Bim is the key mediator of glucocorticoid-induced apoptosis and of its potentiation by rapamycin in human myeloma cells.
Glucocorticoids are widely used in anti-myeloma therapy and their action is potentiated by rapamycin, a mTOR inhibitor. However, the molecular mechanisms underlying these effects remain poorly characterized. We show here that dexamethasone (Dex)-induced apoptosis in MM.1S and OPM-2 cells is characterized by Bax and Bak conformational changes, DeltaPsi(m) loss, cytochrome c release and caspase-3 activation. Rapamycin, which had minimal cytotoxic effect by itself, strongly potentiated Dex-induced apoptosis. Apoptotic gene expression profiling showed an increase in mRNA levels of Bim in MM.1S cells after Dex treatment and further increases in both cell lines when co-treated with rapamycin. Western blot analysis revealed a moderate increase in Bim protein levels in both MM.1S and OPM-2 cells. Immunoprecipitation experiments revealed that most Bim was complexed to Mcl-1 in untreated cells. Upon treatment with Dex, and specially Dex plus rapamycin, Bim-Mcl-1 complex was disrupted and Bim was found associated to a CHAPS-insoluble fraction. Overexpression of Mcl-1 stabilized Bim-Mcl-1 complexes upon treatment with Dex or Dex+rapamycin and fully prevented apoptosis. Gene silencing of Bim inhibited for the most part Dex-induced apoptosis and, to a large extent, apoptosis induced by Dex plus rapamycin. These results, taken together, indicate that Bim protein is the key mediator of apoptosis induced by Dex and also responsible for the potentiating effect of rapamycin, providing molecular criteria for the use of glucocorticoids combined with mTOR inhibitors in myeloma therapy.
López-Royuela
Nuria
N
Department Bioquimica y Biologia Molecular y Celular, Universidad de Zaragoza, 50009 Zaragoza, Spain.
Balsas
Patricia
P
Galán-Malo
Patricia
P
Anel
Alberto
A
Marzo
Isabel
I
Naval
Javier
J
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D045744:Cell Line, Tumor / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology* / Q000627:therapeutic use; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009101:Multiple Myeloma / Q000188:drug therapy / Q000378:metabolism*; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D020123:Sirolimus / Q000494:pharmacology* / Q000627:therapeutic use; D058570:TOR Serine-Threonine Kinases
D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D005938:Glucocorticoids; D007166:Immunosuppressive Agents; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C546842:MTOR protein, human; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus