Anti-tumor effect of Radix Paeoniae Rubra extract on mice bladder tumors using intravesical therapy

Abstract

Radix Paeoniae Rubra (RPR) is the dried root of Paeonia lactiflora Pallas and Paeonia veitchii Lynch, and is a herbal medicine that is widely used in traditional Chinese medicine for the treatment of blood-heat and blood-stasis syndrome, similarly to Cortex Moutan. The present study identified the same three components in RPR and Cortex Moutan extracts. In addition, it has been reported that RPR has an anti-cancer effect. Bladder cancer is the seventh most common type of cancer worldwide. Due to the high recurrence rate, identifying novel drugs for bladder cancer therapy is essential. In the present study, RPR extract was evaluated as a bladder cancer therapy in vitro and in vivo. The present results revealed that RPR extract reduced the cell viability of bladder cancer cells with a half maximal inhibitory concentration of 1-3 mg/ml, and had an extremely low cytotoxic effect on normal urothelial cells. Additionally, RPR decreased certain cell cycle populations, predominantly cells in the G1 phase, and caused a clear sub-G increase. In a mouse orthotopic bladder tumor model, intravesical application of RPR extract decreased the bladder tumor size without altering the blood biochemical parameters of the mice. In summary, the present results demonstrate the anti-proliferative properties of RPR extract on bladder cancer cells, and its anti-bladder tumor effect in vivo. Compared to Cortex Moutan extract, RPR extract may provide a more effective alternative therapeutic strategy for the intravesical therapy of superficial bladder cancer.

Keywords: Radix Paeoniae Rubra; bladder cancer; gallic acid; intravesical therapy; methyl gallate; mouse orthotopic bladder tumor model; paeoniflorin.

Figure 1.

High-performance liquid chromatography analysis of RPR and Cortex Moutan extracts. RPR and Cortex Moutan extracts were prepared using RPMI-1640 medium. The solvents for the standards are (A) ethanol (gallic acid and methyl gallate), methanol (paeoniflorin) and (B) RPMI-1640 medium. Peak 1, gallic acid; Peak 2, methyl gallate; Peak 3, paeoniflorin. Arrows indicate the peaks of gallic acid, methyl gallate and paeoniflorin in the two extracts. RPR, Radix Paeoniae Rubra.

Figure 2.

Cytotoxicity of RPR extract in bladder cancer and normal urothelial cells. The cytotoxicity was analyzed subsequent to RPR treatment for 24 and 48 h. (A) Cytotoxicity of RPR extract in human bladder papillary transitional cell carcinoma BFTC 905 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (B-D) Cytotoxicity of RPR extract in (B) BFTC 905, (C) mice bladder carcinoma MB49 and (D) human SV40-transformed uroepithelium SV-HUC-1 cells using direct cell counting. Control cells were treated with the same volume of medium but without RPR. The cell number for the control was regarded as 100%. Data are represented as the mean ± standard error of the mean. The experiment was repeated three times. RPR, Radix Paeoniae Rubra; SV, simian virus.

Figure 3.

RPR extract induces cell cycle alterations. Cell cycle distribution was analyzed following RPR treatment. (A) Human bladder papillary transitional cell carcinoma BFTC 905 and (B) mice bladder carcinoma MB49 cells were treated with RPMI-1640 medium alone (0 mg/ml RPR) or RPR extract dissolved in RPMI-1640 medium (0.5, 1, 2, 3 and 3.5 mg/ml) for 24 h and 48 h, and subsequently collected for cell cycle analysis. Data are represented as the mean ± standard error of the mean. The experiment was repeated three times. *P<0.05; **P<0.01; ***P<0.001. RPR, Radix Paeoniae Rubra.

Figure 4.

Anti-tumor effect of RPR extract in a mouse orthotopic bladder tumor model. (A) Mouse body weight and drug schedule. MB49 cells stably transformed with the luciferase gene were implanted in the mice on day 1. Following tumor implantation, RPR extract was applied intravesically nine times. Mouse body weights were recorded prior to every intravesical application. (B) Following euthanization of the mice, the bladders were collected. Intravesical application of RPR extract decreased the bladder tumor growth of the RPR-treated mice. Bladder volume (mm³) = (length × wide²) / 2. Data are represented as the mean ± standard error of the mean. ***P=0.001.