Figure 1.
Osthole Inhibited Cell Proliferation of HCC Cell Lines.
(A) Chemical Structure of osthole (B) Viability of SK-HP-1, SMMC-7721, HepG-2 and Hepa1-6 cells treated with osthole. MTT assay was performed to measure cell growth inhibition rate at 48 h after osthole treatment. (C)(D) Viability of SMMC-7721 and Hepa1-6 cells treated with osthole. MTT assay was performed to measure cell growth inhibition rate at 24 h, 48 h and 72 h after osthole treatment. Data shown were representatives of three experiments.
Figure 2.
Effects of Osthole on Cell Cycle of HCC Cells.
Cell cycle analysis of SMMC-7721 and Hepa1-6 cells following 123.0 µM osthole treatment for 24 h by flow cytometry.
Figure 3.
Effects of Osthole on apoptosis of HCC cells.
(A) Induction of apoptosis of SMMC-7721 and Hepa1-6 cells after osthole treatment. SMMC-7721 and Hepa1-6 cells were treated with osthole at doses of 0, 41.0, 84.0, 123.0, 164.0 and 205.0 µM for 48 h. Apoptosis was measured by flow cytometry. (B) Statistical analysis of the percentages of the apoptotic cells. Data shown were representatives of three experiments.
Figure 4.
Effect of Osthole Treatment on the Tumorigenicity of HCC Cells.
A total of 2×106 SMMC-7721 or Hepa1-6 cells were inoculated subcutaneously (s.c.) into the right flank of nude mice or C57/BL6 mice. Mice were randomized into five groups including osthole treatment (244 mg/kg, 122 mg/kg and 61 mg/kg), corn oil alone as the blank control and cisplatin (5 mg/kg) as the positive control on Day8 and were treated once every other day for 2 weeks. (A) The tumor volumes of the nude mice inoculated with SMMC-7721 cells were measured and calculated once every three days. The tumor sizes on day 21 were shown in the inserted figure. (B) Tumor weights of the nude mice inoculated with SMMC-7721 cells were measured on day21. (C) The tumor volumes of the C57/BL6 mice inoculated with Hepa1-6 cells were measured and calculated once every three days. (D) Tumor weights of the C57/BL6 mice inoculated with Hepa1-6 cells were measured on day21. (E) The body weights of nude mice inoculated with SMMC-7721 cells were weighed on day 21. The (F) spleen weights and (G) thymus weights of the C57/BL6 mice inoculated with Hepa1-6 cells were weighed on day21. Each data point represented the mean±S.D. of 10 mice. Data shown were the representatives of three experiments.
Figure 5.
Effects of Osthole Treatment on Caspase-3 expression and NF-κB activation.
(A) SMMC-7721 cells were treated with osthole (123.0 µM) for 12 h, 24 h and 48 h. The nuclear proteins were prepared and analyzed for NF-κB expression by EMSA. (B) SMMC-7721 cells were treated with osthole at doses of 0, 41.0, 82.0, 123.0, 164.0 and 205.0 µM for 24 h. The nuclear proteins were prepared and analyzed for NF-κB expression by EMSA. (C) SMMC-7721 cells were treated with osthole at doses of 0, 41.0, 82.0, 123.0, 164.0 and 205.0 µM for 48 h. The cell lysates were prepared and analyzed for caspase-3 expression by Western blot analysis. Equal loading was confirmed by stripping immunoblots and reprobing for β-actin. Data shown were representatives of three experiments. (D) Statistical analysis of caspase-3 quantification. * p<0.05, ** p<0.01.
Figure 6.
Effects of Osthole on Expressions of Apoptosis-Related Genes.
SMMC-7721 cells were treated with osthole (123.0 µM) for 48 h. The expressions of apoptosis-related genes were analyzed using RT2 Profiler PCR Arrays. Increased expressions of two folds or more were shown. Data shown were representatives of two experiments.