The Effect And Mechanisms Of Diterpenoid B Derived From Rabdosia Excise On Cell Cycle Of Mouse Prostate Cancer Cell Line

The prostate cancer is common in the old age man. With the aging of population and the change of life style and food ingredient, the morbidity of PCa has been increasing year by year in our country. our center had been assumed the responsibility of JICA project. And the first carried out the basic epidemiological study of PCa's early discovery , early diagnosis and prevention and cure. It is thus clear that Prostate cancer are becoming one of the serious diseases gradually threatened our countries gerontal men.The main therapy manages are surgical operation, radiotherapy, and the castration operation etc In early days, prostate cancer seldom have clinical symptom, so it is often detected in later period or after metastasis. The operation was traditional way to cure the PCa and made a good effected. There are no effective treatment for the late period of prostate cancer. Therefore, it has been introduced for us an imminent and urgently solved topic to study the substances with low level of toxicity and therapeutic tool of PCa. Recently researchers have found active component of Rabdosia excise,it could exert antitumor function.Objective: The aim of this paper is to determine the effect of DB on the cell line of prostatic cancer (RM-1) and its related mechanisms. Methods: cell line in culture medium in vitro was treated with different concentrations of DB. MTT assay was used to detect the cell growth inhibitory rate, and morphologic changes of cells was observed by phase contrast microscope. the cell cycle is detected by FCM, the expression of P53,CDK2,CHK1,GRIM-19 and STAT3mRNA is detected by RT-PCR..Immunocytochemistry stain was done to detect the expression of P53 ,CDK2 and GRIM- 19 protein.Results:1. MTT experiment showed that DB can inhibit RM-1 cell growth in a dose- and time- dependent manner.2. We observed the changes of cells under the confocal microscopy , DB treated cells showed retarded proliferation and less division, suspension cell and collapse cell also appeared.3.We used FCM to examine cell cycle in experiment ,the result revealed that the percentage of G1 phase cells increased obviously after treated with DB, the percentage of G1 phase cells was 51.39%.4. After the treatment of DB ( 2μg/ml,4μg/ml,8μg/ml) for 12h, 24h,48h,The mRNA expression of P53, CHK1, GRIM-19 in drug treated groups are higher than control, meanwhile the level of CDK2 ,STAT3 mRNA was less than control(P<0.05);5. After the treatment of DB ( 2μg/ml,4μg/ml,8μg/ml) for 12h, 24h, 48h,Immunocytochemistry stain showed that the expression level of P53, GRIM-19 protein was up regulated in drug treated groups, the expression of CDK2 protein was down regulated . (P<0.05);Discussion: DB can inhibit RM-1 cell growth in a dose- and time- dependent manner. DB inhibits RM-1 cells proliferation and arrests themin G1 phase. P53-CDK2,play an important role of regulating cell cycle.P53 is a critical gene of G1 check point.When the cell go through G1 phase to S phase mainly depends CyclineE-CDK2,as a result, CDK2 can effect the switching of G1 phase to S phase on cell cycle.When DNA damage was found,P53 can bind with definit part,then up regulated P21, P21 can inhibited activity of CDK2,as a result,cell cycle stop.DB can activate P53,inhibit activation of CDK2, arrest cell cycle in G1 phase. In this study,After the treatment of DB ( 2μg/ml,4μg/ml,8μg/ml) for 12h, 24h,48h, The mRNA expression of GRIM-19 in drug treated groups are higher than control, meanwhile the level of STAT3 mRNA was less than control,it was reported STAT3 has an effect on CDK2, we presume DB induced the expression of GRIM-19→blocked the transcription of STAT3→regulated he expression of CDK2,DB inhibit the proceeding of cell cycle ,and carry out anti–tumour effect.Conclusion:1.DB can inhibit RM-1 cell growth in a dose- and time- dependent manner.2.DB can inhibits cell proliferation The mechanisms of it is arrests cell cycle in G1 phase.3.The blocking mechanism of G1 phase is regulating the expression of P53, CDK2. The expression of P53 in drug treated groups is higher than control, meanwhile the level of CDK2 was less than control.4. The expression of GRIM-19 in drug treated groups is higher than control, the level of STAT3 was less than control.it may also attend in the blockage of cell cycle.