| Background:PSCA is a glycosylphosphatidylinositol(GPI)-anchored 123 amino acid cell surface glycoprotein that belongs to the Thy-1/Ly-6 family,and is predominantly prostate cancer-specific.Increasing evidences indicate an important role of PSCA in human tumorigenesis.However,its function and the underlying molecular mechanisms in prostate cancer(PCa)have not been fully elucidated.Objective:In the study,we investigated the effects of PSCA on proliferation and the role of PSCA in the cell cycle and apoptosis of PCa.material and method:The stable cell lines DU145-shRNA and stable PSCA up-regulated cells LNCAP-PSCA were astablised previously.30 nude mice were randomly divided into six groups of five mice each,including DU145,DU145-NC,DU145-shRNA,LNCAP,LNCAP-vector and LNCAP-PSCA groups.preliminary,we investigated effects of PSCA on proliferation in vitro experimental method including MTS and Colony-forming assay,and vivo experimental(mouse xenograft experiment).Flow cytometry were applied to detcect the change of cell cycle and apoptosis,furter,RT-PCR and weston blot analysis check the expression of cell cecyle-associated gene,as well as the LNCAP transfected by c-cmyc-siRNA plasmid.Immunohistochemical analysis was used to detcect to expression of PSCA and cell cycle-assocciated gene in xenograft and clinical prostate cancer tissue.The clinical data of patients who had initial treatments of prostate cancer before December 2013 must meet following criteria: All cases were pathologic diagnosis of prostatic adenocarcinoma after prostate biopsy or surgical perform without metastasis evidence;Patients didn’t received hormonal therapy or chemical therapy before prostate biopsy or prostatectomy.Cases included shoud contain pre-operative PSA,Gleason score,description of margin and lymph,as well as a complete follow-up data(survial and biochemical recurrence)were collected by telephone interview and medical record.Results:MTS assay results show : Compared with control group,DU145 shRNA cell proliferation was slower significantly(P = 0.000)after 72 hours incubation;And LNCAP-PSCA cells proliferation ability significantly enhanced after 48 hours(P = 0.000).than the control group.Plate clone formation assay showed DU145-shRNA formated less and smaller clonies than the control(P=0.000);LNCAP-PSCA formated more and bigger clonies than the control(P=0.000).Mouse xenograft experiment showed tumor size of LNCAP-PSCA were significantly bigger than the control after 2-week inoculation and heavier after 4-week(P=0.000).The opposite showed in the size(P=0.000)and weigh(P=0.000)in DU145-shRNA.Flow cytometry shows knockdown of PSCA in DU145-shRNA cells increased the percentage of cells in G1/G0 phase(60.32% ±1.2%,P=0.000),compared with DU145 cells(48.56%±2.0%,P=0.000),and DU145-NC cells(49.47%±2.5%,P=0.000);Moreover,PSCA overexpression in LNCAP-PSCA cells decreased the percentage of cells in G1/G0 phase(57.13% ±1.2%),compared with LNCAP cells(67.54%±2.0%,P=0.000),and LNCAP-vector cells(68.22%±2.5%,P=0.000).Knockdown of PSCA in DU145-shRNA cells showed no significantly difference in apoptosis rate(4.87% ± 0.48%),compared with DU145 cells(4.23% ± 0.53%,P=0.223)and DU145-NC(4.70% ± 0.61%,P=0.725).Also,PSCA overexpression in LNCAP-PSCA cells indicated no statistical significance in apoptosis rate(1.89% ± 0.37%)as compared with LNCAP cells(2.56% ± 0.18%,P=0.057)and LNCAP-vector cells(2.26% ± 0.13%,P=0.195).Western blotting and qRT-PCR showed expression of c-myc,Cycling D1 and E2 was down-regulated in DU145-shRNA cells compared with DU145-NC cells(P=0.000).Likewise,the expression levels of c-myc,Cycling D1 and Cycling E2 in LNCAP-PSCA cells were higher than in LNCAP-vector cells.we transfected c-myc siRNA into LNCAP-PSCA cells and found that c-myc siRNA markedly reduced cell proliferation and downstream gene cyclin D1,E2 expression in those cells(P=0.000),To investigate the signal path way involved upper the c-myc,We observed a significant decrease in p-AKT protein level in DU145-shRNA cells as compared with DU145-NC cells(P=0.000).Consistent with the results,LNCAP-PSCA cells showed a significantly increased p-AKT protein level compared with LNCAP-vector cells(P=0.000).However,we found that there were no statistical significance of p-ERK1/2 expression,either PSCA knockdown(P=0.439)or PSCA overexpression cells(p=0.886).Furthermore,we observed an AKT inhibitor MK2206 could significantly reduce PSCA-enhanced cell proliferation and c-myc,cyclinD1 and E2 expression in LNCAP-PSCA(P=0.000).We collected 78 cases with available paraffin-embedded tissue blocks and clinical data met the criteria above.High expression of PSCA is correlated with high expression of c-myc(P=0.000).The overexpressions of PSCA and c-myc were both significantly associated with high Gleason score(P = 0.046 and P = 0.001,respectively),positive BCR(P = 0.000 and P = 0.000,respectively)and OS(P = 0.000 and P = 0.000,respectively),but not with age(P =0.894 and p=0.537,respectively),Serum PSA levels(P=0.083 and p=0.359 respectively).The association of the expression levels of PSCA and c-myc with the BCR and OS of PCa patients was analyzed using Kaplan–Meier method(P<0.05).The Cox regression analysis showed that PSCA and c-myc were independent predictors for BCR of PCa(HR 2.834,95 % CI 1.124-7.141,P =0.027 and HR 3.232,95 % CI 1.366-7.649,P =0.008,respectively),and independent predictor for OS of PCa(HR 10.559,95 % CI 1.295-86.129,P =0.028 and HR 12.703,95 % CI 1.611-100.183,P =0.016,respectively).These results suggest PSCA may be used as an independent prognostic predictor for PCa patients.conclusion:PSCA promoted the proliferation and cell cycle progression from G0/G1 to S in PCa LNCAP and DU145 cells by up-regulating c-myc/Cycling D1,E2 via PI3K/AKT signaling pathways not by inhibiting apoptosis pathways.Knock down of PSCA can induce arrest of G0/G1 in DU145 and LNCAP.High expression of PSCA is correlated with high expression of c-myc and predict a poor prognosis.PSCA can be a novel therapeutic targets and strategies for the treatment of PCa. |
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