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

Ann Thorac Surg. 2004 Oct;78(4):1207-14; discussion 1207-14. doi: 10.1016/j.athoracsur.2004.04.029.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenocarcinoma / pathology*
  • Animals
  • Antimetabolites, Antineoplastic / therapeutic use
  • Antineoplastic Agents / pharmacology*
  • Antineoplastic Agents / therapeutic use
  • Apoptosis / drug effects*
  • Boronic Acids / pharmacology*
  • Boronic Acids / therapeutic use
  • Bortezomib
  • Carcinoma, Non-Small-Cell Lung / pathology*
  • Cell Cycle / drug effects
  • Cell Line, Tumor / drug effects
  • Cell Line, Tumor / enzymology
  • Cell Line, Tumor / pathology
  • Deoxycytidine / analogs & derivatives*
  • Deoxycytidine / pharmacology*
  • Deoxycytidine / therapeutic use
  • Drug Screening Assays, Antitumor
  • Gene Expression Profiling
  • Gene Expression Regulation, Neoplastic / drug effects
  • Humans
  • Lung Neoplasms / pathology*
  • Mice
  • Mice, Nude
  • NF-kappa B / analysis
  • NF-kappa B / metabolism
  • Neoplasm Proteins / antagonists & inhibitors*
  • Neoplasm Proteins / biosynthesis
  • Neoplasm Proteins / genetics
  • Proteasome Inhibitors*
  • Pyrazines / pharmacology*
  • Pyrazines / therapeutic use
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transcription Factor RelA
  • Transcription, Genetic / drug effects
  • Tumor Stem Cell Assay
  • Xenograft Model Antitumor Assays

Substances

  • Antimetabolites, Antineoplastic
  • Antineoplastic Agents
  • Boronic Acids
  • NF-kappa B
  • Neoplasm Proteins
  • Proteasome Inhibitors
  • Pyrazines
  • Transcription Factor RelA
  • Deoxycytidine
  • Bortezomib
  • gemcitabine