Synergistic induction of oxidative injury and apoptosis in human multiple myeloma cells by the proteasome inhibitor bortezomib and histone deacetylase inhibitors.
OBJECTIVE
The purpose of this study was to examine interactions between the proteasome inhibitor bortezomib (Velcade) and the histone deacetylase (HDAC) inhibitors sodium butyrate and suberoylanilide hydroxamic acid in human multiple myeloma (MM) cells that are sensitive and resistant to conventional agents.


METHODS
MM cells were exposed to bortezomib for 6 h before the addition of HDAC inhibitors (total, 26 h), after which reactive oxygen species (ROS), mitochondrial dysfunction, signaling and cell cycle pathways, and apoptosis were monitored. The functional role of ROS generation was assessed using the free radical scavenger N-acetyl-l-cysteine.


RESULTS
Preincubation with a subtoxic concentration of bortezomib markedly sensitized U266 and MM.1S cells to sodium butyrate- and suberoylanilide hydroxamic acid-induced mitochondrial dysfunction; caspase 9, 8, and 3 activation; and poly(ADP-ribose) polymerase degradation; resulting in synergistic apoptosis induction. These events were associated with nuclear factor kappaB inactivation, c-Jun NH(2)-terminal kinase activation, p53 induction, and caspase-dependent cleavage of p21(CIP1), p27(KIP1), and Bcl-2, as well as Mcl-1, X-linked inhibitor of apoptosis, and cyclin D1 down-regulation. The bortezomib/HDAC inhibitor regimen markedly induced ROS generation; moreover, apoptosis and c-Jun NH(2)-terminal kinase activation were attenuated by N-acetyl-l-cysteine. Dexamethasone- or doxorubicin-resistant MM cells failed to exhibit cross-resistance to the bortezomib/HDAC inhibitor regimen, nor did exogenous interleukin 6 or insulin-like growth factor I block apoptosis induced by this drug combination. Finally, bortezomib/HDAC inhibitors induced pronounced lethality in primary CD138(+) bone marrow cells from MM patients, but not in the CD138(-) cell population.


CONCLUSIONS
Sequential exposure to bortezomib in conjunction with clinically relevant HDAC inhibitors potently induces mitochondrial dysfunction and apoptosis in human MM cells through a ROS-dependent mechanism, suggesting that a strategy combining these agents warrants further investigation in MM.
Pei
Xin-Yan
XY
Department of Medicine,Virginia Commonwealth University, Medical College of Virginia, Richmond, Virginia 23298, USA.
Dai
Yun
Y
Grant
Steven
S
D000111:Acetylcysteine / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival; D019938:Cyclin D1 / Q000096:biosynthesis; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D007425:Intracellular Membranes / Q000473:pathology; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D009101:Multiple Myeloma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000096:biosynthesis; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011506:Proteins / Q000378:metabolism; D011509:Proteoglycans / Q000096:biosynthesis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology*; D017382:Reactive Oxygen Species; D012978:Sodium Oxybate / Q000494:pharmacology; D053668:Syndecan-1; D053667:Syndecans; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D000077337:Vorinostat; D051636:X-Linked Inhibitor of Apoptosis Protein
D000970:Antineoplastic Agents; D001897:Boronic Acids; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D008562:Membrane Glycoproteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D011480:Protease Inhibitors; D011506:Proteins; D011509:Proteoglycans; D019253:Proto-Oncogene Proteins c-bcl-2; D011719:Pyrazines; D017382:Reactive Oxygen Species; C508241:SDC1 protein, human; D053668:Syndecan-1; D053667:Syndecans; D025521:Tumor Suppressor Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D000077337:Vorinostat; D000069286:Bortezomib; D012978:Sodium Oxybate; D010100:Oxygen; D000111:Acetylcysteine