Kinetic and dose effects of TNF plus WA/Cel on cellular proteasome activity in MDA-MB-231 cells.

<p>Cells were treated with TNF-α (10 ng/ml) or TNF-α (10 ng/ml) plus WA or Cel for the indicated times and doses, followed by measurement of the proteasomal CT-like activity using Z-GGL-AMC or Western blotting for ubiquitinated proteins and IκBα. (A) Kinetic effects of TNF-α plus WA on CT-like activity, ubiquitinated proteins and IκBα. Cells were treated with WA (2.5 µM) with or without TNF-α for the indicated times. (B) Kinetic effects of TNF-α plus Cel on CT-like activity, ubiquitinated proteins and IκBα. Cells were treated with Cel (1 µM) with or without TNF-α for the indicated times. (C) Dose effects of TNF-α plus WA on proteasomal activity, ubiquitinated proteins and IκBα. Cells were treated with the indicated doses of WA with or without TNF-α for 24 hours. (D) Dose effects of TNF-α plus Cel on proteasomal activity, ubiquitinated proteins and IκBα. Cells were treated with the indicated doses Cel with or without TNF-α for 24 hours. Data are shown as mean ± SD of three experiments. (*<i>P</i><0.05, as compared with the untreated control. #<i>P</i><0.05, WA/Cel treated versus WA/Cel+TNF-α).</p

WA and Cel sensitize MDA-MB-231 cells to TNF-α resulting in inhibition of cell proliferation.

<p>(A) Concentration dependent effect of WA or Cel with or without TNF-α on cell viability. MDA-MB-231 cells were treated with different concentrations of WA (upper) or Cel (lower) with or without TNF-α (10 ng/ml) for 48 hours, followed by measurement of cell viability by MTT assay. IC₅₀ was calculated by Origin. (B) Colony formation assays of cells treated with WA or Cel with or without TNF-α. WA (left) or Cel (right) at shown concentrations with or without TNF-α (10 ng/ml) were added to the cells for 24 hours. The medium was subsequently removed and the cells were maintained in culture for a further 10 days. (C) Scratch wound-healing assay for cells treated with WA or Cel with or without TNF-α at shown concentrations. Photographs were taken at 24 hours after treatment and scratching of the well. (D) Colony forming efficiency was calculated. (E) Actual migration speed was calculated by Image-Pro plus. Data are shown as mean ± SD of three experiments. (*P<0.05, versus the untreated group. #P<0.05, WA/Cel treated versus WA/Cel+TNF-α).</p

WA or Cel sensitize MDA-MB-231 cells to TNF-α-induced apoptosis.

<p>(A, B) Cells were treated with different concentrations of WA or Cel with (right) or without (left) TNF-α (10 ng/ml) for 48 hours, followed by measurement of cell viability by flow cytometry. (C) Cell death inducing abilities of WA and TNF-α or Cel and TNF-α combination in a dose responsive manner. Cells were treated with different concentrations of WA (upper) or Cel (lower) as shown with or without TNF-α (10 ng/ml) for 24 hours, followed by detection of PARP by Western blotting. Data are shown as mean ±SD of three experiments. (*<i>P</i><0.05, as compared with the untreated control. #<i>P</i><0.05, WA/Cel treated versus WA/Cel+TNF-α).</p

Inhibition of NF-κB target gene expression by WA/Cel and small interfering RNA.

<p>(A) MDA-MB-231 cells were transfected with <i>si</i>RNA against NF-κBp65 for 6 hours. As a negative control, cells were transfected with the same amount of non targeting control -<i>si</i>RNA. Following transfection, cells were treated with TNF-α (10 ng/ml) for 24 hours. Cells that were not transfected were treated with TNF-α (10 ng/ml) plus WA (5 µM) or Cel (2.5 µM) for 24 hours. The mRNA levels of NF-κB-p65, XIAP and cIAP1/2 were detected by RT-qPCR. (B) MDA-MB-231 cells were transfected with <i>si</i>RNA against NF-κBp65 for 6 hours. The negative control was treated with the same amount of vector-<i>si</i>RNA. Following transfection, cells were treated by TNF-α (10 ng/ml) for 48 hours. Cells that were not transfected were treated with TNF-α (10 ng/ml) plus WA (5 µM) or Cel (2.5 µM) for 48 hours. The levels of NF-κBp65, XIAP and cIAP1/2 proteins were detected by Western blotting. Data are shown as mean ± SD of three experiments.</p

Dose effects of TNF-α plus WA or Cel on caspase-3 and -9 in MDA-MB-231 cells.

<p>(A) Concentration dependent effect of WA and TNF-α or Cel and TNF-α combination on caspase-3 and -9 expression. Cells were treated with the indicated concentrations of WA (upper) or Cel (lower) with or without TNF-α (10 ng/ml) for 24 hours, followed by detection of caspase-3 and -9 by Western blotting. (B) Dose effect of WA and TNF-α or Cel and TNF-α combination on caspase-3 activity. Cells were treated with the indicated concentrations of WA (upper) or Cel (lower) with or without TNF-α (10 ng/ml) for 24 hours, followed by detection of caspase-3 activity by adding specific substrate Ac-Asp-Glu-Val-Asp-AMC. (C) Kinetic effects of WA and TNF-α or Cel and TNF-α combination on caspase-3 and -9. Cells were treated with TNF-α (10 ng/ml) or TNF-α (10 ng/ml) plus WA (1 µM) (upper), or TNF-α (10 ng/ml) plus Cel (1 µM) (lower) for the indicated times, followed by detection of caspase-3 and -9 by Western blotting. (D) Kinetic effect of TNF-α plus WA or TNF-α plus Cel on caspase-3 activity. Cells were treated with TNF-α (10 ng/ml) or TNF-α (10 ng/ml) plus WA (1 µM) (upper), or TNF-α (10 ng/ml) plus Cel (1 µM) (lower) for the indicated times, followed by detection of caspase-3 activity via adding the specific substrate Ac-Asp-Glu-Val-Asp-AMC. Data are shown as mean ± SD of three experiments. (*<i>P</i><0.05, as compared with the untreated control. #<i>P</i><0.05, WA/Cel treated versus WA/Cel+TNF-α).</p

Effect of the combination of TNF-α and WA/Cel on nuclear translocation of NF-κBp65 in cells.

<p>(A, B) The effects of WA (A) or Cel (B) on NF-κBp65 activation. MDA-MB-231 cells were treated with different concentrations of WA or Cel with or without TNF-α (10 ng/ml) for 24 hours. The levels of NF-κBp65 in cytoplasmic and nuclear fractions were analyzed by Western blotting. β-actin and Histone H3 were used as the cytoplamsic and nuclear marker respectively. (C) Inhibition of NF-κB binding activity by WA or Cel in TNF-α-stimulated MDA-MB-231cells. Cells were treated with TNF-α (10 ng/ml) with or without WA (2.5 µM) or Cel (1 µM) for 1.5 h. Nuclear translocation of NF-κBp65 was observed by fluorescence microscope. Data are shown as mean ± SD of three experiments.</p