Article Figures & Data
Figures
- FIGURE 1.
Ursolic acid inhibits constitutively active STAT3 in U266 cells. A. The structure of the pentacyclic triterpenoid ursolic acid. B. U266 cells (2 × 106/mL) were treated with the indicated concentrations of ursolic acid for 4 h and analyzed for nuclear STAT3 levels by EMSA. C. U266 cells (2 × 106/mL) were treated with 50 μmol/L ursolic acid for the indicated durations and analyzed for nuclear STAT3 levels by EMSA. D. Nuclear extracts from U266 cells were incubated with STAT3 antibody and an unlabeled STAT3 oligo probe. Nuclear extracts from untreated myeloid leukemia (KBM-5) cells were taken alone. They were then assayed for STAT3 DNA binding by EMSA. E. Ursolic acid suppresses phospho-STAT3 levels in a dose-dependent manner. U266 cells (2 × 106/mL) were treated with the indicated concentrations of ursolic acid for 4 h, after which whole-cell extracts were prepared and 30 μg of protein were resolved on 7.5% SDS-PAGE gel, electrotransferred onto nitrocellulose membranes, and probed for phospho-STAT3. F. Effect of AG490 on STAT3 phosphorylation in U266 cells. U266 cells (2 × 106) were treated with indicated concentrations of AG490 for 8 h and whole-cell extracts were prepared. Thirty micrograms of whole-cell extract were resolved on 7.5% SDS-PAGE, electrotransferred to a nitrocellulose membrane, probed for phospho-STAT3 (top), stripped, and reprobed for STAT3 (bottom). G. Ursolic acid suppresses phospho-STAT3 levels in a time-dependent manner. U266 cells (2 × 106/mL) were treated with the 50 μmol/L ursolic acid for the indicated time points, after which Western blotting was done as described for (E). The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. H. Ursolic acid had no effect on phospho-STAT5 and STAT5 protein expression. U266 cells (2 × 106/mL) were treated with 50 μmol/L ursolic acid for the indicated time points. Whole-cell extracts were prepared, fractionated on SDS-PAGE, and examined by Western blotting with antibodies against phospho-STAT5 and STAT5. I. Ursolic acid causes inhibition of translocation of STAT3 to the nucleus. U266 cells (1 × 105/mL) were incubated with or without 50 μmol/L ursolic acid for 4 h and then analyzed for the intracelullar distribution of STAT3 by immunocytochemistry. The same slides were counterstained for nuclei with Hoechst (50 ng/mL) for 5 min.
- FIGURE 2.
Ursolic acid down-regulates IL-6–induced phospho-STAT3. A. MM1.S cells (2 × 106/mL) were treated with IL-6 (10 ng/mL) for the indicated time points, whole-cell extracts were prepared, and phospho-STAT3 was detected by Western blot as described in Materials and Methods. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. B. MM1.S cells (2 × 106/mL) were treated with 50 μmol/L ursolic acid for the indicated time points and then stimulated with IL-6 (10 ng/mL) for 15 min. Whole-cell extracts were then prepared and analyzed for phospho-STAT3 by Western blotting. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. Representative of three independent experiments. C. MM1.S cells (4 × 106/mL) were treated with IL-6 (10 ng/mL) for the indicated time points. Whole-cell extracts were prepared and 500 μg of sample were incubated with JAK2 antibody overnight. Immunocomplex was precipitated with protein A/G-agarose beads and then fractionated on 10% SDS-PAGE. Western blot analysis was done with anti–phospho-JAK2 antibody. D. MM1.S cells (4 × 106/mL) were treated with 50 μmol/L ursolic acid for the indicated time points and then stimulated with IL-6 (10 ng/mL) for 15 min. Whole-cell extracts were prepared and 500 μg of sample were incubated with JAK2 antibody overnight. Immunocomplex was precipitated with protein A/G-agarose beads and then fractionated on 10% SDS-PAGE. Western blot analysis was done with anti–phospho-JAK2 antibody. The same samples were analyzed for JAK2 protein. Ursolic acid–induced inhibition of STAT phosphorylation is reversible. U266 cells (2 × 106) were treated with 50 μmol/L ursolic acid for the indicated durations (E) or treated for 1 h and washed with PBS twice to remove ursolic acid before resuspension in fresh medium. Cells were removed at indicated time points and lysed to prepare the whole-cell extract (F). Thirty micrograms of whole-cell extracts were resolved on 7.5% SDS-PAGE, electrotransferred to a nitrocellulose membrane, probed for the phosphorylated-STAT3, and stripped and reprobed with STAT3 antibodies. Representative of three independent experiments.
