| OBJECTIVE:Androgen deprivation therapy(ADT)is currently a main treatment strategy for prostate cancer treatment,but persistent effects may lead to toleration and progress to refractory castration-resistant prostate cancer(CRPC).Though Androgen receptor antagonists,like other chemotherapy drugs,can delay this progression,they eventually fail due to drug resistance.Aberrant caveolin-1(Cav-1)expression has been reported in multiple tumor cell lines,it is unknown whether it is responsible for the progression of castration-resistant prostate cancer(CRPC).Therefore,the aim of this study was to explore whether Cav-1 could be used as a key molecule in the prevention and treatment of CRPC,and to understand its mechanism of action in CRPC,so as to find a new therapeutic strategies to delay the progression of CRPC.METHODS:(1)Collect tissue specimens of patients who came to treatment andinvolved in surgery or punctured at the Department of Urology,the First Affiliated Hospital of Chongqing Medical University from January 2010 to August 2018,including 45 cases of PPC specimens and 36 cases of CRPC specimens.The proteins expression of Cav-1 and related factors in Ras signaling pathways was detected by immunohistochemistry(IHC),and the correlation between H-Ras,K-Ras and PLC? and Cav-1 were analyzed using spearman's correlation analysis.At the same time,the correlation between Cav-1 and the demographic and clinical characteristics and survival of these patients was also be analyzed.(2)A total of 70 PPC and 56 CRPC serum samples were collected from patients prior to surgery between December 2016 and August 2018,at The First Affiliated Hospital of Chongqing Medical University.Enzyme-linked immunosorbent assay(ELISA)was used to analyze the expression of Cav-1 in serum,and the sensitivity and specificity of Cav-1as a diagnostic marker for CRPC was determined by receiver operating curve(ROC)and area under the curve(AUC).The relationship between the level of Cav-1 in CRPC patients and the clinical parameters of patients was also analyzed.(3)Castration-resistant derivatives of LNCaP cells [specifically,bicalutamide-resistant cells(Bic-R)and enzalutamide-resistant cells(En-R)]were constructed.Western blot and RT-qPCR were used to detect the expression of Cav-1 in LNCaP cells,PC3 cells,DU145 cells,Bic-R andEn-R cells.The Cav-1 knockdown plasmid was constructed and transfected into cells.Western blot was used to detect the expression of factors associated with invasion and migration.Transwell and wound-healing assays were used to detect cell invasion and migration.The CCK-8 assays were used to detect the proliferation of Bic-R and En-R cells and the 50%inhibitory concentration of bicalutamide and enzalutamide on cells after knockdown of Cav-1.(4)Take PC3 cells and En-R cells as an example.(1)Cells were divided in blank group,negative control group and Cav-1 knockdown group.Total protein,detergent insoluble membrane protein(DRF)and detergent soluble protein(DSF)were extracted.The expression of Ras isoforms,H-Ras and K-Ras in DRF and its downstream factor PLC? were detected by Western blotting(2)The cells was treatment with H-RasG12 V plasmid with or without Cav-1 knockdown plasmid,Western blot were used to detect the expression of PLC? which is the downstream factor the Ras signal in DRF;(3)The cells were treated with PLC? knockdown lentivirus with or without Cav-1 knockdown plasmid,the expression of factors(such as MMP2,MMP9,Snail)associated with invasion and migration were detected by Western blot.The invasion ability of cells was detected by transwell.In Bic-R and En-R cells,CCK8 was used to detecte the effect of bicalutamide or Enzaluamin in Bic-R and En-R cells,such as in Cav-1 knockdown group with or without H-RasG12 V overexpressiongroup.(5)Take representative PC3 cells and En-R cells as an example.(1)Cells were cultured in serum-free conditions,which removed the effects of exogenous cholesterol and treated cells with different concentrations of drugs(simvastatin,10 ?M;methyl-?-cyclodextrin,10 mM;cholesterol,10?M),Western blot and immunofluorescence were used to detect the expression of Cav-1.(2)Cells were treated with different concentrations of simvastatin(0 ?M,1 ?M,10 ?M,20 ?M),and the expression of HMGCoAR and Cav-1 was detected by Western blot.Different concentrations of bicalutamide(enzuramide)(2.5 ?M,5 ?M,10 ?M,20?M,30 ?M,40 ?M),simvastatin(2.5 ?M,5 ?M,10 ?M,20 ?M,30 ?M,40 ?M),were treated in Bic-R and En-R cells.or simvastatin combined with bicalutamide(enzuramide)(1:1),CCK8 was used to detect the cell viability.RESULTS:(1)Compared with PPC tissue samples,the expressions of Cav-1,H-Ras,K-Ras and PLC? in CRPC were significantly upregulated(P=0.002,P=0.0272,P=0.0377 and P=0.0364,respectively),and there was a positive correlation between Cav-1 expression levels and H-Ras(r=0.4151,P=0.0118),K-Ras(r=0.3443,P=0.0398)and PLC? expression levels(r=0.4338,P=0.0082)in CRPC.At the same time,the positive Cav-1expression were found to be positively correlated with bone metastasis(PPC: P=0.021;CRPC: P=0.006)among demographic and clinical characteristics of these patients.Kaplan-Meier survival analysis showed that in CRPC patients,median recurrence-free survival(RFS)was 36 months in Cav-1 negative patients,while median RFS in Cav-1 positive patients was 25 months,suggesting Cav-1 Positive tumor tissue associated with shorter RFS(P=0.013).Univariate Cox proportional hazards regression analysis suggested that Cav-1 was an independent risk factor for CRPC recurrence after ADT(HR=2.65,95% CI=1.162-6.029,P =0.02).