Circumvention of anti-apoptotic Bcl-2 function by agents that modulate protein kinase C

Bcl-2 inhibits apoptosis induced by a wide spectrum of antitumor drugs, including doxorubicin and daunorubicin, and is thus a major impediment to successful cancer chemotherapy. However, both resistance and toxicity to healthy tissue, particularly cardiac tissue, have restricted the clinical efficacy of the anthracyclines. A new class of anthracyclines with substitutions at the C14 position have the significant changes in biological activity that result from changes in chemical structure. N-benzyladriamycin-14-valerate (AD 198) is representative of this new class of 14-O-acyl anthracyclines. AD 198 displays perinuclear localization and is able to circumvent P-gp, MRP, and at-MDR in vitro and in vivo, without exhibiting cardiotoxicity in vivo. In addition, these agents induce rapid apoptosis in cells despite Bcl-2 or Bcl-X_L overexpression. This family of compounds binds to the C1 regulatory domain of PKC, inhibits phorbol ester-stimulated PKC activity in cell-free studies, and induces PKC translocation in intact cells. PKC-δ has an established role as a pro-apoptotic protein through association of the holoenzyme or catalytic fragment with mitochondria. In proliferating 32D.3 myeloid cells, or in 32D.3 cells engineered to overexpress Bcl-2, substantial levels of PKC-δ are associated with mitochondria. However, following only a 1 h exposure to 5 μM AD 198, loss of mitochondrial membrane potential (ΔΨ_m), cytochrome c release, caspase-3 cleavage, PARP cleavage, and DNA fragmentation were observed. In addition, caspase-mediated cleavage of PKC-δ to the catalytic fragment occurs within 1 h after drug treatment. Pretreatment of 32D.3 cells with the specific PKC-δ inhibitor, rottlerin, but not the PKC-α and -β inhibitor Gö 6976, delayed the 50% cell kill to >24 h for control and Bcl-2 overexpressing 32D.3 cells treated with 5 μM AD 198. The general PKC inhibitor GF 109203X caused a partial delay at a reduced AD 198 concentration. Furthermore, rottlerin delayed ΔΨ _m, cytochrome c release, and PKC-δ and PARP cleavage to >20 h post-drug exposure. In addition, the caspase inhibitor Z-VAD-fmk delayed cytotoxicity, ΔΨ_m, and PARP and PKC-δ cleavage. These results suggest that AD 198 induces mitochondrial-dependent apoptosis in 32D.3 cells by stimulating PKC-δ activation which, in turn, overrides the anti-apoptosic effects of Bcl-2.