering RNA. PS-341-induced cytochrome c release and phosphatidylserine externalization were attenuated by Z-VDVAD-FMK and partially by R-3032. PS-341 activated the BH3-only proteins Bik and Bim and down-regulated Bcl-2 and Bcl-x L mRNA and protein expression. Taken together, PS-341 induces lysosomal cathepsin B redistribution upstream of caspase-2. Caspase-2 activation regulates PS-341-induced mitochondrial depolarization and apoptosis, suggesting that caspase-2 can serve as a link between lysosomal and mitochondrial permeabilization.

Proteasome Inhibitor PS-341 Induces Apoptosis in Cisplatin-resistant Squamous Cell Carcinoma Cells by Induction of Noxa

“…Although we did not observe that PS-341 induced other BH3-only proteins by gene expression profile (3), it is possible that PS-341 may induce the accumulation of BH3-only proteins by inhibiting proteasomal degradations in SCC cells. Several studies have reported that PS-341 could induce the level of Bik proteins to promote apoptosis (31)(32)(33). Thus, in the future, it will be interesting to examine whether PS-341 promotes Bik accumulation by inhibiting its degradation in SCC cells.…”

Lysosomal membrane permeabilization in cell death

“…Moreover, Z-VDVAD-FMK (a chemical inhibitor of caspase-2) or depletion of caspase-2 with an siRNA attenuated MOMP. Thus, caspase-2 emerges as a new link between lysosomal and mitochondrial permeabilization (Yeung et al, 2006). It remains to be determined in which specific circumstances Bid and caspase-2 play predominant roles in mediating post-LMP events such as MOMP.…”

Caspase-2 is required for cell death induced by cytoskeletal disruption

“…In the case of Bid, caspase-2 can directly cleave full-length Bid to activated tBid (Guo et al, 2002;Bonzon et al, 2006). Caspase-2 activation has been implicated in apoptosis in response to a range of signals, including DNA damage, withdrawal of trophic support, heat shock, treatment with chemotherapeutic agents and tumor necrosis factor-related apoptosis-inducing ligand (Kumar, 1995;Troy et al, 2001;Lassus et al, 2002;Panaretakis et al, 2005;Shin et al, 2005;Bonzon et al, 2006;Yeung et al, 2006;Mhaidat et al, 2007). Caspase-2 has also been shown to associate with CD95 DISC and is activated within this complex but seems not to be essential for CD95-mediated cell death (Lavrik et al, 2006).…”