Article Figures & Data
Figures
- Figure 1.
Bortezomib induces STAT3 in HNSCC cells. A, cells (1483, UM-22A, and UM-22B) were left untreated or were treated for varying lengths of time with bortezomib (1483: 80 nmol/L; UM-22A and UM-22B: 20 nmol/L). As a control, cells were treated with 0.1% DMSO. After treatment, whole-cell lysates were subjected to immunoblotting for total STAT3 protein, and probing for β-actin was used to show equal loading. B, cells were treated with bortezomib for the indicated lengths of time, followed by immunoblotting with anti–phospho-STAT3 (Tyr705). Experiments were done thrice with similar results each time.
- Figure 2.
Bortezomib induces cellular STAT3 activity. UM-22B cells were stably transfected with pLucTKSIE, which contains two copies of the STAT3-responsive hSIE element upstream from a luciferase reporter gene. The UM-22B/pLucTKSIE cells were treated with bortezomib (20 nmol/L) for the indicated lengths of time, followed by luciferase assays. The data are plotted as fold induction of luciferase activity relative to untreated cells. Columns, mean of three independent experiments; bars, SE.
- Figure 3.
DA STAT3 inhibits bortezomib-induced cell death whereas DN STAT3 enhances bortezomib action. A, UM-22B cells stably expressing the pLucTKSIE STAT3 reporter construct were transiently transfected with RcCMV/Neo (empty vector) or constructs encoding DA STAT3 or DN STAT3. Simultaneously, the cells were cotransfected with a construct (phRL-TK) directing constitutive expression of Renilla luciferase. After normalization for Renilla luciferase activity, the transfected cells were then left untreated or were treated for 24 h with 10 nmol/L bortezomib. Luciferase assays were done to verify enhancement of cellular STAT3 activity by DA STAT3 and inhibition by DN STAT3. The fold induction of luciferase activity relative to untreated empty vector cells is shown. Columns, mean of three independent experiments; bars, SE. B, UM-22B or 1483 cells transfected with empty vector or constructs encoding DA STAT3 or DN STAT3 were left untreated or were treated for 24 h with 10 nmol/L bortezomib. After treatment, MTS assays were done on triplicate wells, and cellular metabolic activities were plotted relative to untreated empty vector cells. Bars, SD of triplicate wells. Similar results were obtained in two independent experiments. C, UM-22B or 1483 cells were transfected and treated as in B, followed by determination of cell viabilities with the use of trypan blue exclusion assays. For each data point, triplicate wells were plated, and a minimum of 300 cells per well were examined. Columns, mean of three independent experiments; bars, SE. D, UM-22B cells transfected with the indicated constructs were left untreated or were treated for 24 h with 10 nmol/L bortezomib. Immunoblotting with anti-PARP was done to detect cleavage of full-length PARP (116 kDa) to an 89-kDa fragment. Probing with anti–β-actin was used to verify equal protein loading. The experiment was done thrice with similar results.
- Figure 4.
STAT3 decoy enhances bortezomib-induced loss of clonogenic survival. UM-22B cells were mock transfected or were transfected for 4 h with 6.3 nmol/L of mutant control decoy or 6.3 nmol/L of STAT3 decoy. Mock-transfected cells were subsequently incubated for 24 h with 0.1% DMSO or 10 nmol/L bortezomib. Cells transfected with mutant control decoy or STAT3 decoy were incubated for 24 h in the absence or presence of 10 nmol/L bortezomib. After treatment, cells were washed twice in PBS, detached from the plate, diluted, and replated in medium containing 10% FBS in six-well plates. After 2 wk, cells were stained with crystal violet. Top, a representative experiment; bottom, the colonies consisting of >50 cells in each well were counted. Data represent the percent of colonies relative to mock-transfected, DMSO-treated controls. Columns, mean of four independent experiments; bars, SE.
- Figure 5.
STAT3 decoy enhances bortezomib-induced apoptosis signaling. UM-22B cells were subjected to mock transfection or transfection with mutant control decoy or STAT3 decoy as described in Fig. 4. Mock-transfected cells were then incubated for 24 h with 0.1% DMSO or 10 nmol/L bortezomib, whereas cells transfected with mutant control decoy or STAT3 decoy were incubated for 24 h in the absence or presence of 10 nmol/L bortezomib. The treated cells were subjected to immunoblotting with antibodies directed against PARP or the active form of caspase-3. Probing with anti–β-actin was used to verify equal loading. Similar results were obtained in three independent experiments.
- Figure 6.
Guggulsterone synergizes with bortezomib against HNSCC cells. UM-22A (A) and UM-22B (B) cells were seeded at 5,000 per well in 96-well plates, grown overnight, and then treated for 48 h with the indicated concentrations of bortezomib alone, guggulsterone alone, or bortezomib plus guggulsterone. MTS assays were done, and the data were plotted as the percent of metabolic activity relative to untreated control cells. Points, mean of triplicate wells; bars, SD. Combination indexes were determined as described in Materials and Methods. Similar results were obtained in four independent experiments. C, UM-22B cells were left untreated or were treated for 48 h with 0.1% DMSO, 3.5 nmol/L bortezomib alone, 10 μmol/L guggulsterone alone, or bortezomib (3.5 nmol/L) plus guggulsterone (10 μmol/L). After treatment, clonogenic assays were done and quantified as described for Fig. 4. Columns, mean of three independent experiments; bars, SE. D, UM-22B cells were left untreated or were treated for 24 h with 0.1% DMSO, 3.5 nmol/L bortezomib alone, 10 μmol/L guggulsterone alone, or bortezomib (3.5 nmol/L) plus guggulsterone (10 μmol/L). Immunoblotting was done to identify cleaved PARP and the active/processed form of caspase-3. The experiment was done thrice with similar results.
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Volume 8, Issue 8
