Proteasome inhibition sensitizes non-small-cell lung cancer to gemcitabine-induced apoptosis.
BACKGROUND
My colleagues and I have previously shown 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 G2/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.
Department of Thoracic and Cardiovascular Surgery, University of Virginia, Charlottesville, Virginia 22908-0679, USA.
Chadrick E
CE
Denlinger
Brian K
BK
Rundall
Michael D
MD
Keller
David R
DR
Jones
D000230:Adenocarcinoma / Q000473:pathology*; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D001897:Boronic Acids / Q000494:pharmacology* / Q000627:therapeutic use; D000069286:Bortezomib; D002289:Carcinoma, Non-Small-Cell Lung / Q000473:pathology*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology* / Q000627:therapeutic use; D004354:Drug Screening Assays, Antitumor; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology*; D051379:Mice; D008819:Mice, Nude; D016328:NF-kappa B / Q000032:analysis / Q000378:metabolism; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D061988:Proteasome Inhibitors; D011719:Pyrazines / Q000494:pharmacology* / Q000627:therapeutic use; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051996:Transcription Factor RelA; D014158:Transcription, Genetic / Q000187:drug effects; D014410:Tumor Stem Cell Assay; D023041:Xenograft Model Antitumor Assays; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D001897:Boronic Acids; D016328:NF-kappa B; D009363:Neoplasm Proteins; D061988:Proteasome Inhibitors; D011719:Pyrazines; D051996:Transcription Factor RelA; D003841:Deoxycytidine; D000069286:Bortezomib; D000093542:Gemcitabine