Figure 1.
Effect of andrographolide (Andro) and taxifolin (Taxi) on the growth of DU145 cells.
The cells were treated with a range of concentrations of (A) Andro and (B) Taxi for the indicated period of time and cell proliferation was monitored by MTT assay. The data from three independent experiments were expressed as percentage of viability compared to the vehicle control (DMSO). Values are mean ± s.d.
Figure 2.
Change in the cell cycle distribution of DU145 cells treated with andrographolide (Andro).
(A) Cells were exposed to 0–40 µM of Andro for 48 h; (B) cells were exposed to 40 µM of Andro for 0–72 h. Cellular DNA was stained with propidium iodide (PI) and analyzed by flow cytometry. The percentage of cells in each specific phase of the cell cycle was calculated with the ModFit program and expressed as the mean ± s.d. (n = 3 or 4). (C&D) Mitotic index of DU145 cells treated with Andro. Cells were treated with 0, 20, and 40 µM of Andro for 24 h and were fixed and stained with anti-phospho-histone H3 (Ser10) antibody, FITC-conjugated secondary antibody and PI, and analyzed by flow cytometry. (C) A representative density plot of the cells labeled with phosphohistone H3 against the DNA content in the cells. Mitotic index (M) of the cell population is indicated in the boxed area. (D) Mitotic index and percentage of G2/M cells after treatment with two different concentrations of Andro expressed as mean ± s.d. (n = 4). * indicates significantly different from the control (p<0.01).
Figure 3.
Apoptosis of DU145 cells treated with andrographolide (Andro).
(A&B) After treating for 48 h with 0–80 µM of Andro, the cells were collected and doubly stained with Annexin V-FITC antibody and PI before analyzed by flow cytometry. (A) One set of representative dot plots indicates the percentage of early apoptotic cells (Annexin V-FITC+/PI−, lower right hand panel) and the percentage of late apoptotic and dead cells (Annexin V-FITC+/PI+, upper right panel). (B) The plot of the percentage of early apoptotic and of late apoptotic and dead cells under different Andro concentrations expressed as mean ± s.d. (n = 4). (C) The cells were exposed to 40 µM of Andro for 0–72 h. The plot of the percent of early apoptotic and of late apoptotic and dead cells against the time of treatment expressed as mean ± s.d. (n = 4).
Figure 4.
Effect of andrographolide (Andro) on the expression of cell cycle regulators in DU145 cells.
(A) The cells were incubated with 0–80 µM of Andro for 24 or 48 h. 40 µg of the cell lysate was resolved by electrophoresis in a 12% polyacrylamide gel, transferred to nitrocellulose membrane, and immunoblotted against the indicated cell-cycle regulator antibodies. Data are the quantification of the western blots of three independent experiments. (B) The change in cyclin B1 level compared with the control (0 µM) from the quantification of the western blots was plotted against the concentration of Andro at 24 and 48 h. (C) The change in the levels of phosphorylated Cdc25c was plotted against the concentration of Andro. (D) The change in the levels of p21 was plotted against the concentration of Andro. Standard deviations of three independent experiments are shown.
Figure 5.
Time course of Andro treatment on caspases and Bcl-2 family proteins expressed in DU145 cells.
The cells were incubated with 0–80 µM of Andro for 24 or 48 h, harvested and lysed. 40 µg of the cell lysate was separated in 12% polyacrylamide gel, transferred to a nitrocellulose membrane, and immunoblotted with antibodies against (A) caspase-7, -9, and -3 and substrate PARP; and (B) Bcl-2 family proteins, including Bcl-XL, Bcl-2, and Bax. Data are representative of three independent experiments.
Figure 6.
Taxifolin (Taxi) enhances the growth inhibitory effect of Andro on DU145 cells.
(A) The cells were treated with 0–40 µM of Andro and 0, 100 or 200 µM of Taxi for 48 h, and cell proliferation was determined by MTT assay. Data from four independent experiments were expressed as percentage of viability compared to the vehicle control (DMSO) with standard deviation (mean ± s.d., n = 4). * indicates significantly difference from the control (p<0.01). (B) The cells were treated with 20 µM of Andro for 48 h in the presence or absence of 100 µM of Taxi, and observed under differential interference contrast microscopy. Bar = 50 µm. (C) A comparison of the viability by MTT assay of DU145, PNT2, and Hs-27 cells after the treatment with 20 µM of Andro and 100 µM of Taxi. The experiments were performed in triplicate and the standard deviations are indicated.
