Combined proteasome and histone deacetylase inhibition in non-small cell lung cancer

Objective: Inhibitors of histone deacetylases are potent inducers of cell-cycle arrest and apoptosis in certain malignancies. We have previously demonstrated that chemotherapy activates the antiapoptotic transcription factor nuclear factor kappaB in non-small cell lung cancer and fails to induce significant levels of apoptosis. We hypothesize that nuclear factor kappaB inhibition with the proteasome inhibitor bortezomib (formerly known as PS-341) will sensitize non-small cell lung cancer cells to histone deacetylase inhibitor-mediated apoptosis. Methods: Tumorigenic non-small cell lung cancer cells (A549, H358, and H460) were treated with bortezomib, followed by the histone deactylase inhibitor sodium butyrate. After treatment, nuclear factor kappaB transcriptional activity was measured by using a luciferase reporter assay and transcription of the nuclear factor kappaB-dependent gene IL8. Apoptosis was determined on the basis of caspase-3 activation and DNA fragmentation. Western blot analyses for the cell-cycle regulatory proteins p21 and p53 were performed, and cell-cycle alterations were determined by means of FACS analysis. Experiments were performed in triplicate, and statistical significance was determined by using impaired t tests. Results: Butyrate increased nuclear factor KB transcriptional activity 4-fold relative to that seen in control cells (P = .05) in all non-small cell lung cancer cell lines. Treatment with bortezomib reduced butyrate-induced activation of nuclear factor K13 to baseline levels. The proteins p21 and p53 were stabilized after treatment with bortezomib, correlating with a G(2)/M cell-cycle arrest. Treatment with butyrate alone resulted in minimal apoptosis, but combined histone deacetylase and proteasome inhibition increased apoptosis 3- to 4-fold (P = .02). Conclusions: Combined molecular targeting of histone deacteylases and proteasomes synergistically induced apoptosis in non-small cell lung cancer. Pharmacologic nuclear factor kappaB suppression through proteasome inhibition, followed by treatment with histone deacetylase inhibitors, might represent a novel treatment strategy for patients with non-small cell lung cancer.