since SENP1 enhances the c-Jun dependent transcription via deSUMOylation of the CRD1 domain of p300 [18]. In our study, Triptolide shows potent efficacy in suppressing PCa cell proliferation and in inducing apoptosis. We observed that Triptolide down-regulates SENP1, c-Jun and AR expression which are over-expressed in PCa and play a pro-proliferation role. We assume that Triptolide anti-PCa effect could be through down-regulating expression of SENP1, c-Jun and AR. The knockdown and rescue experiments confirm our hypothesis. We found that down-regulation of SENP1, c-Jun or AR expression suppress PCa cell viability, which consist with previous reports that SENP1, c-Jun and AR have pro-proliferation roles in PCa and further suggest that Triptolide anti-PCa effect could be through down-regulation of SENP1, c-Jun and AR expression. Triptolide treatment of PCa cells with silenced SENP1, c-Jun or AR further decreased viable cells while the viability ratios were increased. It is possible that knockdown of SNEP1, c-Jun or AR in PCa cells could dilute targets for Triptolide action. Since Triptolide further decreases cell proliferation in these proteins knockdown PCa cells, which suggests that there could be other genes or proteins involved in Tiptolide function. Over-expression experiments also support above conclusion. Ectopic expression of SENP1, c-Jun or AR increase the viability ratio in Triptolide treated PCa cells, as does the Triptolide binding protein XPB over-expression. Furthermore, co-expression of SENP1, c-Jun and AR induces higher cell viablity ratio. All data indicate that SENP1, c-Jun and AR are potential targets of Triptolide, and down-regulation of these proteins is important for Triptolide anti-PCa toxicity. We notice that individual over-expression or co-expression of SENP1, c-Jun or AR do not suppress completely Triptolide effect. This could result from the possibility that Triptolide suppresses expression of SENP1, c-Jun or AR down-stream target genes or proteins, which disturbs action of over-expressed SENP1, c-Jun and AR. However, we could not exclude that other genes or proteins could be involved in Triptolide induced cell death. Triptolide suppresses expression of many genes or proteins in treated cells, including p53, NF-kB, Bcl-2, Mcl-1 [59]. Our study allows adding three new proteins, SENP1, AR and c-Jun, to the list. Understanding the mechanisms underlying these regulations is of major interest. Based on our data and others studies, we propose that Triptolide suppresses PCa progression through multiple pathways (Fig. 8). Triptolide could inhibit SENP1 transcription and decreases SENP1 mRNA and protein levels that are elevated in PCa. Consequently, the cellular SUMOylation is enhanced which could restore the balance of SUMOylation and deSU- MOylation, and suppress the functions of some key proteins involved in PCa progression. For example, suppression of SENP1 expression and decrease activity of deSUMOylation on AR and HDAC1 in PCa enhance AR SUMOylation and deacetylation by HDAC1, leading to inhibition of AR-mediated transcription. c- Jun mediated transcription is inhibited by the cis repression of CRD1 domain of p300 which could be released by SENP1- mediated deSUMOylation of CRD1 domain. Triptolide could inhibit directly AR expression, inducing suppression of AR mediated transcription. AR down-regulation also inhibits SENP1 expression, enhancing the cellular SUMOylation activities. Triptolide could inhibit directly c-Jun expression to reduce AP-1 and c-Jun mediated transcription. The non-transcriptional func- tion of c-Jun as co-activator also was suppressed. Furthermore, Triptolide could influence other genes expression or target other proteins to disturb these genes or proteins’ abnormal functions in PCa. As a result, inhibition of AR and c-Jun mediated transcription, suppression of other targets functions by SENP1 deSUMOylation and interruption of other important molecular Triptolide Inhibits PCa Cells Proliferation functions contribute to inhibit PCa proliferation and progression, and to facilitate apoptosis. The reason how Triptolide suppresses many genes and proteins expression remains unknown. It’s reported that Triptolide could bind a 90 kD unknown nuclear protein [60], which is revealed to be a subunit of the transcription factor TFIIH XPB [61]. Triptolide covalently binds to XPB and inhibits its DNA- dependent ATPase activity, leading to the inhibition of RNA polymerase II–mediated transcription. Triptolide was also found to induce phosphorylation of Rpb1, another subunit of RNA polymerase II, and subsequent proteasome-dependent degrada- tion, which results in a global transcription inhibition [62]. Both XPB and Rpb1 as targets of Triptolide may account for its suppression effect on expression of many genes and its high efficacy on cancer cells. Further studies are required to determine whether Triptolide down-regulates SENP1, c-Jun and AR through targeting XPB and Rpb1. In summary, our study showed that Triptolide, an active component extracted from Chinese medicinal herb, is an effective agent against prostate cancer. Its anti-tumor activity may be attributed to mechanisms involving down-regulation of SENP1 that restores SUMOylation and deSUMOyaltion balance and negative regulation of AR and c-Jun expression that inhibits the AR and c-Jun mediated transcription in PCa. Materials and Methods Ethics statement This study was approved by The Ethics Committee for Animal Experiments of the Fourth Military Medical University (Permit Number: 11004) and the experimental protocol was carried out in strict accordance with the institutional guidelines and the criteria outlined in the ‘‘Guide for Care and Use of Laboratory Animals’’. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Reagents Purified Celastrol (.98%) and Triptolide (.98%) were purchased from p-p Technologies, Inc (Shenzhen, China) or Sigma-Aldrich (C0869 & T3652), which were dissolved in DMSO (Wolsen) at a stock concentration of 50 mM and stored at 4uC. Trypan blue was from Sigma-Aldrich. Thiazolyl Blue Tetrazolium Bromide (MTT, M6494), Vybrant Apoptosis Assay Kit #3 (V13242), Trizol Reagent and LipofectamineTM LTX with PlusTM Reagent were obtained from Invitrogen. High-Capacity cDNA Reverse Transcription Kits was supplied by Applied Biosystems. SYBRH PrimeScriptTM RT-PCR Kit II was from TaKaRa (Dalian, China). DharmaFECTH 2 transfection reagent (T200201) was from Dharmacon RNAi Technologies. Antibodies for SENP1 (sc-46634), AR (sc-7305), a-tubulin (sc-5286), b-actin (C4), SUMO-1 (sc-5308) were from Santa Cruz Biotechnology. Another SENP1 antibody (ab58417) was from Abcam. Antibodies for Caspase-3 (#9662), PARP (#9532) and c-Jun (#9165) were from Cell Signaling Technology. Antibodies for Flag (CW0090) and HA (CW0260) tags were from CWBIO (Beijing, China). Rabbit polyclonal SUMO-1 antibody was generated by our laboratory using recombinant SUMO-1according to standard protocol. Secondary antibodies for goat anti-mouse IR Dye 680 (926–32220) and 800 cw (926–32210), donkey anti-goat IR Dye 680 (926–32224) and 800 cw (926–32214), and goat anti-rabbit IR Dye 680 (926–32221) and 800 cw (926–32211) were from LI- COR Biosciences. PLoS ONE | www.plosone.org 13 May 2012 | Volume 7 | Issue 5 | e37693