Z-FA-fmk inhibits effector caspases but not initiator caspases 8 and 10, and demonstrates that novel anticancer retinoid-related molecules induce apoptosis via the intrinsic pathway.
Synthetic retinoid-related molecules (RRMs) have been described that show strong antiproliferative activity and induce apoptosis in cancer cells. These RRMs induce caspase activity independently of the retinoid receptors in Jurkat T cells. We observed that the inhibitor of cathepsins B and L Z-FA-fmk blocks the induction of DEVDase activity, DNA fragmentation, and externalization of phosphatidylserine by selective RRMs. Z-FA-fmk can inhibit caspase activity in vitro and selectively inhibits recombinant effector caspases 2, -3, -6, and -7. In contrast, purified initiator caspases 8 and 10 are not affected, whereas the apoptosome-associated caspase 9 is only partially inhibited by Z-FA-fmk in vitro. These data correlate with the covalent binding of biotinylated Z-FA-fmk to the active large subunit of effector caspases. This selective targeting of effector caspases is also observed in Jurkat cells and has been used to demonstrate that RRMs induce apoptosis through the mitochondrial pathway and activate caspase 8 in a Z-FA-fmk-sensitive manner. Thus, Z-FA-fmk fails to inhibit Fas-mediated activation of caspase 8, but completely inhibits RRM-induced processing of caspase 8. Z-FA-fmk does not prevent the autoproteolytic cleavage of caspase 9 in Jurkat cells and partially inhibits the processing and full maturation of effector caspases induced by the RRMs. Moreover, Z-VAD-fmk and Z-FA-fmk have no effect on the release of cytochrome c induced by the RRMs. Other cathepsin inhibitors elicit no effect on RRM-induced apoptosis in Jurkat cells, suggesting that caspases are the major effectors of RRM action.
Lopez-Hernandez
Francisco J
FJ
Sidney Kimmel Cancer Center, San Diego, California 92121, USA.
Ortiz
Maria A
MA
Bayon
Yolanda
Y
Piedrafita
F Javier
FJ
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017304:Annexin A5 / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D019921:Biotinylation; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002403:Cathepsins / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D045304:Cytochromes c / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology*; D006801:Humans; D019169:Jurkat Cells; D007659:Ketones / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D010768:Phosphoserine / Q000378:metabolism; D012176:Retinoids / Q000494:pharmacology; D019014:fas Receptor / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D017304:Annexin A5; D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D007659:Ketones; D010446:Peptide Fragments; D012176:Retinoids; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; D010768:Phosphoserine; C057383:MDL 201053; D045304:Cytochromes c; D002403:Cathepsins; D010447:Peptide Hydrolases; D020169:Caspases; C108711:DEVDase