N-benzyladriamycin-14-valerate (AD 198) activates protein kinase C-d holoenzyme to trigger mitochondrial depolarization and cytochrome c release independently of permeability transition pore opening and Ca influx

Unlike nuclear-targeted anthracyclines, the extranucleartargeted doxorubicin congener, N-benzyladriamycin-14valerate (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 (Dcm) 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-d inhibitor, rottlerin, and transfection with a transdominant-negative PKC-d expression vector both inhibit AD 198 cytotoxicity through inhibition of Dcm and cytochrome c release. While the pan-caspase inhibitor Z-VAD-FMK blocks AD 198-induced PKC-d cleavage, however, it does not inhibit Dcm and cytochrome c release, indicating that AD 198 induces PKC-d holoenzyme activation to achieve apoptotic mitochondrial effects. AD 198-mediated Dcm 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-d holoenzyme, resulting in Dcm 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. Anti-Cancer Drugs 17:495–502 !c 2006 Lippincott Williams & Wilkins.