Inhibition of nuclear factor kappaB chemosensitizes non-small cell lung cancer through cytochrome c release and caspase activation.

ABSTRACT

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.