ABSTRACT
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 kappa B in non-small cell lung cancer and fails to induce significant levels of apoptosis. We hypothesize that nuclear factor kappa B 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 kappa B transcriptional activity was measured by using a luciferase reporter assay and transcription of the nuclear factor kappa B-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 unpaired t tests. RESULTS: Butyrate increased nuclear factor kappa B 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 kappa B 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 kappa B 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.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Apoptosis/drug effects , Boronic Acids/administration & dosage , Bortezomib , Butyrates/administration & dosage , Carcinoma, Non-Small-Cell Lung/physiopathology , Cell Cycle/drug effects , Cell Survival/drug effects , Cysteine Endopeptidases/administration & dosage , Histone Deacetylases/administration & dosage , Humans , Interleukin-8/genetics , Lung Neoplasms/physiopathology , Multienzyme Complexes/administration & dosage , NF-kappa B/drug effects , NF-kappa B/metabolism , Protease Inhibitors/administration & dosage , Proteasome Endopeptidase Complex , Pyrazines/administration & dosage , Transcription, Genetic/drug effects , Transcriptional Activation/drug effects , Treatment Outcome , Tumor Cells, CulturedABSTRACT
OBJECTIVE: Although we have previously shown that inhibition of nuclear factor kappaB sensitizes non-small cell lung cancer cells to chemotherapy-mediated cell death, the apoptotic pathways mediating this process are unknown. The purpose of this study was to determine whether chemosensitivity after the inhibition of nuclear factor kappaB in non-small cell lung cancer cells is a mitochondrial and caspase-mediated process and whether it is dependent on nuclear factor kappaB transcriptional activity. METHODS: Previously described H157 non-small cell lung cancer cells were treated with gemcitabine, and DNA fragmentation was determined. Caspase 3, 6, 7, 8, and 9 activity in cytoplasmic extracts was determined fluorometrically. The mitochondrial permeability index and cytosolic cytochrome c levels were also determined. The caspase inhibitor Boc-D, as well as nuclear factor kappaB-regulated gene products A1, c-IAP-2, and Bcl-X(L), were added to H157 cells lacking nuclear factor kappaB and the degree of apoptosis assessed. All experiments were performed in triplicate, and data significance was determined by means of analysis of variance. RESULTS: Non-small cell lung cancer cells lacking functional nuclear factor kappaB (H157I) underwent more apoptosis after chemotherapy than vector control cells (H157V). There was an increase in the mitochondrial permeability index and cytochrome c release after chemotherapy in the H157I cells. H157I cells also had more activation of caspases 3 and 9 than control cells. Inhibition of caspase activity or transfection with nuclear factor kappaB-regulated gene products rescued cell death after the inhibition of nuclear factor kappaB. CONCLUSION: Chemosensitization by means of inhibition of nuclear factor kappaB in non-small cell lung cancer cells occurs through increased cytochrome c release and caspase 3 and 9 activation. Inhibition of nuclear factor kappaB or its gene products in addition to chemotherapy warrants further study as a treatment strategy in patients with advanced-stage non-small cell lung cancer.
