mitochondrial cytochrome c release and caspases activation. These results indicate that mitochondria-mediated pathway and caspase cascade are involved in Triptolide-induced apoptosis. However, Tan et al [36] reported that potent caspase inhibitor zVAD-fmk causes significant reduction of focal adhesion kinase (FAK) and PARP cleavage, but fails to prevent Triptolide-induced cell death in breast cancer cells MCF-7, suggesting that Triptolide-induced apoptosis is probably mediated through multiple mechanisms and pathways in addition to mitochondria-mediated pathway and caspase cascade. It seems that Triptolide induces apoptosis regardless of p53 status of target cells as both LNCap (p53 intact) and PC-3 (p53 deficient) cells are responsive to Triptolide. The previous reported studies have shown that Triptolide induces apoptosis in these PCa cells without affecting p53 expression [31], and that Triptolide may function differently in various cells with different p53 status [37], pointing the potential efficacy of Triptolide on tumor types with p53 mutation/deletion. Taken together, these data indicate that Triptolide is capable to trigger massive apoptosis in cancer cells through multiple mechanisms and pathways. Epigenetic alterations play a key role in cancerogenesis and progression [38]. These alterations include not only DNA methylation and covalent histone modification, but also nonhis- tone protein postmodification like phosphorylation, acetylation, ubiquitylation and SUMOylation, the altered expression of enzymes of these postmodification and the imbalance of these reversible covalent modifications. Since epigenetic aberrations are reversible and can be restored to their normal state, epigenetic therapy to reduce the tumorigenicity promises a novel approach against cancer and is becoming attractive [39–41]. For example, successful use of bortezomib, a 26S proteasome inhibitor, for the treatment of multiple myeloma is based on its targeting in ubiquitin/proteasome-mediated pathways [42–44]. The therapeu- tic efficacy of arsenic trioxide in acute promyelocytic leukemia is dependent on the SUMOylation of PML–RARa oncoprotein [21]. These evidences indicate that ubiquitination and SUMOyla- tion play important roles in human cancer progression and could be effective therapy targets. SUMOylation has been linked to tumorigenesis and cancer metastasis [45,46]. Altered expression of SENPs has been detected in several carcinomas [47]. Balance of SUMOylation and DeSUMOylation is important for cell to maintain normal physiological process. The imbalance perturbs cell fate and initiate cancer development [47]. SENP1, one of SUMO-specific protease which processes the pro-SUMO to mature SUMO and deconjugates the modified proteins is elevated in PCa and plays a regulatory role in PCa development [15]. Although the mechanism underlining the function of overex- pressed SENP1 in PCa progression is not clear, SENP1 has been demonstrated to promote many proteins activities playing roles in PCa development. SENP1 reverses the ligand-induced SUMOyla- tion of AR and promotes AR-dependent transcription [48]. SENP1 also promotes AR-mediated transcription via its deSU- MOylation on HDAC1 which inhibits the HDAC1 deacetylase activity and transcription suppression ability [17]. SENP1 enhances the c-Jun dependent transcription via deSUMOylation of the CRD1 domain of p300, releasing the cis repression of CRD1 domain of p300 [18]. SENP1 is essential for stabilization of hypoxia inducible factor 1 (HIF1a) through its deSUMOylation during the hypoxia-induced process [12]. SENP1 is also essential in endothelial cells as a positive regulator of hypoxia-driven VEGF production and angiogenesis [49]. In the present study, we demonstrated that Triptolide significantly down-regulates SENP1 expression at both mRNA and protein levels and results in an enhanced cellular SUMOylation in PCa cells. The potent efficacy Triptolide Inhibits PCa Cells Proliferation of Triptolide on PCa may be mediated by the down-regulation of SENP1 expression and restoration of cellular SUMOylation level. We observed that Triptolide suppressed AR expression at mRNA and protein levels and inhibited AR-mediated transcrip- tion in AR-positive LNCaP cells. Androgens are critical for the normal prostate development and AR transduces the androgen signals to regulate the network of androgen response genes [50]. On the other hand, androgens are involved in PCa development under pathological situation. The androgen ablation therapy remains a standard treatment for advanced PCa. Most patients show positive response to the treatment but nearly 20% cases develop to hormone-refractory (HR) PCa in 1–2 years. Once the cancer progresses to HRPC stage, the PCa turns more malignant with poor prognostics. Throughout prostate cancer progression including HRPC, AR overexpresses or undergoes mutations [51,52]. Most identified mutated AR in PCa still retain their transcription activities and are transcriptionally active in response to ligands in addition to androgens. AR co-activators such as SRC- 1, SRC-3, TIF-2 [53,54] which could enhance the transcriptional activity of AR and contribute to the sensitization of AR to low levels of androgen concentrations are also elevated in prostate cancer. These observations suggest that PCa may result from dysregulation of AR activity, pointing that AR could be an important target for PCa therapy. Therefore, it is highly probable that Triptolide inhibits PCa proliferation via suppression of AR expression and consequent break of androgen response network functions. That explains why AR positive LNCaP cells are more sensitive to Triptolide than AR-negative PC-3 cells. Interestingly, SENP1 modulates the AR transcriptional activities via its deSUMOylation on AR and HDAC1 [17]. A positive feedback loop exists between AR and SENP1 in which SENP1 enhances the AR-dependant transcription, while AR potentiates the SENP1 expression by directly binding to the SENP1 promoter [55]. Disruption of this loop significantly blunts proliferation of androgen-dependent PCa cells [55]. Triptolide may disturb the AR and SENP1 loop by suppressing SENP1 and AR expression and consequently inhibiting AR-mediated transcription. We further demonstrated that Triptolide suppressed c-Jun expression at mRNA and protein levels and c-Jun mediated transcription in PCa cells. c-Jun, in combination with Fos subfamily proteins, forms the early response transcription factor AP-1. AP-1 regulates target genes expression through binding to the AP-1 element in the promoter of these genes that involved in various cellular events including development, cellular prolifera- tion, differentiation, transformation, inflammation, apoptosis and cellular migration [56]. Plenty of evidence indicate that c-Jun is a bona fide oncoprotein. c-Jun was found highly expressed in many types cancers such as sarcomas [57], classical Hodgkin’s disease and anaplastic large cell lymphoma (ALCL) [58], in which c-Jun promotes cancer cell proliferation and suppresses apoptosis. c-Jun is elevated in PCa associated with cellular proliferation and invasion [25]. In addition, c-Jun acts as an AR co-activator to stimulate AR transactivation by mediating receptor dimerization and subsequent DNA binding [25]. Since the important implica- tion of c-Jun in carcinogenesis and tumor progression, it could be a novel target in cancer treatment. It is highly possible that Triptolide inhibits PCa cell proliferation through down regulation of c-Jun expression and consequent inhibition of c-Jun and AP-1 mediated transcription. The AR-mediated transcription suppres- sion may be partially caused via Triptolide-induced inhibition of c- Jun co-activation. Analysis of c-Jun target genes expression showed the c-Jun mediated transcription was inhibited by Triptolide as a result of c-Jun expression suppression. The Triptolide-induced suppression of SENP1 expression may also involve in this process PLoS ONE | www.plosone.org 12 May 2012 | Volume 7 | Issue 5 | e37693