- FIGURE 3.
A. Ursolic acid suppresses phospho-Src levels in a time-dependent manner. U266 cells (2 × 106/mL) were treated with 50 μmol/L ursolic acid, after which whole-cell extracts were prepared and 30-μg aliquots of those extracts were resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with phospho-Src antibody. The same blots were stripped and reprobed with Src antibody to verify equal protein loading. B. Ursolic acid suppresses phospho-JAK1 expression in a time-dependent manner. U266 cells (2 × 106/mL) were treated with 50 μmol/L ursolic acid, after which whole-cell extracts were prepared and 30-μg portions of those extracts were resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with phospho-JAK1 antibody. The same blots were stripped and reprobed with JAK1 antibody to verify equal protein loading. C. Ursolic acid suppresses phospho-JAK2 expression in a time-dependent manner. U266 cells (4 × 106/mL) were treated with 50 μmol/L ursolic acid for the indicated time intervals. Whole-cell extracts were prepared and 500 μg of sample were incubated with JAK2 antibody overnight. Immunocomplex was precipitated with protein A/G-agarose beads and then fractionated on 10% SDS-PAGE. Western blot analysis was done with anti–phospho-JAK2 antibody. The same samples were analyzed for JAK2 protein. D. U266 cells (2 × 106/mL) were treated with 50 μmol/L ursolic acid for the indicated time intervals, after which whole-cell extracts were prepared and 30-μg portions of those extracts were resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with phospho-ERK1/2 antibody. The same blots were stripped and reprobed with ERK1/2 antibody to verify equal protein loading. E. U266 cells (2 × 106/mL) were treated with indicated concentrations of ursolic acid, after which whole-cell extracts were prepared and 30-μg portions of those extracts were resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with phospho-Akt antibody. The same blots were stripped and reprobed with Akt antibody to verify equal protein loading. F. Pervanadate reverses the phospho-STAT3 inhibitory effect of ursolic acid. U266 cells (2 × 106/mL) were treated with the indicated concentration of pervanadate and 50 μmol/L ursolic acid for 4 h, after which whole-cell extracts were prepared and 30-μg portions of those extracts were resolved on 7.5% SDS-PAGE gel, electrotransferred onto nitrocellulose membranes, and probed for phospho-STAT3 and STAT3. G. Ursolic acid induces the expression of SHP-1 protein in U266 cells. U266 cells (2 × 106/mL) were treated with indicated concentrations of ursolic acid for 4 h, after which whole-cell extracts were prepared and 30-μg portions of those extracts were resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with SHP-1 antibody. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. H. Ursolic acid induces SHP-1 gene expression. U266 cells (4 × 106/mL) were treated with indicated concentrations of ursolic acid for 4 h, and total RNA was extracted and examined for expression of SHP-1 by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to show equal RNA loading. I. Effect of SHP-1 knockdown on ursolic acid–induced expression of SHP-1. U266 cells (2 × 106/mL) were transfected with either SHP-1 siRNA or scrambled siRNA (50 and 100 nmol/L). After 24 h, cells were treated with 50 μmol/L ursolic acid for 4 h and whole-cell extracts were subjected to Western blot analysis for SHP-1. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. J. Transfection with SHP-1 siRNA reverses ursolic acid–induced suppression of STAT3 activation. U266 cells (2 × 106/mL) were transfected with either SHP-1 siRNA or scrambled siRNA (50 and 100 nmol/L). After 24 h, cells were treated with 50 μmol/L ursolic acid for 4 h and whole-cell extracts were subjected to Western blot analysis for phosphorylated STAT3. The same blots were stripped and reprobed with STAT3 antibody.
