PS-341 (bortezomib) induces lysosomal cathepsin B release and a caspase-2-dependent mitochondrial permeabilization and apoptosis in human pancreatic cancer cells.
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-xL 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.
Yeung
Bonnie H Y
BH
Division of Gastroenterology, Mayo Clinic and Mayo College of Medicine, Rochester, Minnesota 55905, USA.
Huang
Der-Chen
DC
Sinicrope
Frank A
FA
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D000072224:Bcl-2-Like Protein 11; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D053143:Caspase 2; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism*; D024101:Mitochondrial Proteins; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D010539:Permeability; D010718:Phosphatidylserines / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism
D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; C096820:BIK protein, human; D000072224:Bcl-2-Like Protein 11; D001897:Boronic Acids; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D010718:Phosphatidylserines; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011719:Pyrazines; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D051020:bcl-X Protein; D000069286:Bortezomib; D045304:Cytochromes c; C490360:CASP2 protein, human; D053143:Caspase 2; D003546:Cysteine Endopeptidases; D002401:Cathepsin B