Proteasome inhibition sensitizes non-small cell lung cancer to histone deacetylase inhibitor-induced apoptosis through the generation of reactive oxygen species.
OBJECTIVE
The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces apoptosis in some malignancies through mitochondrial injury and generation of reactive oxygen species. Histone deacetylase inhibitors also activate the antiapoptotic transcription factor nuclear factor kappaB. We hypothesize that proteasome inhibition with bortezomib (Velcade; Millennium Pharmaceuticals, Inc, Cambridge, Mass)will inhibit nuclear factor kappaB activation, enhance suberoylanilide hydroxamic acid-induced mitochondrial injury, and sensitize non-small cell lung cancer cells to apoptosis.


METHODS
Four tumorigenic non-small cell lung cancer cell lines were treated with nothing, suberoylanilide hydroxamic acid, bortezomib, or both drugs. Nuclear factor kappaB-dependent transcription was determined by reporter gene assays and endogenous interleukin 8 transcription. Reactive oxygen species were quantified by using the fluorophore H 2 DCFDA. Cell viability was determined on the basis of clonogenic survival, and apoptosis was measured by quantifying caspase-3 activity and DNA fragmentation. Apoptosis and cell-survival assays were repeated in similarly treated cells incubated in the presence or absence of N-acetyl cysteine. Statistical significance was determined by means of analysis of variance.


RESULTS
Suberoylanilide hydroxamic acid significantly enhanced interleukin 8 and nuclear factor kappaB-dependent reporter gene transcription, and these effects were inhibited by bortezomib ( P < or = .01). Combined treatment with suberoylanilide hydroxamic acid and bortezomib induced greater reactive oxygen species generation, more apoptosis ( P < or = .02), and more cell death ( P < or = .001) than either drug alone. N-acetyl cysteine diminished the induction of apoptosis and enhanced cell survival ( P < or = .04).


CONCLUSIONS
Suberoylanilide hydroxamic acid and bortezomib synergistically induce reactive oxygen species generation in non-small cell lung cancer, and this plays a critical role in the induction of apoptosis after treatment. Combined treatment with suberoylanilide hydroxamic acid and bortezomib might be an effective treatment strategy for non-small cell lung cancer.
Denlinger
Chadrick E
CE
Department of Thoracic and Cardiovascular Surgery, University of Virginia, Charlottesville, VA 22908, USA.
Rundall
Brian K
BK
Jones
David R
DR
D017209:Apoptosis / Q000187:drug effects*; D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000503:physiopathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000503:physiopathology; D008928:Mitochondria / Q000187:drug effects; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D061988:Proteasome Inhibitors; D011719:Pyrazines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D000077337:Vorinostat
D001897:Boronic Acids; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D016328:NF-kappa B; D061988:Proteasome Inhibitors; D011719:Pyrazines; D017382:Reactive Oxygen Species; D000077337:Vorinostat; D000069286:Bortezomib