Cortex Moutan Induces Bladder Cancer Cell Death via Apoptosis and Retards Tumor Growth in Mouse Bladders

Abstract

Cortex Moutan is the root bark of Paeonia suffruticosa Andr. It is the herbal medicine widely used in Traditional Chinese Medicine for the treatment of blood-heat and blood-stasis syndrome. Furthermore, it has been reported that Cortex Moutan has anticancer effect. In this study, the Cortex Moutan extract was evaluated in bladder cancer therapy in vitro and in vivo. Cortex Moutan extract reduces cell viability with IC50 between 1~2 mg/ml in bladder cancer cells, and it has lower cytotoxicity in normal urotheliums. It arrests cells in G1 and S phase and causes phosphatidylserine expression in the outside of cell membrane. It induces caspase-8 and caspase-3 activation and poly(ADP-ribose) polymerase degradation. The pan caspase inhibitor z-VAD-fmk reverses Cortex Moutan-induced cell death. Cortex Moutan also inhibits cell invasion activity in 5637 cells. In mouse orthotopic bladder cancer model, intravesical application of Cortex Moutan decreases the bladder tumor size without altering the blood biochemical parameters. In summary, these results demonstrate the antiproliferation and anti-invasion properties of Cortex Moutan in bladder cancer cells and its antibladder tumor effect in vivo. Cortex Moutan may provide an alternative therapeutic strategy for the intravesical therapy of superficial bladder cancer.

Figure 1

The cytotoxicity of CM extract in bladder cancer cells and normal urotheliums. The cytotoxicity was analyzed after CM treatment for 24 h and 48 h. The cell number of control was regarded as 100%. Data represent the mean ± SEM of quadruplicate. The experiment was repeated for three times.

Figure 2

CM extract induces cell apoptosis. (a) Cell cycle distribution after CM treatment. Cells were treated with medium or CM extract (0, 0.5, 1, 2, 3, 3.5 mg/mL) for 24 h and 48 h then collected for cell cycle analysis. Data represent the mean ± SEM of triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control. (b) Apoptosis analysis by Annexin-V-PI staining assay. The apoptosis percentage implied the Annexin-V-positive and PI-negative staining cells. Data represent the mean ± SEM of triplicate. ***P < 0.001 compared with control. (c) Western blot analysis of apoptotic proteins including PARP (original 116 kDa and degraded 89 kDa forms), original caspase-8, activated caspcas-3, and GAPDH (internal control). (d) z-VAD-fmk reverses CM-induced cell death. z-VAD-fmk was added in medium 1 h before CM treatment, and cell number was counted after CM treatment for 24 h. Data represent the mean ± SEM of quadruplicate. *P < 0.05, and **P < 0.01 compared with each other. All the experiments were repeated for three times.

Figure 3

CM extract inhibits cell invasion. After medium or CM treatment for 24 h, migrated 5637 cells were counted. The cell number of control was regarded as 100%. Data represent the mean ± SEM of triplicate. ***P < 0.001 compared with control. The experiment was repeated for three times.

Figure 4

The antitumor effect of CM extract in a mouse orthotopic bladder tumor model. (a) Mouse body weight and drug schedule. MB49 cells were implanted at Day 1. Mouse body weight was recorded weekly or every operation. (b) Intravesical application of CM extract retards mouse bladder tumor growth and muscle invasion. After tumor implantation, CM extract was applied intravesically for 5 times. After sacrifice, each bladder volume (mm³) was calculated by (length × wide²)/2. Data represent the mean ± SEM. **P < 0.01 compared with control. In the HE stain slide photos, M: muscle, U: urothelium, double-head arrow: muscle layer, and single arrow in control: the tumor invading into muscle layer.