Oridonin Induces Apoptosis And Cell Cycle Arrest Of Gallbladder Cancer Cells And Its Mechanism

Background and objective: Gallbladder cancer, the most frequent malignancy of the bile duct, is a highly aggressive, lethal neoplasm that is associated with high mortality and extremely poor prognosis. Due to the lack of specific clinical symptoms and signs, most cases were detected at an advanced stage and lost the chance of operation. Because of the high post-operative recurrence and metastasis, the prognosis is poor. The tumor is not sensitive to the current conventional chemotherapy. Therefore, it’s an urgent need to explore a new treatment for gallbladder cancer. Oridonin, an active diterpenoid compound purified from the Chinese herb Rabdosia rubescens, has been reported to have various pharmacological and physiological effects, such as anti-inflammatory, anti DNA mutation and antioxidant. Studies have shown that oridonin induces apoptosis in cells derived from a variety of cancers. In this study, we investigated whether oridonin induced growth inhibition, cell cycle arrest in gallbladder cancer cells in vitro and in vivo, and we explored the possible mechanisms of action, which could provide experimental evidence for the potential application of oridonin as a new natural anti-tumor medicine for gallbladder cancer.Methods: 1. Oridonin induced apoptosis and cell cycle arrest of gallbladder cancer cells The anti-tumor activity of oridonin on SGC996 and NOZ cells was assessed by the MTT and colony forming assays. Cell cycle changes were detected by flow cytometric analysis. Apoptosis was detected by annexin V/PI double-staining and Hoechst 33342 staining assays. The in vivo efficacy of oridonin was evaluated using a NOZ xenograft model in athymic nude mice. 2. The mecanism of apoptosis and cell cycle arrest induced by oridonin The loss of mitochondrial membrane potential was observed by Rhodamine 123 staining. Activation of caspases(caspase-3,-8and-9) was measured by caspases activity assay. The expression of cell cycle- and apoptosis-related proteins in vitro and in vivo was analyzed by western blot analysis. 3. Oridonin induced apoptosis of gallbladder cancer cells by down-regulation of HER2 The expression of HER2 in gallbladder cancer cells after treatment of oridonin was detected by Real-Time PCR and Western blot analysis. Apoptosis was detected by MTT assay and annexin V/PI double-staining after overexpression of HER2. The expression of apoptosis-related proteins in vitro and in vivo was analyzed by western blot analysis. Surgical specimens, as well as clinicopathological data of 77 cases of gallbladder cancer and 70 cases of cholecystitis patients were collected. To investigate the relationship between overexpression of HER2 and clinicopathological/prognosis data in gallbladder cancer patients, the positive rate of HER2 expression between two groups was detected by immunohistochemistry. The correlation between expression of HER2 and Ki-67 was also analyzed.Results: Oridonin had obvious growth inhibition effect in a dose and time-dependent manner. With the increase of oridonin concentration, the early and late apoptosis rate increased significantly. The typical apoptotic morphology changes and loss of mitochondrial membrane potential were also observed at the same time. We observed the activation of caspase-3,-9 activities, while the activity of caspase-8 had no significant changes. In contrast, our study show the inhibition of NF-κB nuclear translocation and an increase Bax/Bcl-2 ratio accompanied by activated caspase-3, caspase-9 and PARP-1 cleavage after treatment with oridonin, which indicate that the mitochondrial pathway is involved in oridonin-mediated apoptosis. After treatment with oridonin, the percentage of G0/G1-phase cells dramatically decreased, whereas the percentage of cells in S-phase dramatically increased compared to control cells. Oridonin treatment down-regulated cyclin A and cyclin B1 and up-regulated cyclin D1, indicating that oridonin induces S-phase arrest in these cells. Intraperitoneal injection of oridonin(5, 10, or 15 mg/kg) for 3 weeks significantly inhibited the growth of NOZ xenografts in athymic nude mice. Furthermore, our study found that oridonin could significantly down regulate the expression of HER2 in gallbladder cancer cells. Overexpression of HER2 partially reversed the anti-tumor effect of oridonin. The positive rate of HER2 in gallbladder cancer patients was significantly higher than that in the cholecystitis patientes(46.7% vs. 0%). The expression of HER2 in gallbladder cancer patients was correlated with pathologic stage. The positive rate of HER2 in patients with III-IV was significantly higher than that in patients with 0-II(59.6% vs. 26.7%). The expression of Ki-67 was positively correlated with HER2 protein expression(Kappa=0.376, P=0.001). The median survival time of gallbladder cancer patients with HER2 positive and HER2 negative was 18.5 months and 23.5 months; 1 year and 3 year survival rate was 27.7%, 16.9% and 63.4%, 25.1%. The prognosis of patients with HER2 negative was better than those of HER2 positive. The expression of HER2 and pathological stage were two independent risk factors for prognosis in gallbladder cancer patients.Conclusion: Oridonin induced apoptosis and S phase arrest by down-regulation of HER2 expression and activation of mitochondrial pathway in gallbladder cancer cells. Therefore, oridonin has potential as a novel anti-tumor therapy for the treatment of gallbladder cancer.