ABSTRACT
Bcr-Abl activity in chronic myelogenous leukemia (CML) results in dysregulated cell proliferation and resistance against multiple cytotoxic agents due to the constitutive activation of proliferative signaling pathways. Currently, the most effective treatment of CML is the inhibition of Bcr-Abl activity by imatinib mesylate (Gleevec). Imatinib efficacy is limited by development of resistance through either expression of Bcr-Abl variants that bind imatinib less avidly, increased expression of Bcr-Abl, or expression of multidrug transport proteins. N-Benzyladriamycin-14-valerate (AD 198) is a novel antitumor PKC activating agent that triggers rapid apoptosis through PKC-delta activation and mitochondrial depolarization in a manner that is unaffected by Bcl-2 expression. We demonstrate that Bcr-Abl expression does not confer resistance to AD 198. Further, AD 198 rapidly induces Erk1/2 and STAT5 phosphorylation prior to cytochrome c release from mitochondria, indicating that proliferative pathways are active even as drug-treated cells undergo apoptosis. At sub-cytotoxic doses, AD 198 and its cellular metabolite, N-benzyladriamycin (AD 288) sensitize CML cells to imatinib through a supra-additive reduction in the level of Bcr-Abl protein expression. These results suggest that AD 198 is an effective treatment for CML both in combination with imatinib and alone against imatinib-resistant CML cells.
Subject(s)
Antibiotics, Antineoplastic/therapeutic use , Apoptosis/drug effects , Fusion Proteins, bcr-abl/metabolism , Piperazines/therapeutic use , Pyrimidines/therapeutic use , Benzamides , Cytochromes c/metabolism , Doxorubicin/analogs & derivatives , Doxorubicin/therapeutic use , Drug Resistance, Neoplasm , Drug Synergism , Fusion Proteins, bcr-abl/genetics , HL-60 Cells/drug effects , HL-60 Cells/metabolism , HL-60 Cells/pathology , Humans , Imatinib Mesylate , Immunoblotting , K562 Cells/drug effects , K562 Cells/metabolism , K562 Cells/pathology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , Protein Kinase C/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , STAT5 Transcription Factor/metabolismABSTRACT
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.
