Figure 1.
Chemical structure of betulinic acid (BA).
Molecular weight = 456.
Figure 2.
Effect of betulinic acid (BA) on hypoxia-induced HIF-1α activation in PC-3 cells.
(A) PC-3 cells were treated with various concentrations of BA (0, 12.5, 25, 50 or 100 µM) for 24 h. Cell viability was analyzed by MTT assay. (B) Cells were exposed to normoxia or hypoxia for 0.5, 2, 4, 6, 8, 12 or 24 h. Cell lysates were prepared and subjected to Western blotting to determine the expression of HIF-1α. (C) Cells were treated with or without BA (5 or 10 µM) under normoxic or hypoxic condition for 4 h. Cell lysates were prepared and subjected to Western blotting to determine the expression of HIF-1α. (D) Nuclear extract was prepared from the cells treated with BA (0, 10, 20 or 40 µM) under normoxia or hypoxia for 4 h. HIF-1α transcription activity was measured by using TransAM HIF-1 transcription factor assay kit. Data represent means ± S.D. ##, p<0.01 vs normoxia control, and *, p<0.05 and ** < 0.01 vs hypoxia control.
Figure 3.
Effect of betulinic acid (BA) on hypoxia-induced STAT3 activation in PC-3 cells.
PC-3 cells were treated with or without BA (5 or 10 µM) under normoxic or hypoxic condition for 4 h. (A) Cell lysates were prepared and subjected to Western blotting for phospho-STAT3 and STAT3. (B) Nuclear extracts were prepared and applied to EMSA to analyze the STAT3-DNA binding activity. (C) Cells were treated with or without BA (10 µM) under hypoxia. Immunocytochemistry was performed for STAT3. DAB (brown) and hematoxylin-eosin was used as a substrate and a counterstaining, respectively.
Figure 4.
Effect of betulinic acid (BA) on hypoxia-induced angiogenesis.
(A and B) PC-3 cells were treated with 0, 5 or 10 µM BA for 24 h. (A) VEGF levels in the culture supernatants were measured by using a Quantikine VEGF ELISA kit. (B) Cell lysates were prepared and subjected to Western blotting to determine VEGF expression. Graphs represent relative band intensities of VEGF/β-actin. Data represent means ± S.D. ##, p<0.01 vs normoxia control, and *, p<0.05 and ** <0.01 vs hypoxia control. (C) HUVECs were treated with VEGF (20 ng/ml) as positive control or the culture supernatant from PC-3 cells treated with or without BA (10 µM) under normoxia or hypoxia. Tube formation assay was performed using growth factor reduced Matrigel. Cells were fixed with Diff-Quick solution, photographed randomly under an Axiovert S 100 light microscope at ×100 magnification and counted.
Figure 5.
Effect of betulinic acid (BA) on STAT3 binding on the VEGF promoter in hypoxic PC-3 cells.
(A) PC-3 cells were treated with or without BA (10 µM) under normoxia or hypoxia for 4 h. The immunoprecipitated DNA with rabbit normal IgG, HIF-1α or STAT3 antibody was amplified by PCR analysis for VEGF promoter. (B) Cells were transiently transfected with siRNA for scramble or STAT3 for 24 h and treated with or without BA (10 µM) for 18 h under hypoxia. VEGF levels in the culture supernatants were measured by using a Quantikine VEGF ELISA kit. Data represent means ± S.D. #, p<0.05 vs control, and *, p<0.05 vs control siRNA. Cell lysates were subjected to Western blotting for phospho-STAT3, STAT3 and HIF-1α.