Inhibitors of cysteine cathepsin and calpain do not prevent ultraviolet-B-induced apoptosis in human keratinocytes and HeLa cells.
Caspases, members of the cysteine protease family, execute UVB-induced apoptosis in several cell lines and keratinocytes. Several researchers investigating UVB-induced apoptosis have demonstrated a dose-dependent protective effect of the synthetic peptide caspase inhibitor zVAD-fmk. However, zVAD-fmk displays a dose-dependent protective effect against UVB-induced apoptosis, even at doses higher than those required to block all known proapoptotic caspases. In addition, it is known that zVAD-fmk also inhibits other cysteine proteases including cathepsins and calpains, and these proteases have recently been demonstrated to play a role in the execution of programmed cell death induced by other stimuli, e.g. TNF-alpha. The purpose of the present study was therefore to investigate whether inhibitors of cysteine cathepsins and calpains could prevent UVB-induced apoptosis in HeLa cells and keratinocytes. This was done by investigating the effect of the irreversible cysteine protease inhibitor zFA-fmk, the cathepsin B inhibitor CA-074-Me and the calpain inhibitor ALLN on the viability of UVB-irradiated human keratinocytes and HeLa cells. At concentrations of 10 microM and above zVAD-fmk conferred partial dose-dependent protection against UVB-induced apoptosis in HeLa cells and keratinocytes. Moreover, caspase-3 activity was completely blocked at zVAD-fmk concentrations of 1 microM in HeLa cells. This indicates that caspase-independent mechanisms could be involved in UVB-induced apoptosis. However, the protease inhibitors zFA-fmk, CA-074-Me and ALLN all failed to prevent UVB-induced apoptosis in HeLa cells and keratinocytes. In conclusion, the protective effect of zVAD-fmk at high concentrations indicates that other proteases than caspases are active in the execution of UVB-induced apoptosis but further studies are needed to identify these proteases.
Bang
Bo
B
Department of Dermatology, Gentofte Hospital, Copenhagen, Denmark. bb22@bbh.hosp.dk
Baadsgaard
Ole
O
Skov
Lone
L
Jäättelä
Marja
M
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology / Q000528:radiation effects; D002154:Calpain / Q000037:antagonists & inhibitors; D061945:Caspase Inhibitors; D002403:Cathepsins / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004151:Dipeptides / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000528:radiation effects; D007659:Ketones / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism; D014466:Ultraviolet Rays
D000590:Amino Acid Chloromethyl Ketones; C400541:CA 074 methyl ester; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D007659:Ketones; D007976:Leupeptins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C063261:acetylleucyl-leucyl-norleucinal; C057383:MDL 201053; D010741:Phospholipases A; D002403:Cathepsins; D002154:Calpain