Abstract
The role of Bim in synergistic interactions between UCN-01 and MEK1/2 inhibitors in human multiple myeloma cells was investigated. Exposure of U266 or RPMI8226 cells to UCN-01 resulted in ERK1/2 activation-associated Bim(EL) phosphorylation/down-regulation, events abrogated by MEK1/2 inhibitors. Enforced activation of ERK1/2 by transfection with constitutively active MEK1 diminished the capacity of PD98059 but not PD184352 to block UCN-01-mediated Bim(EL) phosphorylation and to potentiate apoptosis. Cotreatment with MEK1/2 inhibitors increased the association of Bim(EL) with both Bcl-2 and Bcl-x(L) in UCN-01-treated cells, leading to Bax/Bak conformational change and Bax mitochondrial translocation. Down-regulation of Bim(EL) by shRNA substantially diminished UCN-01/MEK inhibitor-mediated Bax/Bak activation and apoptosis. Furthermore, transfection of cells with S65A Bim, a mutant resistant to UCN-01-mediated phosphorylation, significantly sensitized cells to UCN-01 lethality. Conversely, ectopic expression of either Bcl-2 or Bcl-x(L) did not alter UCN-01/MEK1/2 inhibitor-mediated modifications in Bim(EL) phosphorylation but largely prevented cell death. Finally, IL-6 or IGF-1 failed to prevent MEK1/2 inhibitors from blocking UCN-01-induced Bim(EL) phosphorylation/degradation or cell death. Collectively, these findings argue that UCN-01-mediated ERK1/2 activation leads to Bim(EL) phosphorylation/inactivation, resulting in cytoprotection, and that interference with these events by MEK1/2 inhibitors plays a critical role in synergistic induction of apoptosis by these agents.
Publication types
-
Research Support, N.I.H., Extramural
-
Research Support, Non-U.S. Gov't
-
Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
-
Antineoplastic Agents / pharmacology*
-
Apoptosis / drug effects*
-
Apoptosis Regulatory Proteins / antagonists & inhibitors
-
Apoptosis Regulatory Proteins / genetics
-
Apoptosis Regulatory Proteins / metabolism*
-
Bcl-2-Like Protein 11
-
Benzamides / pharmacology
-
Blotting, Western
-
Drug Synergism
-
Enzyme Inhibitors / pharmacology*
-
Humans
-
Immunoprecipitation
-
Insulin-Like Growth Factor I / pharmacology
-
Interleukin-6 / pharmacology
-
MAP Kinase Kinase 1 / antagonists & inhibitors*
-
MAP Kinase Kinase 1 / metabolism
-
MAP Kinase Kinase 2 / antagonists & inhibitors*
-
MAP Kinase Kinase 2 / metabolism
-
Membrane Proteins / antagonists & inhibitors
-
Membrane Proteins / genetics
-
Membrane Proteins / metabolism*
-
Multiple Myeloma / drug therapy*
-
Multiple Myeloma / metabolism
-
Multiple Myeloma / pathology
-
Phosphorylation / drug effects
-
Protein Conformation
-
Protein Kinase C / antagonists & inhibitors
-
Protein Transport
-
Proto-Oncogene Proteins / antagonists & inhibitors
-
Proto-Oncogene Proteins / genetics
-
Proto-Oncogene Proteins / metabolism*
-
Proto-Oncogene Proteins c-bcl-2 / metabolism
-
RNA, Small Interfering / pharmacology
-
Staurosporine / analogs & derivatives*
-
Staurosporine / pharmacology
-
Subcellular Fractions
-
bcl-2 Homologous Antagonist-Killer Protein / metabolism
-
bcl-2-Associated X Protein / metabolism
-
bcl-X Protein / metabolism
Substances
-
2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide
-
Antineoplastic Agents
-
Apoptosis Regulatory Proteins
-
BAK1 protein, human
-
BAX protein, human
-
BCL2L1 protein, human
-
BCL2L11 protein, human
-
Bcl-2-Like Protein 11
-
Benzamides
-
Enzyme Inhibitors
-
IL6 protein, human
-
Interleukin-6
-
Membrane Proteins
-
Proto-Oncogene Proteins
-
Proto-Oncogene Proteins c-bcl-2
-
RNA, Small Interfering
-
bcl-2 Homologous Antagonist-Killer Protein
-
bcl-2-Associated X Protein
-
bcl-X Protein
-
Insulin-Like Growth Factor I
-
7-hydroxystaurosporine
-
MAP2K2 protein, human
-
Protein Kinase C
-
MAP Kinase Kinase 1
-
MAP Kinase Kinase 2
-
MAP2K1 protein, human
-
Staurosporine