Mechanisms Of Triptolide On The Expression Of Androgen Receptor In Human Prostate Cells

PART I Effects of triptolide on the expression of androgen receptor in human prostate cells and its mechanisms in transcription levelObjectiveThe incidence rate of prostate cancer (PCa) showed a rising trend in China. Triptolide (TP) is one of the major extracts from traditional Chinese medicine Tripterygium wilfordii, it shows strong growth inhibition and induced apoptotic effects on PCa cells with unclear mechanism. Androgen receptor (AR) is a key target in the treatment of prostate cancer, and our previous study showed that TP can downregulate the expression of AR in PCa cells. Therefore this study is to explore the mechanisms in TP induced downregulation of AR in PCa cells in transcription level.MethodsLNCaP cells were cultured and treated by TP, qRT-PCR and Western blot were used to detect AR and its downstream target genes mRNA and protein expression. The restriction-free cloning method was used to construct a series of pGL3-AR promoter reporter gene vectors which were transfected into LNCaP cells to investigate the effects of TP on the transcriptional activity of AR promoter. NF-kB inhibitor and Western blot were used to explore whether PI3K/AKT/NF-kB pathway is involved in the regulation of AR by TP. The data was analysed by GraphPad Prism 5.Results1. The mRNA and protein expression of AR was dose dependent downregulated by TP in LNCaP cells, AR target gene PART1 and prostate specific antigen (PSA) was also downregulated.2. A series of pGL3-AR promoter reporter vectors were successfully constructed and validated by sequencing and luciferase activity.3. The promoter reporter gene assay showed that TP could down regulate the expression of AR at the transcriptional level, and its regulation site located in-965/+265 bp of AR promoter.4. PI3K/AKT/NF-κB pathway associated with AR promoter activity was drowregulated by TP.ConclusionsOur results demonstrated that the transcriptional activity of AR in LNCaP cells was downregulated by TP, and PI3K/AKT/NF-κB pathway may be involved in the regulation mechanism.PART Ⅱ The mechanisms of triptolide on regulating the expression of androgen receptor in prostate cancer cells in post-transcription levelObjectiveThe regulatory effect of TP on AR may be achieved through the regulation on AR protein stability, and AR degradation by calcium related calpain may be the major mechanism. This study is to investigate the effect of TP on AR protein degradation, and the function of protease involved.MethodsLNCaP cells were treated by TP, cycloheximide (CHX), protease inhibitors or reagents can change intracellular Ca2+concentration, Western blot was used to detect the expression of calpain-1, calpain-2, calpastatin, and AR.Results1. Using CHX to inhibit the protein synthesis of LNCaP cells, then treat cells by TP, the results confirmed that TP can downregulate the expression of AR in LNCaP cells at the level of post transcription.2. After treating LNCaP cells with specific calpain inhibitor ALLM, the degradation of AR induced by TP was found to be reversed. This indicated that the effect of TP on the stability of AR protein might be mediated by calpain.3. TP had no effect on the protein expression of calpain-1, calpain-2 and calpastatin in LNCaP cells.4. Using Ca2+chelator and Ca2+ionophore can down or up-regulated Ca2+concentration in LNCaP cells, respectively. This results indicated increased intracellular Ca2+concentration can down regulate the expression of AR protein. The effect of TP on downregulation of AR protein expression was via upregulating the intracellular Ca2+concentration and activating the calpain activity.ConclusionsTP can downregulate the expression of AR in LNCaP cells at post transcription level, which may be realized by upregulation of intracellular Ca2+concentration and activation of calpain.PART III The expression of calpain-1, calpain-2, calpastatin, calmodulin and AR in PCa tissuesObjectiveCalpain, calpastatin and calmodulin (CaM) are calcium related proteins and are all related to the stability of AR protein. The study detect the protein expression of AR and calcium related proteins above-mentioned in prostate tissues, analysis the correlation of protein expression and clinicopathological features of PCa patients, to investigate the relationship of calcium related proteins in PCa development.MethodsCollect tissue samples meeting the research requirements in the department of urology of clinical medical college of Yangzhou University from January 2013 to June 2015, including 25 cases of radical prostatectomy specimens and 25 cases of benign prostatic hyperplasia (BPH) specimens after TURP. Immunohistochemistry was used to detect the protein expression of calpain-1, calpain-2, calpastatin, CaM and AR. Image-pro-plus 6 software was used to measuere the optical density of the gland dyed by IHC, and the data were analyzed using SPSS 17.0 software.Results1. The expression of calpain-1, calpain-2, calpastatin, CaM and AR was found in both BPH (n=25) and PCa (n=25) tissues. The expression of five proteins in PCa tissues were all significantly higher than that in BPH, respectively (P<0.001).2. Only calpastatin and CaM had the positive correlation with each other in both BPH and PCa tissues.3. ROC curve analysis showed that the AUC of calpain-1, calpain-2, calpastatin, CaM and AR were 0.8112,0.9808,0.9824,1.000 and 0.9680, respectively.4. The expression of the five proteins was not associated with age, prostate volume, serum PSA, Gleason score, and nerve invasion.ConclusionsCalpain may play a promoting role in the development and progression of PCa, while the overexpression of calpastatin and CaM may be related to the inhibitory effect of calpain activity, leading to the high expression of AR. Calpain, calpastatin and CaM have the potential to be tumor markers of PCa.