Involvement of cathepsin D in chemotherapy-induced cytochrome c release, caspase activation, and cell death.
Treatment of cells with chemotherapy drugs activates the intrinsic mitochondrial pathway of apoptosis and the caspase protease cascade. Recently, the lysosomal protease cathepsin D has been implicated in apoptosis caused by oxidative stress, inhibition of protein kinase C, and stimulation of the TNFR1 and Fas death receptors. However, the role of cathepsin D in chemotherapy-induced cell death has remained largely unexplored. In this report, we show that treatment of U937 leukemia cells with the chemotherapy drug etoposide (VP-16) results in cathepsin D release into the cytosol within 4 hours after initiation of drug treatment. VP-16-induced cathepsin D release was not inhibited by z-VAD-FMK or pepstatin A, suggesting that it occurred independently of the activities of caspase proteases or cathepsin D. Down-regulation of cathepsin D expression in suspension U937 cells or adherent HeLa cells using cathepsin D small interfering RNA partially inhibited cell death resulting from treatment of cells with tumor necrosis factor-alpha, tumor necrosis factor-related apoptosis inducing ligand, or the chemotherapy drugs VP-16, cisplatin, and 5-fluorouracil. Moreover, cathepsin D down-regulation significantly delayed cytochrome c release and caspase-3 activation in response to chemotherapy treatment. Incubation of isolated mitochondria with cathepsin D-treated cytosolic extracts resulted in potent release of cytochrome c, indicating that a cytoplasmic substrate mediates the effects of cathepsin D on mitochondria. Together, these findings show that cathepsin D plays an important role in chemotherapy-induced cell death, and that cathepsin D lies upstream of cytochrome c release and caspase-3 activation in the chemotherapy-induced execution pathway.
Emert-Sedlak
Lori
L
Department of Medicine, University of Pittsburgh Cancer Institute, Hillman Cancer Center Research Pavilion, Room 2.18c, 5117 Centre Avenue, Pittsburgh, PA 15213-2582, USA. johnsond@pitt.edu
Shangary
Sanjeev
S
Rabinovitz
Asaf
A
Miranda
Michelle B
MB
Delach
Scott M
SM
Johnson
Daniel E
DE
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002402:Cathepsin D / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002945:Cisplatin / Q000494:pharmacology; D045304:Cytochromes c / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects*; D005047:Etoposide / Q000494:pharmacology*; D005472:Fluorouracil / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000494:pharmacology; D020298:U937 Cells
D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D000972:Antineoplastic Agents, Phytogenic; D034741:RNA, Small Interfering; D005047:Etoposide; D045304:Cytochromes c; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D002402:Cathepsin D; D002945:Cisplatin; D005472:Fluorouracil