title: Novel extranuclear-targeted anthracyclines override the antiapoptotic functions of Bcl-2 and target protein kinase C pathways to induce apoptosis.
abstract: 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-delta 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-delta are associated with mitochondria. However, after a 1-h exposure to 5 microM AD 198, cytochrome c release, caspase-3 activation, poly(ADP-ribose) polymerase (PARP) cleavage, PKC-delta cleavage, and DNA fragmentation are observed. Pretreatment of 32D.3 cells with the selective PKC-delta inhibitor, rottlerin, but not the general PKC inhibitor, GF 109203X, or PKC-alpha and -beta inhibitor, Gö 6976, delayed the 50% cell kill to >24 h for control and Bcl-2 overexpressing 32D.3 cells treated with 5 microM AD 198. Rottlerin delayed PKC-delta 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-CH2F blocked PKC-delta 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-delta holoenzyme on mitochondria, which, in turn, overrides the antiapoptotic effects of Bcl-2.
authors.affiliation: Department of Pharmacology, The University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Barrett
authors.forename: Felicia L
authors.initials: FL
authors.lastname: Lewis
authors.forename: J Brent
authors.initials: JB
authors.lastname: Roaten
authors.forename: Trevor W
authors.initials: TW
authors.lastname: Sweatman
authors.forename: Mervyn
authors.initials: M
authors.lastname: Israel
authors.forename: John L
authors.initials: JL
authors.lastname: Cleveland
authors.forename: Leonard
authors.initials: L
authors.lastname: Lothstein
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000627:therapeutic use*; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis; D001692:Biological Transport; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D003574:Cytochrome c Group / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000031:analogs & derivatives; D006801:Humans; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D008956:Models, Chemical; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D015398:Signal Transduction; D013347:Subcellular Fractions; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000903:Antibiotics, Antineoplastic; D000970:Antineoplastic Agents; D003574:Cytochrome c Group; D007527:Isoenzymes; D019253:Proto-Oncogene Proteins c-bcl-2; D004317:Doxorubicin; D011065:Poly(ADP-ribose) Polymerases; C497259:Prkcd protein, mouse; C496628:PRKCA protein, human; C497258:PRKCD protein, human; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases