Novel Extranuclear-targeted Anthracyclines Override the Antiapoptotic Functions of Bcl-2 and Target Protein Kinase C Pathways to Induce Apoptosis 1 Supported in part by grants from The Susan G. Komen Breast Cancer Foundation (to L. L.) and NIH (to J. L. C.), and by the American Lebanese Syrian Associated Charities (ALSAC) of St. Jude Children’s Research Hospital.1

Bcl-2 inhibits apoptosis induced by numerous antitumor drugs, including doxorubicin and daunorubicin and is, thus, a major impediment to successful cancer chemotherapy. Here, we report the ability of a novel family of nonnuclear targeted anthracyclines to induce rapid apoptosis in cells despite Bcl-2 or Bcl-X L expression. Typified by N -benzyladriamycin-14-valerate (AD 198) and N -benzyladriamycin-14-pivalate (AD 445), this family of compounds binds to the C1 regulatory domain of protein kinase C (PKC), competitively inhibits phorbol ester binding in cell-free studies, and induces PKC translocation in intact cells. PKC-δ has an established role as a pro-apoptotic protein through the 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, after a 1-h exposure to 5 μ m AD 198, cytochrome c release, caspase-3 activation, poly(ADP-ribose) polymerase (PARP) cleavage, PKC-δ cleavage, and DNA fragmentation are observed. Pretreatment of 32D.3 cells with the selective PKC-δ inhibitor, rottlerin, but not the general PKC inhibitor, GF 109203X, or 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 μ m AD 198. Rottlerin delayed PKC-δ and PARP cleavage to >20 h post-drug exposure and also delayed mitochondrial membrane depolarization. In contrast, the pan-caspase inhibitor Z-Val-Ala-Asp-CH 2 F blocked PKC-δ and PARP cleavage, but not mitochondrial membrane depolarization. These results suggest that AD 198 induces mitochondrial-dependent apoptosis in 32D.3 cells by activating PKC-δ holoenzyme on mitochondria, which, in turn, overrides the antiapoptotic effects of Bcl-2.