Article Figures & Data
Figures
- Figure 1.
Experimental design to study the effect of silymarin and silibinin on OH-BBN–induced bladder carcinogenesis in male ICR mice. Chemical structure of OH-BBN (A) and chemical structure of silibinin (B). C, 5-wk-old male ICR mice were randomly divided into six groups. Bladder cancer was induced in animals of groups 2 to 4 (30 mice in each group) by administration of OH-BBN (0.05%, w/v) in the drinking water for 6 wk. The drinking water containing carcinogen was changed twice a week. Mice in groups 3 and 4 were also gavaged with silymarin or silibinin (200 mg/kg body weight) in sterile saline, 5 d/wk for a period of 51 wk, starting 1 wk before OH-BBN exposure. Mice in groups 1, 5, and 6 (10 mice in each group) were gavaged with sterile saline or silymarin/silibinin (200 mg/kg body weight) in sterile saline for the same time period. D, in the experiment detailed in C, body weight of each mouse was recorded weekly throughout the experiment. Points, mean body weight per mouse of all the mice from each group, plotted as a function of time (weeks) for each group; bars, SE. SY, silymarin; SB, silibinin.
- Figure 2.
Histopathology of the urothelium of mice in OH-BBN–induced bladder carcinogenesis. At the end of the study detailed in Fig. 1C, urinary bladders were processed for H&E staining, and a representative picture is shown for each group. Normal urothelial mucosa, characterized by epithelium of <3 layers without any anaplasia, is shown in control, silymarin, and silibinin groups (×400). OH-BBN–treated mouse urothelium showing localized cellular proliferation, with invasive carcinoma infiltrating the submucosa or muscle layer with undifferentiated features (×100; subsets, ×400). Silymarin + OH-BBN and silibinin + OH-BBN groups show epithelium of ≥3 layers with diffused mucosal dysplasia.
- Figure 3.
Silymarin and silibinin feeding inhibits the neoplastic progression of OH-BBN–induced bladder carcinogenesis in male ICR mice. Urothelial tissue samples obtained from the experimental groups as detailed in Fig. 1C were randomly analyzed in a double-blinded manner to evaluate the histopathology of urothelium. Urothelium was classified as normal urothelial mucosa, characterized by epithelium of <3 layers without any anaplasia; mucosal dysplasia, characterized by epithelium of ≥3 layers with moderate to severe anaplasia with diffused proliferation; papillary/nodular (PN) dysplasia, characterized by moderate or severe anaplastic epithelial lesion of localized cellular proliferation resulting in nodular or papillary forms; urothelial carcinoma, characterized by invasive carcinoma infiltrating the submucosa or muscle layer with transitional-cell carcinoma or undifferentiated features. χ2 analysis and Fisher exact test were used to compare the incidence of dysplasia, papillary/nodular dysplasia, and carcinoma in OH-BBN and silymarin + OH-BBN or silibinin + OH-BBN groups; P < 0.05 was considered significant. *, P < 0.001; $, P < 0.01, versus OH-BBN group.
- Figure 4.
Effect of silymarin and silibinin feeding on OH-BBN–induced urothelial cell proliferation and mitogenic signaling. Immunohistochemical and immunoblot analyses of urothelial tissue samples obtained from the experiment detailed in Fig. 1C were done to study the molecular biomarkers and events associated with proliferation. A, immunohistochemical staining for PCNA (magnification, ×400) in urothelium was done as detailed in Materials and Methods. Arrows, PCNA-positive cells. B, the proliferating cells were quantified by counting PCNA-positive cells over total cells in five randomly selected fields at ×400 magnification from 10 different samples in each group. The proliferation index was determined as (number of positively stained cells × 100) / total number of cells counted. Columns, mean proliferation index in each group; bars, SE. C, bladder tissue samples were randomly taken from each group and analyzed for PCNA, cyclin D1, and phosphorylated and total ERK1/2 protein levels by immunoblotting. Membrane was stripped and reprobed with β-actin as loading control.
- Figure 5.
Effect of silymarin and silibinin on apoptosis and associated molecular events in OH-BBN–induced urothelium. Immunohistochemical and immunoblot analyses of urothelial tissue samples obtained from the experiment detailed in Fig. 1C were done to study the molecular biomarkers and events associated with apoptosis. A, apoptosis was analyzed by TUNEL staining in urothelium as detailed in Materials and Methods. The apoptotic index was determined as (number of TUNEL-positive cells × 100) / total number of cells counted. Columns, mean in each group; bars, SE. B, bladder tissue samples were randomly taken from each group and analyzed for cleaved caspase-3, cleaved poly(ADP-ribose) polymerase (PARP), and survivin protein levels by immunoblotting. Each membrane was stripped and reprobed with β-actin as loading control. C, immunohistochemical staining for survivin in urothelium was done as detailed in Materials and Methods. The survivin-positive cells were determined as [number of positively stained (brown) cells × 100] / total number of cells counted. Columns, mean in each group; bars, SE. D, nuclear extract was prepared from bladder tissue samples randomly taken from each group and analyzed for phosphorylated and total NF-κB p65 protein levels by immunoblotting. Membrane was stripped and reprobed with histone H1 as loading control.
Volume 6, Issue 12
