Prostate Androgen-regulated Gene: A Novel Potential Target For Androgen-independent Prostate Cancer Therapy

Part OneDetection of differential expression of PAR between androgen-independent prostate cancer cell and androgen-dependentprostate cancer cell Aim: To study the difference of PAR expression betweenandrogen-independent prostate cancer cell and androgen-dependent prostate cancer cell.Methods: The difference of PAR expression between LNCaP and PC3 cells wasdetected by RT-PCR.Results: PAR expression was higher in PC3 cells than in LNCaP cells by 3-fold.Conclusion: PAR gene specifically overexpressed in androgen-independent prostatecancer cell.Part Two Study of the effects of androgen on PAR gene expression in AR-positiveprostate cancer cell and AR-negative prostate cancer cellAim: To investigate the effects of dihydrotestosterone(DHT) on PAR gene expression in AR-positive prostate cancer cell and AR-negative prostate cancer cell.Methods: Effects of DHT at different concentrations on proliferation of LNCaP andPC3 cells were studied by means of cell counts. PAR mRNA of LNCaP cells after DHTstimulation was detected by RT-PCR.Results: DHT at low concerntrations(0.001nM-lnM) stimulated proliferation andupregulated PAR mRNA expression of LNCaP cells. Maximum effect occurred at0.1 nM DHT(P<0.05).Whereas DHT at concemtrations greater than 1nM inhibited theLNCaP cells proliferation and its PAR mRNA expression. DHT had no significant effecton proliferation and PAR mRNA expression of PC3 cells(P>0.05).Conclusion: DHT modulates proliferation and PAR mRNA expression of LNCaPcells in a dose-related manner.Part ThreeStudy of the mechanism of dihydrotestosterone regulating PAR geneexpressionAims: To investigate the mechanism of dihydrotestosterone(DHT) on PARgene expression.Methods: PAR mRNA of LNCaP cells after DHT stimulation with its antagonistflutamide were detected by RT-PCR. DHT effects on PAR expression were evaluated byRT-PCR in PC3 cells stably transfected with vector containing wild-type AR.Results: The androgen receptor (AR) antagonist flutamide inhibited the stimulatoryeffect induced by DHT. Reintroduction of AR into PC3 cells by stable transfectionrestored the androgen effect on PAR up-regulation.Conclusion: DHT regulates PAR mRNA expression in an AR dependent pathway. PARgene is a potential target of gene and drugs therpy for androgen resistant prostate cancer. Part FourStudy of the effects of PAR gene down-regulation on malignant phenotype of prostate cancer cell lineAims: To investigate the effects and mechanism of downregulated PARexpression on proliferation of PC3 cells by using RNA interference (RNAi).Methods: Suppression of PAR expression was achieved by transfection of PC3 cellswith short hairpin RNA (shRNA) expression vectors targeted against PAR, which werenamed as psiRNA-PAR1, psiRNA-PAR2 and psiRNA-PAR3. The inhibitory effectswere confirmed by RT-PCR. The growth characteristics of PC3 transfectants wereanalysed by cell counts, colony formation in soft agar and flow cytometry.Results: The expression of PAR was suppressed by three shRNA expression vectors.PsiRNA-PAR1 was shown to inhibit the PAR expression most efficiently, with theinhibitory rate of 81.18 ± 1.68% peaking at 48 hours after transfection. PC3transfectants exhibited a decreased cell proliferation in cell culture and a low efficiencyof colony formation in soft agar. Flow cytometry revealed a cell cycle G2-M arrest andinduction of apoptosis.Conclusion: Downregulated PAR expression inhibits the growth of PC3 cells, throughinducing G2-M arrest and activating apoptotic pathway. As a potential proto-oncogenewhich trigger and/or maintain malignant proliferation, PAR may be a very importanttarget in the gene therapy in the future.