target genes cyclin D1, vascular endothelial growth factor (VEGF), and urokinase-type plasminogen activator [8]. Triptolide acts either independently of or partly dependently on p53 to inhibit solid xenografted tumors growth in mice [9,10]. However, the efficacy and molecular mechanism of Triptolide on PCa are less studied. SUMOylation is a novel ubiquitin-like post-translational mod- ification. Four different SUMO proteins, SUMO-1, SUMO-2, SUMO-3 and SUMO-4, have been identified [11]. Similar to ubiquitination, SUMOyaltion involves a series of enzymatic processes. The mature SUMO is activated by conjugation to the E1 enzyme (SAE1/SAE2), transferred to the E2 enzyme (Ubc9) and ligated to the specific lysine residue of the target proteins by an E3 enzyme [11]. SUMOylation modulates multiple cell biological processes such as nuclear transport, cell cycle, chromatin remodeling, transcriptional regulation, DNA repair, and altering proteins ubiquitination and degradation [12]. Ample evidence has shown that SUMOylation is involved in development of human diseases including cancer. SUMOylation is a reversible process. The conjugated SUMO molecules can be cleaved by SUMO-specific proteases (SENPs). Six SENP proteins have been identified which deSUMOylate target proteins in different ways. SENP1 and SENP2 can remove all 3 SUMOs from target proteins, whereas other SENPs show specificity for SUMO-2 and SUMO-3 [13]. DeSUMOylation has been demonstrated to involve in the human diseases progression [14] such as PCa [15] and breast cancer [16]. Previously, Cheng et al [15] reported that deSUMOylation plays an important role in the development of PCa. They found that SENP1 was over- expressed in human PCa specimens but not in normal human prostate cells. siRNA inhibition of SENP1 reduced PCa cells growth. In addition, their initial results in vivo in transgenic mice indicated that over-expression of SENP1 leads to the development of prostatic intraepithelial neoplasia (PIN) at an early age. Further, SENP1 markedly enhances the activity of AR–dependent tran- scription by deSUMOylation of HDAC1 [17] and c-Jun– dependent transcription by deSUMOylating the CRD1 domain of p300 [18], which all involved in the tumorigenesis of PCa. Overall, these data suggest SENP1 play an important role in PCa progression and could be a novel PCa marker and therapy target. In the present study, we demonstrated that Triptolide signifi- cantly inhibited PCa cell proliferation in vitro and xenografted PC- 3 tumor progression in vivo, Triptolide also induced apoptosis in PCa cells. The anti-tumor efficacy of Triptolide was more potent than Celastrol. Meanwhile, we found Triptolide significantly down-regulated the SENP1 expression in both mRNA and protein levels, resulting in an enhanced cellular SUMOylation in PCa cells. Furthermore, Triptolide also negatively regulated AR and c- Jun expression and suppressed AR and c-Jun transcription. Overall, our experimental results suggest that Triptolide is a natural potential compound for PCa therapy. Results Triptolide significantly inhibited PCa cell proliferation in vitro Both Triptolide (Fig. 1A) and Celastrol (Fig. 1B) belong to terpenes and are two active components extracted from the Chinese herb Tripterygium wilfordii Hook F [1,19]. Celastrol, as a potent proteasome inhibitor, inhibits PCa cell proliferation and induces cell apoptosis [1]. Triptolide has been reported to suppress cell proliferation in many types of cancer, but its efficacy on PCa and target molecules have not been clearly studied. To determine the anti-tumor activity of Triptolide on PCa cells and compare Triptolide Inhibits PCa Cells Proliferation with Celastrol, we performed growth curve assay and cell viability assay using two PCa cell lines, LNCaP (androgen-dependent) and PC-3 cells (androgen-independent). We confirmed that both Triptolide and Celastrol caused significant inhibition of prolifer- ation in LNCaP and PC-3 cells. In the growth curve assay, Triptolide significantly inhibited the proliferation of PCa cells even at doses of 5 nM in LNCaP cells (Fig. 1C) and 10 nM in PC-3 cells (Fig. 1E). In contrast, Celastrol only suppressed PCa cells growth at high doses such as 1 mM in LNCaP cells (Fig. 1D) and 0.5 mM in PC-3 cell (Fig. 1F). These results showed that Triptolide can efficiently suppress PCa cell proliferation. To further determine the cytotoxic effect of Triptolide on PCa cell and compare with Celastrol, we carried out cell viability assay using MTT method. Both PCa cell lines were treated with broad doses of Triptolide or Celastrol, from 0.01 mM to 5 mM, for 48 h. As shown in Figure 1G, at low dose (such as 0.02 mM), Triptolide was sufficient to kill majority LNCaP cells but residual proportion of viable cells persisted even in the presence of higher concentrations of Triptolide. It was in contrast to the dose-dependent cytotoxic effect of Celastrol. However, a much higher dose of Celastrol was required to attain a similar cytotoxic effect with 0.02 mM Triptolide. Similar pattern was observed in cell viability assay for PC-3 cells (Fig. 1H). To quantify the efficacy of two compounds on PCa cell, we calculated the IC50 value according to the cell viability data. Triptolide had a much lower IC50 values for both cell lines compared to Celastrol (Table 1). These data demonstrated that Triptolide is an effective agent to inhibit PCa cell proliferation and to induce cell death. Triptolide induced apoptosis in PCa cells in vitro We next assessed whether Triptolide- and Celastrol-induced cell death corresponded to an apoptotic response. LNCaP and PC-3 cells were treated with 1 mM Triptolide and Celastrol for 24 h, respectively, stained with fluorescein isothiocyanate (FITC)-con- jugated annexin V (AV) and 50 mg/ml propidium iodide (PI), then analyzed by fluorescence microscopy and by flow cytometry. The simultaneous detection of phosphatidylserine externalization and loss of membrane integrity with AV-FITC and PI, respectively, discriminates between early apoptosis (AV+), late apoptosis eventually leading to secondary necrosis (AV+/PI+) and primary necrosis (PI+). As shown in Figure 2A and Figure 2B, treatment with Triptolide and Celastrol resulted in a higher proportion of cells with positive AV and/or PI staining. Flow cytometric analysis showed that treatment with Triptolide and Celastrol caused respectively 40% and 30% of LNCaP cells to be AV+/PI2, whereas only ,6% of vehicle (DMSO)-treated cells showed an apoptotic response. The two compounds had relatively moderate effect in inducing apoptosis in PC-3 cells, 8.77% and 15.2% respectively, versus 4% in vehicle (DMSO)-treated cells (Fig. 2C and Fig. S1). These results showed that both Triptolide and Celastrol induced apoptosis in PCa cells. In addition, LNCaP cells were more sensitive to Triptolide and Celastrol than PC-3 cells. It has been reported that Triptolide induces apoptosis through caspase cascade [4–6]. To further determine whether caspase is involved in Triptolide-induced apoptosis in PCa cells, we investigated the status of caspase-3 in PCa cells treated with various doses Triptolide or Celastrol for 24 h or with 1 mM Triptolide and Celastrol for desired times. Activation of caspases-3 was monitored by Western blot using a polyclonal antibody that recognize both procaspase-3 and its active cleavage fragments, p17 and p12. As shown in Figure 2D–2E, the cleavage of procaspase-3 was enhanced in Triptolide-treated LNCaP cells, and to a less degree, in Celastrol-treated LNCaP cells. Only very low levels of caspase-3 fragments were detectable in Celastrol or Triptolide- PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e37693