(2)The expression level of Cav-1 in serum of CRPC patient was significantly higher than PPC patients(CRPC: 1.57±0.83 ng/ml vs.PPC:0.64±0.25 ng/ml;P<0.001).The ROC curve indicated that serum Cav-1could be used as a diagnostic biomarker for CRPC(AUC=0.876).When the cutoff value of Cav-1 was 0.68 ng / ml,the diagnostic sensitivity was82.1%,specificity was 80%.At the same time,statistical analysis of relevant clinical parameters and Cav-1 levels in CRPC patients showed that the increase in Cav-1 expression was positively correlated with tumor metastasis(P=0.003).(3)Western blot and RT-qPCR showed that Cav-1 was low in LNCaP cells,and significantly increased in PC3 cells,DU145 cells,Bic-R and En-R cells compared with LNCaP cells.After knocking down Cav-1,Western blot analysis showed that the expression of related factors(MMP2,MMP9,Snail),associated with invasion and migration,was significantly downregulated.Transwell and wound-healing experiments showed that the invasion and migration ability was significantly reduced.CCK-8experiments showed that the proliferation of Bic-R and En-R cells was significantly attenuated after knocking down Cav-1.The inhibitory strength of bical-R and En-R cells was significantly increased by bicalutamide and enzalutamide.This indicates that knockdown of Cav-1 can inhibit CRPC cell metastasis and resistance to AR antagonists.(4)Western blot analysis showed that after knocking down Cav-1,the expression of two isoforms of Ras(H-Ras and K-Ras)and its downstream factor PLC? were significantly downregulated;both H-RasG12 V and K-RasG12 V could upregulated the expression of PLC? in DRFs,but knockdown of Cav-1 could only reverse the upregulated caused by H-RasG12 V,suggested that the Cav-1 regulated the expression of PLC?by H-Ras.At the same time,knockdown of PLC? significantly downregulated the expression of related factors(MMP2,MMP9,Snail),which association with invasion and migration.Simultaneous,knockdown of Cav-1 could only reduce the expression of PLC? in DRF,but not the expression of PLC? in DSF.Consistently,knockdown of Cav-1 in combination with knockdown of PLC? more effectively downregulated the related factors of invasion and migration compared with knockdown of PLC? alone,indicated that knockdown Cav-1 can inhibited the expressionof PLC? in cell membrane lipid rafts so as to affect the invasion and metastasis ability of cells.CCK8 showed that although bicalutamide(enzalutamide)did not affect the cell viability of drug-resistant cells,bicalutamide(enzalutamide)could significantly suppress cell viability when knockdown of Cav-1.At the same time,supplementing H-RasG12 V could moderately reverse the suppression effect caused by Cav-1knockdown or Cav-1 knockdown combined with bicalutamide(enzalutamide),which indicated that knockdown Cav-1 can enhance the therapeutic effect of bicalutamide(or enzalutamide),and this process is partially affected by activated H-Ras.(5)Western blot and immunofluorescence assay showed that two drugs,simvastatin and methyl-?-cyclodextrin,which deplete intracellular cholesterol by different pathways,inhibited the expression of cell membrane Cav-1.Inversely,cholesterol replenishment resulted in increased cell membrane Cav-1 expression.Moreover,the effects of different concentrations of simvastatin on intracellular HMGCoAR and Cav-1 were dose-dependent.At the same time,CCK8 results showed that simvastatin can inhibited the viability of drug-resistant cells,and could synergistically increased the therapeutic effect of bicalutamide(enzuramide)when combined with bicalutamide(enzuramide).CONCLUSION:This study explored the effect of membrane Cav-1/H-Ras/PLC?signaling on the metastasis and drug resistance of CRPC.Cav-1 appears to be a promising predictive biomarker for CRPC and could be used to identify patients requiring more intensive treatment and as a putative candidate therapeutic target.Moreover,simvastatin was identified as an inhibitor of the development of castration resistance and a factor in delaying the progression of resistance to AR antagonists by regulated the expression of Cav-1. |
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