Proteasome inhibition sensitizes non-small-cell lung cancer to gemcitabine-induced apoptosis

Background. My colleagues and I have previouslyshown that chemotherapy activates the antiapoptotic transcription factor nuclear factor (NF)-kappaB in non-small-cell lung cancer (NSCLC). We hypothesized that inhibition of NF-kappaB by using the proteasome inhibitor bortezomib (Velcade) would sensitize NSCLC to gemcitabine-induced apoptosis. Methods. Tumorigenic NSCLC cell lines (H157 and A549) were treated with nothing, gemcitabine, bortezomib, or both compounds. NF-kappaB activity was determined by nuclear p65 protein levels, electrophoretic mobility shift assays, and reverse transcription-polymerase chain reaction of the NF-kappaB-regulated genes interleukin-8, c-IAP2, and Bcl-xL. The p21 and p53 protein levels were determined in similarly treated cells. Cell-cycle dysregulation was assessed by fluorescence-activated cell sorting analysis. Cell death and apoptosis were quantified by clonogenic assays, caspase-3 activation, and DNA fragmentation. NSCLC A549 xenografts were generated and treated as noted previously. Tumor growth was assessed over a 4-week treatment period. Statistical analysis was performed with analysis of variance. Results. Gemcitabine enhanced nuclear p65 levels, NF-kappaB binding to DNA, and transcription of all NF-kappaB-regulated genes. Bortezomib inhibited each of these effects. Combined gemcitabine and bortezomib enhanced p21 and p53 expression and induced S-phase and G(2)/M cell-cycle arrests, respectively. Combined treatment killed 80% of the NSCLC cells and induced apoptosis, as determined by caspase-3 activation (p = 0.05) and DNA fragmentation (p = 0.02). NSCLC xenografts treated with combination therapy grew significantly slower than xenografts treated with gemcitabine alone (p = 0.02). Conclusions. Bortezomib inhibits gemcitabine-induced activation of NF-kappaB and sensitizes NSCLC to death in vitro and in vivo. This combined treatment strategy warrants further investigation and may represent a reasonable treatment strategy for select patients with NSCLC given the current clinical availability of both drugs. (C) 2004 by The Society of Thoracic Surgeons.