Cooperative cytotoxicity of proteasome inhibitors and tumor necrosis factor-related apoptosis-inducing ligand in chemoresistant Bcl-2-overexpressing cells.
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.
Nencioni
Alessio
A
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. A.Nencioni@gmx.net
Wille
Lucia
L
Dal Bello
Giovanna
G
Boy
Davide
D
Cirmena
Gabriella
G
Wesselborg
Sebastian
S
Belka
Claus
C
Brossart
Peter
P
Patrone
Franco
F
Ballestrero
Alberto
A
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*
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