Chadrick E. Denlinger, MD, Brian K. Rundall, DO, Michael D

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Proteasome Inhibition Sensitizes Non–Small-Cell Lung Cancer to Gemcitabine-Induced Apoptosis Chadrick E. Denlinger, MD, Brian K. Rundall, DO, Michael D. Keller, BS, David R. Jones, MD The Annals of Thoracic Surgery Volume 78, Issue 4, Pages 1207-1214 (October 2004) DOI: 10.1016/j.athoracsur.2004.04.029

Fig 1 Non–small-cell lung cancer cells were treated with escalating doses of gemcitabine (1 to 200 μmol/L) for 24 hours. Cell survival was determined by staining with crystal violet dye. The Annals of Thoracic Surgery 2004 78, 1207-1214DOI: (10.1016/j.athoracsur.2004.04.029)

Fig 2 (A) Nuclear extracts from non–small-cell lung cancer (NSCLC) cells treated with nothing, gemcitabine (Gem; 10 μmol/L), bortezomib (Velcade; Vel; 50 nmol/L), or both drugs were analyzed by Western blot analysis probing for the transcriptionally active nuclear factor (NF)-κB subunit p65. Blots were reprobed for RNA pol II as a loading control. (B) DNA binding of NF-κB was determined in the A549 cell line after identical treatment conditions by electrophoretic mobility shift assay. (C) Messenger RNAs from similarly treated NSCLC cells were evaluated by reverse transcription-polymerase chain reaction to determine c-IAP2, Bcl-xL, and interleukin (IL)-8 expression. (GAPDH = glyceraldehyde phosphate dehydrogenase; TNF = tumor necrosis factor.) The Annals of Thoracic Surgery 2004 78, 1207-1214DOI: (10.1016/j.athoracsur.2004.04.029)

Fig 3 (A) Non–small-cell lung cancer cells were treated for 12 hours with nothing, gemcitabine (Gem; 10 μmol/L), bortezomib (Velcade; Vel; 50 nmol/L), or both drugs. Western blot analysis was performed to probe for p21 and p53. Blots were reprobed for β-tubulin as a loading control. (B) Cell-cycle profiles were determined by fluorescence-activated cell sorting analysis in the A549 cells after 12 hours of treatment. The Annals of Thoracic Surgery 2004 78, 1207-1214DOI: (10.1016/j.athoracsur.2004.04.029)

Fig 4 Apoptosis was determined in non–small-cell lung cancer cells after 24 hours of treatment with nothing, gemcitabine (gem; 10 μmol/L), bortezomib (Velcade; 50 nmol/L), or both drugs by quantifying (A) caspase-3 activity and (B) DNA fragmentation (‡p ≤ 0.004; †p ≤ 0.003; *p = 0.02). ▧ = no treatment; ▨ = Velcade; = gemcitabine; ■ = gem/Velcade. (RFU = relative fluorescence units; OD = optical density.) The Annals of Thoracic Surgery 2004 78, 1207-1214DOI: (10.1016/j.athoracsur.2004.04.029)

Fig 5 A549 non–small-cell lung cancer xenografts were developed in athymic nude mice. Treatment was initiated when tumors achieved a volume of 0.5 cm3. Tumors were measured and volumes were calculated every other day over a 4-week treatment period (p ≤ 0.04 versus gemcitabine). (Gem = gemcitabine; Vel = Velcade [bortezomib].) The Annals of Thoracic Surgery 2004 78, 1207-1214DOI: (10.1016/j.athoracsur.2004.04.029)