Figure 7.
Taxi enhances Andro-induced cell cycle arrest in DU145 cells.
The cells were treated with (A) 0–40 µM of Andro for 48 h (B) 20 µM of Andro for 0–48 h in the presence or absence of 100 µM of Taxi. Cellular DNA was stained with PI and analyzed by flow cytometry. The distribution of the cell cycle was calculated with the ModFit program. Mean ± s.d. (n = 4) of percentage of G2/M cell in the presence of Taxi (100 µM) and in the absence of Taxi (Taxi−) were compared. * indicates significant difference between two groups, *p<0.01, **p<0.05. (C) The cells were treated with 20 µM of Andro for 24 h in the presence or absence of 100 µM of Taxi. The treated cells were fixed and doubly stained with anti-phosphohistone H3 (Ser10) antibody and PI, and analyzed with flow cytometry. Mitotic index expressed as mean ± s.d. (n = 3 or 4) in the Taxi− and Taxi 100 µM groups were compared, *p<0.01. (D) Lysates from 40 µg of the cells that had been treated with the indicated Andro and Taxi for 24 h, and then was immunoblotted against antibodies of phosphorylated Cdc25c, cyclin B1, p21, and actin as control. Data are representative of three independent experiments. (E–G) Quantification of the signals from the immunoblots of the cyclin B (E), p-Cdc25c (F), and p21 (G) were plotted under different indicated conditions. The values in the plots are the mean ± s.d. of three independent experiments.
Figure 8.
Taxi enhances Andro-induced apoptosis in DU145 cells.
The cells were treated with (A&B) 0–40 µM of Andro for 48 h, and (C) 20 µM of Andro for 24 or 48 h in the presence or absence of 100 µM of Taxi. (A) The cells were double stained with Annexin V-FITC antibody and PI and then analyzed with flow cytometry. Mean ± s.d. (n = 4) of percent of early apoptotic cell in the presence of Taxi (100 µM) and in the absence of Taxi (Taxi−) were compared. * indicates a significant difference between the two groups, *p<0.01. (B&C) Western blot analysis of caspase-7, caspase-9, and PARP in DU145 cells. 40 µg of the cell lysates were immunoblotted with antibodies against the caspases and PARP. Data are representatives of three independent experiments.
Figure 9.
Immunofluorescence microscopic images of treating DU145 cells with Andro and Taxi on the spindle-chromosome configuration.
The cells were treated with 20 µM of Andro in the presence and absence of 100 µM of Taxi for 24 h. The treated cells were fixed and immunostained with anti-tubulin antibody and FITC conjugated secondary antibody to visualize microtubules (green) and Hoechst 33342 to visualize chromosomes (blue). Representative images of spindle organization in the cells treated with vehicle control (hollow arrow), Andro alone (solid arrows) and combined Andro and Taxi (dashed arrows) are shown. Bar = 10 µm.
Figure 10.
The combined effect of Andro and Taxi on enhancing tubulin assembly in vitro.
Tubulin was incubated at 37°C in the reaction buffer in the presence of (A) 1% DMSO (vehicle control), Andro (20, 40 or 200 µM) or 10 µM of Taxol (positive control); (B) 1% DMSO; 100 µM Taxi; 40 µM Andro; 40 µM Andro+100 µM Taxi; or 10 µM Taxol. Tubulin polymerization was monitored by measuring the optical density at 340 nm for 60 min. Data are representative of three independent experiments. (C and D) The rate of increase in OD, Vmax, of the indicated conditions was plotted. The data represent the mean ± s.d. from three independent experiments.
Figure 11.
The effect of MAD2 knockdown on the mitotic index of DU145 cells treated with Andro and/or Taxi.
Cells were either treated with Andro and/or Taxi for 24 h or transfected with the negative control siRNA or MAD2 specific siRNA for 24 h and later treated with Andro and/or Taxi for additional 24 h. Then cells were fixed and stained with anti-phospho-histone H3 (Ser10) antibody, FITC-conjugated secondary antibody and PI, and analyzed by flow cytometry. Mitotic index is expressed as mean ± s.d. * indicates significant difference between two groups, *p<0.01.