PCI-24781 induces caspase and reactive oxygen species-dependent apoptosis through NF-kappaB mechanisms and is synergistic with bortezomib in lymphoma cells.
OBJECTIVE
We investigated the cytotoxicity and mechanisms of cell death of the broad-spectrum histone deacetylase (HDAC) inhibitor PCI-24781, alone and combined with bortezomib in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines and primary lymphoproliferative (CLL/SLL) cells.


METHODS
Apoptosis, mitochondrial membrane potential, cell cycle analysis, and reactive oxygen species (ROS) were measured by flow cytometry, whereas caspase activation was determined by Western blot. Nuclear factor kappaB (NF-kappaB)-related mRNAs were quantified by reverse transcription-PCR, NF-kappaB-related proteins by Western blotting, and NF-kappaB DNA-binding activity by electromobility shift assay. Finally, gene expression profiling was analyzed.


RESULTS
PCI-24781 induced concentration-dependent apoptosis that was associated with prominent G(0)/G(1) arrest, decreased S-phase, increased p21 protein, and increased ROS in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines. Dose-dependent apoptosis with PCI-24781 was also seen among primary CLL/SLL cells. PCI-24781-induced apoptosis was shown to be ROS- and caspase-dependent. Combined PCI-24781/bortezomib treatment resulted in strong synergistic apoptosis in all non-Hodgkin lymphoma lines (combination indices, 0.19-0.6) and was additive in Hodgkin lymphoma and primary CLL/SLL cells. Further, PCI-24781/bortezomib resulted in increased caspase cleavage, mitochondrial depolarization, and histone acetylation compared with either agent alone. Gene expression profiling showed that PCI-24781 alone significantly down-regulated several antioxidant genes, proteasome components, and NF-kappaB pathway genes, effects that were enhanced further with bortezomib. Reverse transcription-PCR confirmed down-regulation of NF-kappaB1 (p105), c-Myc, and IkappaB-kinase subunits, where NF-kappaB DNA binding activity was decreased.


CONCLUSIONS
We show that PCI-24781 results in increased ROS and NF-kappaB inhibition, leading to caspase-dependent apoptosis. We also show that bortezomib is synergistic with PCI-24781. This combination or PCI-24781 alone has potential therapeutic value in lymphoma.
Division of Hematology/Oncology, Northwestern University Feinberg School of Medicine and the Robert H Lurie Comprehensive Cancer Center, Chicago, IL 60611, USA.
Savita
S
Bhalla
Sriram
S
Balasubramanian
Kevin
K
David
Mint
M
Sirisawad
Joseph
J
Buggy
Lauren
L
Mauro
Sheila
S
Prachand
Richard
R
Miller
Leo I
LI
Gordon
Andrew M
AM
Evens
D000368:Aged; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D001572:Benzofurans / Q000494:pharmacology*; D015153:Blotting, Western; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D005260:Female; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008223:Lymphoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008228:Lymphoma, Non-Hodgkin / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008875:Middle Aged; D016328:NF-kappa B / Q000378:metabolism*; D011719:Pyrazines / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D001572:Benzofurans; D001897:Boronic Acids; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D016328:NF-kappa B; D011719:Pyrazines; D017382:Reactive Oxygen Species; D000069286:Bortezomib; D020169:Caspases; C512352:abexinostat