Experimental Study Of Oridonin And Oridonin Nanosuspension On Human Pancreatic Cancer Cell Line PANC-1

Pancreatic adenocarcinoma is one of the most deadly forms of cancer. The incidence rate of pancreatic cancer is 1% to 3% in human tumors,8% to 10% in gastrointestinal tumors. It is a fast developing disease and is difficult to detect and diagnose. Currently, surgery offers the best possibility for survival, but at time of diagnosis, only 20% of patients are eligible for resection and the 5-year survival rate of those patients remains about 20%～25%.80% of patients are inoperable or metastatic with 2-year survival rate of 5%～18% and 5 year survival rate of 0～5%. Therefore, adjuvant therapy, especially chemotherapy to improve the quality of life for advanced pancreatic cancer patients is more important. At the same time, survival may be extended under certain conditions, and could win the chance of operation.Oridonin, an active diterpenoid isolated from Rabdosia Rubescens, has been reported to have anti-tumor effect. Previous reports have demonstrated that oridonin exhibited remarkable inhibitory effects and was a potent apoptosis inducer in a variety of cancer cells. Because of its poor solubility in water and short biological half-life in vivo, the current anti-cancer agent is not ideal. Therefore, it is extremely critical to solve the problem of poor water solubility of oridonin and improve its bioavailability.Nanosuspension is applied to new formulations of insoluble agents. It is a suspension of drug nanocrystals in a liquid stabilized by a surfactant or polymer. The advantage of nanosuspension is increasing the solubility and dissolution velocity of agents. Compared with the traditional framework of nanosystems, nanosuspension doesn't need carrier material; can effectively avoid the side effects of cosolvent and the size requirements of cyclodextrin on drug molecules. In addition, nanosuspension can also produce bio-adhesion, increase drug stability and enhance drug effectiveness.In this study, we detected and compared the effects of oridonin and oridonin nanosuspension on the growth, apoptosis, and cell cycle of human pancreatic cancer cells line PANC-1.Part 1:Growth inhibitory effects of oridonin and oridonin nanosuspension on PANC-1 cells【Objective】To detect and compare the effect of oridonin and oridonin nanosuspension on proliferation of PANC-1 cell.【Methods】(1) Cellular morphologic changes were observed with optical microscope after PANC-1 cells treated with different concentrations of oridonin or oridonin nanosuspension.(2) MTT assay was used to analyze the effect of oridonin and oridonin nanosuspension on PANC-1 cell viability.【Results】(1) Results of optical microscope observation showed that PANC-1 cells had morphological changes and the cell number gradually decreased in a dose-dependent manner.(2) MTT assay showed that oridonin and oridonin nanosuspension both induced cell death in a dose-and time-dependent manner. However, the inhibitory rate of oridonin nanosuspension was significant higher than that of oridonin.【Conclusion】Oridonin and oridonin nanosuspension both inhibited the growth of PANC-1 cells, and the inhibitory effect of oridonin nanosuspension was more effective. Part 2:Effects of oridonin and oridonin nanosuspension on PANC-1 cell apoptosis【Objective】To investigate and compare the effect of oridonin and oridonin nanosuspension on apoptosis of PANC-1 cell.【Methods】(1) PI staining and Hoechst 33342 staining were used to detect morphologic changes of PANC-1 cells treated with different concentrations of oridonin or oridonin nanosuspension.(2) The percentage of apoptosis was determined by flow cytometer method.(3) Caspase-3 activity was measured by spectrophotometry, after PANC-1 cells were cultured with oridonin or oridonin nanosuspension for different time period.(4) The pro-caspase-3, Bax and Bcl-2 protein expression were determined by Western blot method.【Results】(1) Results of PI staining and Hoechst 33342 staining showed that both oridonin and oridonin nanosuspension induced PANC-1 cells apoptosis.(2) Results of flow cytometer method showed that oridonin and oridonin nanosuspension both induced PANC-1 apoptosis in a dose-dependent manner. Oridonin nanosuspension had a more significant apoptosis-inducing effect.(3) Caspase-3 activity analysis showed that caspase-3 activity increased significantly compared with the control group.(4) Western blot results showed that pro-caspase-3 was activated, the expression of Bax was up-regulated, whereas the expression of Bcl-2 had no obvious change.【Conclusion】Both of oridonin and oridonin nanosuspension could induce apoptosis of PANC-1 cells, and the latter had a better induction effect. The molecular mechanism of apoptosis may be mediated by the expression change of Bax/Bcl-2 ratio and the activation of pro-caspase-3.Part 3:Effects of oridonin and oridonin nanosuspension on PANC-1 cell cycle[Objective]To investigate and compare the effect of oridonin and oridonin nanosuspension on PANC-1 cell cycle distribution.[Methods](1) Cell cycle distribution was detected by flow cytometry analysis.(2) The CyclinBl,Cdc2 and p-cdc2 (T161) protein expression were determined by Western blot method.[Results](1) Oridonin and oridonin nanosuspension both induced G2/M phase cell cycle arrest in PANC-1 cells, and oridonin nanosuspension had a more obvious effect.(2) Western blot results showed that the expression of CyclinB1 and p-cdc2 (T161) was suppressed in a time-dependent manner, whereas the expression of Cdc2 had no obvious change.[Conclusion]Oridonin and oridonin nanosuspension both induced G2/M phase cell cycle arrest in PANC-1 cells. Its molecular mechanism may be related with the decreased expression of CyclinBl and p-cdc2 (T161).In conclusion, research results displayed that both oridonin and oridonin nanosuspension could inhibit the growth of PANC-1 cells possibly by inducing G2/M phase cell cycle arrest and apoptosis, and oridonin nanosuspension had a more significant effect. The molecular mechanism of apoptosis may be mediated by the expression change of Bax/Bcl-2 ratio and the activation of pro-caspase-3. The mechanism of G2/M phase cell cycle arrest may be related with the decreased expression of CyclinBl and p-cdc2 (T161).