Study Of Effect And Mechanisms Of Ursolic Acid On Prolifertion And Apoptosis In Prostate Cancer DU145 Cells In Vitro

Prostate cancer is a common tumor in older men.At present,prostate cancer has become the second most serious cancer in terms of its threat to men's health.Because early prostate cancer patients usually have no obvious clinical symptoms,so most patients go to hospital due to symptoms,bone or distant metastasis have occurred,and the opportunity of radical surgery has lost.The endocrine therapeutic effect of androgen deprivation in the early years to the majority of patients with prostate cancer is obvious,but as the disease progresses,the patient will still inevitably developed into castration resistant prostate cancer stage,seriously influence patient's quality of life and survival time,and eventually lead to death.There is no effective treatment to cure castration resistant prostate cancer,so urgently need to be looking for effective treatment and drugs to slow disease progression.Traditional Chinese Medicine of china has a long history and wide application,it has get more and more attention of the doctor in the field of tumor treatment.And it has the characteristics of low adverse reactions or side effects.and has become the focus of many researchers.Ursolic acid is a pentacyclic triterpene compounds,widely exists in the nature,and is present in the leaves and fruits of Ericaceae bearberry,leaves of Scrophulariaceae paulownia tomentosa and Oleaceae privet.Ursolic acid has extensive biological activities,including cancer resistance,protection liver,anti-inflammation and antiviral activity etc.Recent studies have shown that ursolic acid has inhibition of cell proliferation and induce the apoptosis of action on a variety of malignant tumor cell.Studies have reported that ursolic acid also have good clinical effect on prostate cancer.This experiment uses human prostatic cancer DU145 cells in castration resistant prostate cancer in vitro culture as the research object,research and discuss the apoptosis and reproductive effects of ursolic acid on DU145 cell and study its related mechanisms.Ursolic acid with different concentration on the DU145 cells after different time,observing the effect of proliferation and promoting apoptosis about ursolic acid on DU145 cell by CCK-8 method,application of microscopy,flow cytometry(FCM),spectrophotometry and Western blot methods,and explore its related mechanisms,provide experimental basis for finding new therapic drugs to prostate cancer.Objective Through cultivating androgen independence prostate cancer DU145 cells in vitro,investigate and discussion proliferation and promote apoptosis effect of ursolic acid on DU 145 cell and study its related mechanism.Methods Cultivation of prostate cancer DU145 cells in vitro,experimental group with different concentrations of ursolic acid(10,20,40,80?M)on DU145 cells at different time,compared with the control group without ursolic acid,then measure proliferation inhibition rate of ursolic acid on DU145 cell by Cell Counting kit-8(CCK-8)assay;observe morphology change of DU145 cell through inverted microscope;detect cell apoptosis ratio and cell cycle by Flow cytometry(FCM);detect activity changes of Caspase-3 and Caspase-9 by Spectrophotometry;examine the protein expression of cytochrome C and cofilin-1 in whole cell,cytoplasm and mitochondrial,ROCK and PTEN protein expression in cytoplasm with Western blot analysis.All data are expressed as the mean ± standard deviation.SPSS version 13.0 was used to perform all statistical analyses.P<0.05 was considered to indicate a statistically significant difference.Results1.CCK-8 assay was used to measure proliferation inhibition rate of ursolic acid on DU145 cell,the results show that different concentrations of ursolic acid(10,20,40,80?M)can restrain the proliferation of DU145 cells after different times(24-72h),compared with concrol group(P<0.01),and with the increase of concentration of ursolic acid and the extension of time,its inhibitory effect on DU145 cells gradually strengthen,of which 80?M ursolic acid has most evident inhibition effect after 72 hour,clearly in a time-and dose-dependent manner.2.Observation through microscope,ursolic acid treatment of cell proliferation slowly,and with increasing concentration of ursolic acid and time extension,apoptosis morphology change,including cell volume reduction,intercellular space increased,shape irregularly,cytoplasmic vacuoles,and nuclear condensation,etc.in control group,the cell grow adherent walls well,the form has no obvious change.3.Flow cytometry shows that the different concentration of ursolic acid(10,20,40?M)treatment on DU145 cells after 48 hour,cell apoptosis rate were 8.27±0.72%,12.84±0.75%,21.63±0.97%,respectively,compared with the control group(6.81±0.61%),the apoptosis rates have significantly statistical difference(P<0.01),show ursolic acid can induce the apoptosis of DU145 cells in a dose-dependent manner.Cell cycle detection results show that proportion of cells in G0/G1 phase increasing,and the S phase cells proportion reduced,and the effect of 40 uM ursolic acid group is most obvious,there are statistically significant differences(P<0.01).4.Spectrophotometry on the activity of Caspase-3 and Caspase-9 test results shows that the different concentration of ursolic acid(10,20,40?M)treatment on DU145 cells after 48 hour,the activity of Caspase-3,Caspase-9 obviously rise respectively,compared with the control group,have significantly statistical difference(P<0.01)and both in a dose-dependent manner.5.Western blotting detect Cytochrome c and cofilin-1 protein expression of whole cell,cytoplasm and mitochondrial,the results show that,compared with control group,after 48 hour,20?M and 40?M ursolic acid treatment group can significantly activated Cytochrome c protein expression but reduced cofilin-1 protein expression in the cytoplasm of DU145 cells(P<0.01),and reduced Cytochrome c protein expression but increased cofilin-1 protein expression in mitochondrial(P<0.01),Cytochrome c and cofilin-1 in whole cell remain unchanged.Western blotting detect protein expression of ROCK and PTEN,the results show that,compared with control group,with 20?M and 40?M ursolic acid treatment for 48 hour,ROCK protein expression can significantly reduced in DU145 cells(P<0.01).PTEN protein expression can significantly enhanced in DU145 cells(P<0.01).Conclusion 1.ursolic acid suppresses DU145 cell proliferation,in a time-and dose-dependent manner.2.ursolic acid induces apoptosis and participate in cell cycle regulation,cell blocked in G0/G1 phase and reduce the number of S phase in a dose-dependent manner.3.ursolic acid can activate Caspase-9,Caspase-3 cascade mitochondrial apoptosic pathway induced DU145 cells apoptosis,one of its mechanism may activate ROCK/PTEN signaling pathways mediated cofilin-1 mitochondrial translocation and make the mitochondrial Cytochrome c release into the cytoplasm,thus activate Caspase-9,Caspase-3 cascade mitochondrial apoptotic pathways,induce DU145 cell apoptosis.