A cellular mechanism that reversibly inactivates pancaspase inhibitor zAsp-CH(2)-DCB: a potential pitfall causing discrepancy between in vitro and in vivo caspase assays.
Cell-permeable pancaspase inhibitors such as zAsp-CH2-DCB and zVAD-fmk are widely used to examine the involvement of caspases in cell death models. While examining the caspase-dependence of staurosporine (STS)-induced neuroblastoma cell death, we found that zVAD-fmk but not zAsp-CH2-DCB inhibits apoptosis. Time course analysis revealed that, in contrast to zVAD-fmk which constantly inhibited the processing of endogenous caspase substrates, zAsp-CH2-DCB inhibited substrate processing only for the first few hours after its addition to the culture medium. However, when the caspase activity in lysates prepared from cells treated with STS and zAsp-CH2-DCB was measured in vitro, quite unexpectedly, it was found that zAsp-CH2-DCB completely inhibits the STS-mediated activation of caspases throughout the observation period even when it apparently failed to inhibit the processing of caspase substrates within intact cells. These findings together suggest that there exists a cellular mechanism that inactivates zAsp-CH2-DCB in a reversible manner. This reversible inactivation was an active, intracellular process requiring de novo protein synthesis and was observed in another cell line HeLa and with different apoptotic stimuli such as ultraviolet irradiation. Our results have important implications that require consideration when designing experiments involving the use of caspase inhibitors as well as interpreting their results.
Sakurada
Kaori
K
Biophysics Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
Kitanaka
Chifumi
C
Kokubu
Akiko
A
Tomiyama
Arata
A
Sunayama
Jun
J
Kayama
Takamasa
T
Kuchino
Yoshiyuki
Y
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors*; D061945:Caspase Inhibitors; D020169:Caspases / Q000032:analysis* / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004792:Enzyme Precursors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009447:Neuroblastoma / Q000201:enzymology / Q000473:pathology; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis; D011480:Protease Inhibitors / Q000494:pharmacology*; D012107:Research Design; D019311:Staurosporine / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D004792:Enzyme Precursors; D011480:Protease Inhibitors; C093260:benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D001224:Aspartic Acid; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; D019311:Staurosporine