Proteasome inhibition sensitizes non-small cell lung cancer to histone deacetylase inhibitor-induced apoptosis through the generation of reactive oxygen species

Objectives: The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces apoptosis in some malignancies through mitochondrial injury and generation of reactive oxygen species. Historic 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 less than or equal to .01). Combined treatment with suberoylanilide hydroxamic acid and bortezomib induced greater reactive oxygen species generation, more apoptosis (P less than or equal to .02), and more cell death (P less than or equal to .001) than either drug alone. N-acetyl cysteine diminished the induction of apoptosis and enhanced cell survival (P less than or equal to 5.04). 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.