Sesquiterpens, Isolated From Commiphora Opobalsamum, Inhibit Cell Growth And Downregulate AR Expression And Function In Prostate Cancer Cells

Prostate cancer(PCa)is the most frequently diagnosed male cancer and second leading cause of cancer deaths in the United States,the prevalence of prostate cancer has also been increasing in China.Androgens and androgen receptor(AR)-mediated signaling is crucial for the development and progression of prostate cancer.Naturally occurring phytochemicals that target the AR signaling offer significant protection against this disease.Myrrh is the resinous exudates obtained from trees of Commiphora species.And more than 300 secondary metabolites have been found in this genus,including terpenoids,steroids,flavonoids,carbohydrates,et al.Sesquiterpenoids from myrrh possess diverse biological activities,including antibacterial,anaesthetic,antihyperglycemia et al.Recent studies have shown that,sesquiterpene compounds have the potential anti-tumor activity,but the molecular mechanism of anti-tumor remained unknown.After isolation,two sesquiterpenoids were purified from myrrh, 1(10)E,2R,4R)-2-methoxy-8,12- epoxygermacra-1(10),7,11-trien-6-one(ST1)and 2-methoxy-5-acetoxy-furanogermacr- 1(10)-en-6-one(ST2)respectively,and anti-tumor activities were examined by MTT assay.The result showed us that they had inhibitory effects on the proliferation of tumor cells including of HT-29,PC3, PC3M,DU145,LNCaP,and inhibited LO2 cells growth,a immortalizied liver cell line.The present study was designed to understand the possible mechanisms by which sesquiterpenoids inhibit proliferation of prostate cancer cells.The effect of Myrrh sesquiterpene compounds on prostate cancer cell cycle was firstly detected by flow cytometry.Reverse transcription PCR(RT-PCR)and western blot experiments were employed to detect the expressions of p21 and cyclinD at mRNA and protein levels.Transient transfection of pGL3-cyclinD-promoter into the prostate cancer cells was used to analyze the effect of the two compounds on reporter activity of the cyclinD promoter.It showed that the two sesquiterpenoids had an inhibitory effect on the proliferation of prostate cancer cells,and caused a G₀/G₁ arrest, induced the expression of p21 and produced a reduction of cyclinD.We descovered that although p21 and cyclinD participate in the inhibitory effect on the proliferation of prostate cancer cells,may not be the key factor,because they were changed little significantly.AR plays a pivotal role in in the development and progression of prostate cancer,and there is regulatory relationship between AR and p21,cyclinD,therefore,it's necessary to investigate the effect of sesquiterpenoids on AR.Western blot results showed that androgen could increase AR expression,but this androgen-midiated effect could be reversed by ST1 and ST2,AR nucler localization also be resduced by ST1 and ST2.Immunofluorescence and luciferase analysis were employed to detect the effect of the two compounds on AR expression, the results was in concordance with western blot.In order to analyze the effect of compound on AR function,transient transfection of pGL₃-PSA Promoter,hk2-3ARE-Luc into LNCaP and PC3 cells respectively,luciferase analysis was used to investigate the reporter activity of pGL₃-PSA Promoter,hk2-3ARE-Luc.Moreover PSA is secretory protein,the protein level of PSA in cell culture medium was detected.It showed that ST could decrease the protein level of PSA significantly.AR coregulator can affect AR transcriptional activation directly by interaction with AR.Therefore co-immunoprecipitation was performed to analysis the interaction between AR and AR coactivator.The results showed that androgen could rease the interaction between AR and AR coactivator SRC-1,ARA70,sesquiterpenoids from myrrh could weaken interaction between AR and AR coactivator SRC-1,ARA70.In all,we discovered that the two sesquiterpenoids we isolated from myrrh had inhibitory effect on the proliferation of prostate cancer cells.The possible molecular mechanisms involved cell cycle arrest,reduction of AR,weaken interaction between AR and AR coactivator SRC-1,ARA70,thus to result in degrading function of AR,inhibiting cell proliferation.This study provided information for myrrh anti-tumor activity,and broaded the thought for drag treatment of prostate cancer.