The Study Of Isotan Leaf Emodin And Phloretin Inducing Apoptosis Of Prostate Cancer Cells And Its Molecular Mechanism

By performing FCM,PCR,western-blot and Confocal assays,we investigate the effects of flavonoids isorhapontigenin and phloretin on prostate cancer cells proliferation and apoptosis and related molecular mechanism.The main contents are as follows:1.The molecular mechanism of Isorhapontigenin induced apoptosis on prostate cancer cells:Isorhapontigenin(ISOR)belongs to oligostilbenes and is similar to the structure of resveratrol.It has been reported that ISOR can induce apoptosis in cancer cells.But the molecular mechanism of ISOR in cancer was not well know.To explore the impact of ISOR on prostate cancer cells.We designed assays as follows:(1)We treated PCa cells and normal cells with increasing doses of ISOR to observing cell morphology changes.Resluts revealed ISOR inhibited cell viability.Then analyzed cell viability by performing MTT assay.It indicated ISOR may induced apoptosis in PCa cells.(2)LNCaP and CWR22Rv1 cells were treated with ISOR.Then DAPI staining assay and FCM were carried out to determine the degree of ISOR induced apoptosis.We revealed ISOR treatment can induce the protein levels up-regulation of cleaved PARP-1 and caspase 3,while the protein levels of XIAP and Cyclin DI were downregulated.And ratio of Bax/Bcl-2 were up-regulated after ISOR treatment,indicated that ISOR could induce apoptosis in PCa cells.Then,we revealed that ISOR treatment downregulated AR and PSA at both mRNA and protein levels.Moreover,Sp1 was also down-regulated,which plays an important role in regulating the expression of AR.Then we use MTM-Sp1 inhibitor to treat LNCaP and CWR22Rvl cells.The data indicated that Sp1 played a significant role in ISOR induced AR downregulation.We used CHX or MG132 combine with ISOR to clarify whether ISOR influenced the post-translational level of AR.We found that the proteasome was involved in ISOR-enhanced depletion of AR and AR-Vs protein.Immuniprecipitated AR in the presence of MG 132 and ISOR had once again proved the degradation of AR.The activity of AKT was inhibited by ISOR.LNCaP and CWR22Rvl cells were treated with ISOR or combined with LY294002-AKT inhibitor,results revealed that ISOR treatment significantly induced p-AKT and AR down-regulation.By performing confocal assays,we revealed ISOR treatment apparently induced FOXO1 nuclear translocation,and then induce the expression of some FOXO1 associated target genes.(6)Finally,we found ISOR treatment downregulated p-EGFR and p-ERK1/2 levels.Moreover,LNCaP and CWR22Rvl cells were treated with ISOR for increasing time,or treated with EGF,results revealed p-EGFR was directly inhibited by ISOR.Then the EGFR-AKT and EGFR-ERK signal pathways were inbitited.Taking together,ISOR induced AR levels down-regulation by inhibiting AR transcription and inducing AR protein degradation.ISOR directly down-regulated p-EGFR levels,consequently activated FOXO1,and thus suppressed PCa cell proliferation and induced apoptosis.2.The study of apoptosis induction of prostate cancer cells by phloretin and its molecular mechanism:Phloretin,a dihydrogen flavonoid,has a variety of biological activities.But the effects of phloretin on prostate cancer is still less research.Therefore,we study the effects of phloretin on prostate cancer cell and explore the mechanism of apoptosis induction,to promote the application of phloretin in the field of cancer.(1)The influence of phloretin on WPMY-1,BPH-1,LNCaP,CWR22Rvl,PC3 and DU145 cell was observation by morphological.The results shown that phloretin could inhibit prostate cancer cell proliferation and growth,but had no effect on WPMY-1 and BPH-1 cells.The results of MTT assay was consistent with morphological observation.(2)Whether phloretin induces the apoptosis of prostate cancer cell was analysised by DNA electrophoresis,DAPI staining,FCM and western-blot.The results demonstrated that the apoptosis rate of cancer cell was significantly increased in a dose-dependent manner.Without affected the expression of p53,the cleaved caspase 3 and PARP-1 were upregulated,and the protein level of XIAP was decreased by phloretin.These data suggest that phloretin induced apoptosis in a caspase-dependent manner.(3)Then we found that phospho-AKT(Ser 473)was inhibited by phloretin.Western-blot experiments were performed to confirm that the phosphorylation of FOXO1 was down-regulated by phloretin.In addition,the protein levels of XIAP,Cyclin D1,p-AKT and p-FOXO1 were significantly down-regulated by combined LY294002 and phloretin.(4)PCR experiments were performed to confirm that the expression of FOXO1 and Bim genes were up-regulated by phloretin in a dose-dependent manner.The downstream genes of FOXO1,Bim and p21 were increased,which indicated that the activition of FOXO1 was up-regulated by phloretin.Then,confocal assay found that phloretin induced nuclear localization of FOXO1 in cells.(5)Phloretin caused a decrease in EGFR and ERK1/2 phosphorylation.Time-course experiment was profformed to analysis whether phloretin directly inhibit EGFR activation.The results revealed that the phosphorylation of EGFR was quickly inhibited by phloretin.But EGF-stimulated autophosphorylation of EGFR was also inhibited by phloretin.All these results indicate that phloretin can inhibit the activity of PI3K-AKT and MEK-ERK1/2 signal pathways by inhibiting p-EGFR.Through up-regulation of FOXO1 transcription activity,phloretin promoted the expression of the genes which regulated the cell cycle and apoptosis.These involved in phloretin-induced prostate cancer cell apoptosis.