N-benzyladriamycin-14-valerate (AD 198) activates protein kinase C-delta holoenzyme to trigger mitochondrial depolarization and cytochrome c release independently of permeability transition pore opening and Ca2+ influx.
Unlike nuclear-targeted anthracyclines, the extranuclear-targeted doxorubicin congener, N-benzyladriamycin-14-valerate (AD 198), does not interfere with normal topoisomerase II activity, but binds to the C1b regulatory domain of conventional and novel isoforms of protein kinase C (PKC). The resulting interaction leads to enzyme activation and rapid apoptosis in a variety of mammalian cell lines through a pathway involving mitochondrial events such as membrane depolarization (Deltapsim) and cytochrome c release. Unlike other triggers of apoptosis, AD 198-mediated apoptosis is unimpeded by the expression of Bcl-2 and Bcl-XL. We have further examined AD 198-induced apoptosis in 32D.3 mouse myeloid cells to determine how the anti-apoptotic effects of Bcl-2 are circumvented. The PKC-delta inhibitor, rottlerin, and transfection with a transdominant-negative PKC-delta expression vector both inhibit AD 198 cytotoxicity through inhibition of Deltapsim and cytochrome c release. While the pan-caspase inhibitor Z-VAD-FMK blocks AD 198-induced PKC-delta cleavage, however, it does not inhibit Deltapsim and cytochrome c release, indicating that AD 198 induces PKC-delta holoenzyme activation to achieve apoptotic mitochondrial effects. AD 198-mediated Deltapsim and cytochrome c release are also unaffected by cellular treatment with either the mitochondrial permeability transition pore complex (PTPC) inhibitor cyclosporin A or the Ca chelators EGTA and BAPTA-AM. These results suggest that AD 198 activates PKC-delta holoenzyme, resulting in Deltapsim and cytochrome c release through a mechanism that is independent of both PTPC activation and Ca flux across the mitochondria. PTPC-independent mitochondrial activation by AD 198 is consistent with the inability of Bcl-2 and Bcl-XL expression to block AD 198-induced apoptosis.
Lothstein
Leonard
L
Department of Pharmacology and The UT Cancer Institute, University of Tennessee Health Science Center, Memphis, Tennessee, USA. llothstein@utmem.edu
Savranskaya
Luydmila
L
Barrett
Christina M
CM
Israel
Mervyn
M
Sweatman
Trevor W
TW
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D045304:Cytochromes c / Q000378:metabolism; D004317:Doxorubicin / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D042381:Granulocyte Precursor Cells / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000502:physiology; D000083162:Mitochondrial Permeability Transition Pore; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D004317:Doxorubicin; D045304:Cytochromes c; C046808:N-benzyladriamycin-14-valerate; D051745:Protein Kinase C-delta; D002118:Calcium