Article Figures & Data
Figures
- Figure 1.
Effects of nitrosylcobalamin (NO-Cbl), Apo2L/TRAIL, and their combination on the proliferation of melanoma cell lines A375, WM9, and WM3211 and normal cell lines CMN1, DMN1, and primary HFF fibroblasts. Left panels: Cells were treated with NO-Cbl (open bars), Apo2L/TRAIL (hatched bars), or pre-treated with NO-Cbl followed by Apo2L/TRAIL (solid bars) for three days, and growth was measured by the colorimetric sulforhodamine B assay (44). Data points represent the mean of four replicates±standard error of the mean (SEM). Right panels: Synergy between NO-Cbl and Apo2L/TRAIL was determined by median effect analysis (48), (combination index >1 indicates antagonism, =1 indicates additivity, and <1 indicates synergy). (a) The sequential treatment of NO-Cbl and Apo2L/TRAIL induced synergistic antiproliferative activity in A375, WM9 and WM3211 cells at each combined dose. (b) Normal melanocyte CMN1 and DMN1 cell lines, and normal HFF fibroblasts were completely resistant to simultaneous exposure to NO-Cbl, Apo2L/TRAIL, or the pre-treatment with NO-Cbl followed by Apo2L/TRAIL.
- Figure 2.
Effect of NO-Cbl, Apo2L/TRAIL and the combination on the growth of A375 melanoma xenografts. NCR male athymic nude (nu/nu) mice were injected subcutaneously with 4×106 A375 cells (n=8 per group). Drug treatments began on day 2 after injection of tumor cells. NO-Cbl was administered twice daily for the duration of the study. Apo2L/TRAIL was administered every other day. Control mice received phosphate buffered saline. Tumor volume was measured three times per week. Data points represent the mean tumor volume (mm3)±SEM.
- Figure 3.
TUNEL apoptosis assay. A375 cells were treated with NO-Cbl, Apo2L/TRAIL, and their combination. NO-Cbl and Apo2L/TRAIL were minimally effective as single agents but demonstrated greater apoptosis when administered concomitantly. The highest levels of apoptosis were observed when cells were pre-treated with NO-Cbl for 12 h followed by Apo2L/TRAIL treatment for 24 h. Conversely, the effect of Apo2L/TRAIL followed by NO-Cbl was no different than Apo2L/TRAIL alone.
- Figure 4.
NF-κB-luc reporter assay. NF-κB-luc transfected A375 cells were pre-treated with NO-Cbl for 1 h followed by Apo2L/TRAIL or TNF-α for 4 h. Renilla luciferase was co-transfected to normalize samples for transfection efficiency. Cell lysates were analyzed for NF-κB-luc reporter activity. NO-Cbl pre-treatment inhibited Apo2L/TRAIL- and TNF-α-induced activation of the NF-κB-luc reporter.
- Figure 5.
Western blot analysis of IκBα and phospho-IκBα. A375 cells were pre-treated for 16 h with NO-Cbl followed by Apo2L/TRAIL or TNF-α stimulation. IκBα and phospho-IκBα protein levels were determined in A375 whole-cell lysates. (a) After stimulation with Apo2L/TRAIL (30 min) or TNF-α (15 min), IκBα was almost totally degraded. NO-Cbl efficiently blocked IκBα degradation following Apo2L/TRAIL, but only partially blocked IκBα degradation following TNF-α. (b) After 1 h, cellular levels of IκBα are restored as a result of re-synthesis. NO-Cbl blocks the phosphorylation of newly translated IκBα. Band retardation of IκBα is evident following Apo2L/TRAIL or TNF-α stimulation. Phospho-IκBα migrates slower than IκBα (compare middle two lanes to other four lanes). c, NO-Cbl, NOC-18, and SNAP pre-treatment all inhibited Apo2L/TRAIL-induced IκBα phosphorylation.
- Figure 6.
IκB kinase (IKK) activity. IKK activity was assessed using recombinant GST-IκBα(1-54) and γ32P-ATP as substrates. The phosphorylated GST fusion protein was detected by autoradiography. (a) IKK activity was determined in A375 cells pre-treated with NO-Cbl followed by Apo2L/TRAIL or TNF-α stimulation for 30 min and 15 min, respectively. NO-Cbl treatment inhibited IKK activity more effectively when Apo2L/TRAIL was the stimulus, compared to stimulation by TNF-α. Anti-β-actin antibody served as the irrelevant antibody with no phosphorylation of GST-IκBα(1-54) observed. (b) Coomassie blue-stained gel shows equal loading of GST-IκBα(1-54) substrate. (c) Immunoblot analysis shows the presence of equal amounts of total IKK in the lysates. β-actin was used as a loading control.
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