Oleanolic acid and ursolic acid inhibit proliferation in transformed rat hepatic oval cells

Figure 1 H₂O₂ promoted WB-F344 cell proliferation. A: The effect of H₂O₂ on cell proliferation. WB-F344 cells were exposed to 7 × 10^-4-7 × 10^-9 mol/L H₂O₂ for 6, 9, 12, 15, and 18 h. Cell proliferation was evaluated using the MTT assay. The data represent the mean ± SD derived from three independent experiments; B: Flow cytometric analyses of the cell cycle in H₂O₂-treated WB-F344 cells. The cells in the control and the H₂O₂ groups were stained with 10 μg/mL propidium iodide, and the DNA content was analyzed as described in the Materials and Methods; C: Histograms indicating the percentages of WB-F344 cells in the control and H₂O₂ exposure groups in the G₁, G₂/M and S phases. The data represent the mean ± SD derived from three independent experiments. ^aP < 0.05 vs control group.

Figure 2 H₂O₂ induced WB-F344 malignant transformation. A: The morphology of H₂O₂-treated WB-F344 cells. The cells were cultured in complete medium (control) or treated with 7 × 10^-7 mol/L H₂O₂ 12 h per day for 21 d. Cell morphology was observed under microscope (× 400). An increasing nucleus to cytoplasm ratio was observed (green arrow), as were many mitotic cells (yellow arrow), prokaryotes (blue arrow) and even tumor giant cells (red arrow); B: The tumorigenicity of transformed WB-F344 cells as determined using the methylcellulose culture assay. Cell colony formation was observed under microscope (× 400); C: H₂O₂ increased WB-F344 cell aneuploidy. The cells were cultured in complete medium (control) or treated with 7 × 10^-7 mol/L H₂O₂ 12 h per day for 21 d and harvested for flow cytometry. DNA was stained with propidium iodide (10 μg/mL), and a BD FACS Calibur was used to analyze cellular DNA content. The population of > 4N cells represent aneuploidy cells. Histograms indicating the aneuploidy cells among the control cells and the H₂O₂-treated cells. The data represent the mean ± SD derived from three independent experiments.

Figure 3 Effect of oleanolic acid and ursolic acid on cell proliferation. A, B: The inhibitory effect of oleanolic acid (OA) and ursolic acid (UA) on H₂O₂-induced cell proliferation. Cell proliferation was evaluated using the MTT assay (see Materials and Methods). The data represent the mean ± SD derived from three independent experiments; C: The effects of OA and UA on quiescent BRL rat liver epithelial cell proliferation. BRL cells were exposed to 4 μmol/L OA or 8 μmol/L UA for 72 h. Cell proliferation was evaluated using the MTT assay. The data represent the means ± SD derived from three independent experiments; D: The effects of OA and UA on quiescent WB-344 cells proliferation. WB-344 cells were exposed to 4 μmol/L OA or 8 μmol/L UA for 72 h. Cell proliferation was evaluated using the MTT assay. The data represent the mean ± SD derived from three independent experiments.

Figure 4 Growth inhibitory effects of oleanolic acid and ursolic acid on H₂O₂-treated WB-F344 cells. A: Flow cytometric analyses of the cell cycle of H₂O₂-treated WB-F344 cells following treatment with oleanolic acid (OA) (4 μmol/L), ursolic acid (UA) (8 μmol/L) or 5-FU (0.05 mmol/L, positive control). After 72 h, the cells were harvested and stained with 10 μg/mL propidium iodide (PI), and the DNA content was analyzed, as described in the Materials and Methods; B: Cell cycle quantitative analysis. Histograms indicating the fraction of H₂O₂-treated WB-F344 cells and those that were treated with OA, UA or 5-FU in the G₁, G₂/M and S phases. The data represent the means ± SD derived from three independent experiments. ^aP < 0.05 vs H₂O₂ model group.