The investigation of a traditional Chinese medicine, Guizhi Fuling Wan (GFW) as an intravesical therapeutic agent for urothelial carcinoma of the bladder

Abstract

Background: The high risk of recurrence faced by patients with bladder cancer has necessitated the administration of supplemental intravesical chemotherapy; however, such treatments often result in severe side effects. As a result, novel intravesical agents with enhanced efficacy and minimal toxicity are urgently required for the treatment of bladder cancer.

Methods: Guizhi Fuling Wan (GFW) is a traditional Chinese medicine shown to inhibit the growth of hepatocellular carcinoma. This study evaluated the growth inhibition of GFW using normal human urothelial cells and bladder cancer cells; the efficacy of GFW treatment was further compared with mitomycin C, epirubicin, and cisplatin. We also examined the progression of cell cycle and apoptosis in bladder cancer cells in response to GFW treatment. CCK-8 was employed to analyze cell viability and flow cytometry was used to study the cell cycle and apoptosis. The mechanisms underlying GFW-induced cell cycle arrest were determined by Western blot analysis.

Results: Our data demonstrate the potent inhibitory effect of GFW in the proliferation of bladder cancer cell lines, BFTC 905 and TSGH 8301. GFW presented relatively high selectivity with regard to cancer cells and minimal toxicity to normal urothelial cells. Our results also demonstrate that GFW interferes with cell cycle progression through the activation of CHK2 and P21 and induces apoptosis in these bladder cancer cells.

Conclusions: Our results provide experimental evidence to support GFW as a strong candidate for intravesicle chemotherapy against bladder cancer.

Figure 1

HPLC analysis: (A) HPLC chromatograms of (1) amygdalin, (2) paeoniflorin, (3) cinnamaldehyde, and (4) paeonol detected at (a) 215 nm, (b) 232 nm, (c) 280 nm, (d) 275 nm; (B) HPLC chromatograms of (1) amygdalin, (2) paeoniflorin, (3) cinnamaldehyde, and (4) paeonol in GFW detected at (a) 215 nm, (b) 232 nm, (c) 280 nm, (d) 275 nm.

Figure 2

Cytotoxicity of GFW and Cinnamomi Ramulus against normal human urothelial cell line, HUC 4449: Cells were initially seeded in 96-well plates at 1 × 10⁴cells per well and cultured for 24 h. The cells were subsequently starved in medium supplemented without FBS for 24 h, and then treated with various concentrations of agents for 24 h. Cell viability was detected using the Cell Counting Kit-8 (CCK-8). Data are presented as mean ± SEM (n=6). Significant differences from the no treatment control is indicated by * * (p<0.01), as determined by one-way ANOVA and Dunnett’s comparison test.

Figure 3

Cytotoxicity of GFW, cisplatin, Epirubicin and mitomycin against normal human urothelial cell line, HUC 4449 and bladder cancer cell lines, TSGH 8301 and BFTC 905: Cells were initially seeded in 96-well plates at 1 × 10⁴cells per well and cultured for 24 h. The cells were subsequently starved in medium supplemented without FBS for 24 h and then treated with various concentrations of agents for 24 h. Cell viability was detected using the Cell Counting Kit-8 (CCK-8). Data are presented as mean ±SEM (n=6). Significant differences from the no treatment control is indicated by * * (p<0.01), as determined by one-way ANOVA and Dunnett’s comparison test.

Figure 4

Effects of GFW on cell cycle of bladder cancer cells: (A) BFTC 905 and (B) TSGH 8301 cells. Cells were cultured in 6-well plates at 2 × 10⁵ cells per well for 24 h, followed by starvation in medium without FBS for 24 h. The cells were then treated with 0, 0.5, or 1 mg/ml of GFW for 24 h. Cells were harvested and stained using propidium iodide staining solution for 30 min in the dark and analyzed by flow cytometry. Data are presented as the mean ± SEM (n = 4). Significant differences from the no treatment control are indicated by * * (p<0.01), as determined by one-way ANOVA and Dunnett’s comparison test. (C) Western blots of phosphorylated CHK2, CHK2, and P21. The expression levels of phosphorylated CHK2 increased in both BFTC 905 and TSGH 8301 cells following treatment with GFW for 2 h. A substantial increase in p21 was observed in BFTC 905 cells following treatment with GFW for 4–6 h. A high basal level of P21 prior to treatment and a mild, gradual increase in p21 level following treatment with GFW for 6 h was observed in TSGH 8301 cells.

Figure 5

Effects of GFW on apoptosis of bladder cancer cells: (A) BFTC 905 and (B) TSGH 8301 cells were treated with GFW at concentrations of 0.5 and 1 mg/ml for 24 h. Cells were then analyzed by FACS to determine the relative% of apoptotic Annexin V/PI cells. The lower left quadrant (Q3) represents viable cells; the upper left quadrant (Q1) represents necrotic cells; the lower right quadrant (Q4) represents early apoptotic cells; and the upper right quadran (Q2) represents nonviable late apoptotic cells. The relative percentages of apoptotic cells including early and late apoptotic cells were determined. Data are represented as mean ± SEM of two independent experiments performed in triplicate. Significant differences from the no treatment control are indicated by * * (p<0.01), as determined by one-way ANOVA and Dunnett’s comparison test.