- FIGURE 4.
A. Ursolic acid enhances the expression of proapoptotic Bax and Bak proteins. U266 cells (2 × 106/mL) were treated with 50 μmol/L ursolic acid for the indicated time intervals, after which whole-cell extracts were prepared and 30-μg portions of those extracts were resolved on 10% SDS-PAGE and probed against Bax and Bak antibodies. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. B. Ursolic acid suppresses STAT3 regulated antiapoptotic gene products. U266 cells (2 × 106/mL) were treated with 50 μmol/L ursolic acid for the indicated time intervals, after which whole-cell extracts were prepared and 30-μg portions of those extracts were resolved on 10% SDS-PAGE, membrane sliced according to molecular weight, and probed against cyclin D1, Bcl-2, Bcl-XL, survivin, and VEGF antibodies. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. C. Ursolic acid suppresses the expression of Mcl-1 protein. U266 cells (2 × 106/mL) were treated with 50 μmol/L ursolic acid for the indicated time intervals, after which whole-cell extracts were prepared and 30-μg portions of those extracts were resolved on 10% SDS-PAGE and probed against Mcl-1 antibodies. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. D. Ursolic acid inhibits gene expression. U266 cells (4 × 106/mL) were treated with ursolic acid (50 μmol/L) for the indicated time points, and total RNA was extracted and examined for expression of cyclin D1, Bcl-2, and Bcl-xL by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control to show equal RNA loading.
- FIGURE 5.
Ursolic acid suppresses proliferation, causes accumulation of cells in G0-G1 phase, does not affect IL-6Rα expression, and activates caspase-3. A1 to A3. U266, MM1.S, and RPMI 8826 cells were plated in triplicate, treated with 25 μmol/L ursolic acid, and then subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on day 2, 4, or 6 to analyze proliferation of cells. B. U266 cells (2 × 106/mL) were synchronized by incubation overnight in the absence of serum and then treated with 50 μmol/L ursolic acid for the indicated time points, after which the cells were washed, fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. C. Ursolic acid does not modulate cell-surface expression of IL-6Rα in U266 cells. Cells were harvested and labeled with mouse anti-human IL-6Rα FITC-conjugated antibody and analyzed by flow cytometry. D. U266 cells (1 × 106/mL) were treated with 50 μmol/L ursolic acid for the indicated time intervals at 37°C. Cells were incubated with anti–Annexin V antibody conjugated with FITC and then analyzed with a flow cytometer for early apoptotic effects. E. U266 cells were treated with 50 μmol/L ursolic acid for the indicated time points and whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blotting against caspase-3 antibody. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. F. U266 cells were treated with 50 μmol/L ursolic acid for the indicated time points and whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blot against PARP antibody. The same blot was stripped and reprobed with β-actin antibody to show equal protein loading. Representative of three independent experiments. G. Caspase inhibitor suppresses the ursolic acid–induced apoptosis of U266 cells. U266 cells (1 × 106/mL) were preincubated with 50 μmol/L ursolic acid and 10 and 50 μmol/L zVAD-FMK alone or in combination for 48 h at 37°C. Cells were incubated with anti–Annexin V antibody conjugated with FITC and then analyzed with a flow cytometer for early apoptotic effects.
- FIGURE 6.
A. Ursolic acid potentiates the apoptotic effect of thalidomide and bortezomib. U266 cells (1 × 106/mL) were treated with 25 μmol/L ursolic acid and 10 μg/mL thalidomide or 20 nmol/L bortezomib alone or in combination for 24 h at 37°C. Cells were stained with a live/dead assay reagent for 30 min and then analyzed under a fluorescence microscope as described in Materials and Methods. The results shown are percent apoptosis and are representative of three independent experiments. B. Quantitative analysis of live/dead assay done in (A). Bars, SD between the triplicates.
Volume 5, Issue 9
