title: Cooperative cytotoxicity of proteasome inhibitors and tumor necrosis factor-related apoptosis-inducing ligand in chemoresistant Bcl-2-overexpressing cells.
abstract: OBJECTIVE
  Bcl-2 overexpression is frequently detected in lymphoid malignancies, being associated with poor prognosis and reduced response to therapy. Here, we evaluated whether Bcl-2 overexpression affects the cytotoxic activity of proteasome inhibitors taken alone or in association with conventional anticancer drugs or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).
  
  
  METHODS
  Jurkat cells engineered to overexpress Bcl-2 were treated with proteasome inhibitors (MG132, epoxomicin, and bortezomib), anticancer drugs (etoposide and doxorubicin), TRAIL, or combinations of these compounds. Cell death and loss of mitochondrial transmembrane potential were detected by flow cytometry. Cytosolic relocalization of cytochrome c and SMAC/Diablo, caspase cleavage, and Bcl-2 and Mcl-1 levels were determined by immunoblotting. Nuclear factor-kappaB inhibition was done by retroviral transduction with a dominant-negative mutant of IkappaBalpha.
  
  
  RESULTS
  Bcl-2 overexpression results in significant inhibition of apoptosis in response to proteasome inhibitors, antiblastics, and TRAIL. Addition of TRAIL to proteasome inhibitors results in a synergistic cytotoxic effect in Bcl-2-overexpressing cells, whereas this result is not reproduced by the combination of proteasome inhibitors with antiblastic drugs. Importantly, proteasome inhibitors plus TRAIL induce mitochondrial dysfunction irrespective of up-regulated Bcl-2. Bcl-2 cleavage to a fragment with putative proapoptotic activity and elimination of antiapoptotic Mcl-1 may both play a role in proteasome inhibitors-TRAIL cooperation. Conversely, nuclear factor-kappaB inhibition by proteasome inhibitors is per se insufficient to explain the observed synergy.
  
  
  CONCLUSIONS
  Combined proteasome inhibitors and TRAIL overcome the apoptotic threshold raised by Bcl-2 and may prove useful in the treatment of chemoresistant malignancies with up-regulated Bcl-2.
authors.affiliation: Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. A.Nencioni@gmx.net
authors.forename: Alessio
authors.initials: A
authors.lastname: Nencioni
authors.forename: Lucia
authors.initials: L
authors.lastname: Wille
authors.forename: Giovanna
authors.initials: G
authors.lastname: Dal Bello
authors.forename: Davide
authors.initials: D
authors.lastname: Boy
authors.forename: Gabriella
authors.initials: G
authors.lastname: Cirmena
authors.forename: Sebastian
authors.initials: S
authors.lastname: Wesselborg
authors.forename: Claus
authors.initials: C
authors.lastname: Belka
authors.forename: Peter
authors.initials: P
authors.lastname: Brossart
authors.forename: Franco
authors.initials: F
authors.lastname: Patrone
authors.forename: Alberto
authors.initials: A
authors.lastname: Ballestrero
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D005047:Etoposide / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D007976:Leupeptins / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D016328:NF-kappa B / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D011719:Pyrazines / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D001897:Boronic Acids; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008562:Membrane Glycoproteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D016328:NF-kappa B; D009363:Neoplasm Proteins; D009842:Oligopeptides; D061988:Proteasome Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D011719:Pyrazines; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D000069286:Bortezomib; D005047:Etoposide; D004317:Doxorubicin; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; C078846:epoxomicin