Triptolide Inhibits PCa Cells Proliferation experiments. * indicates P,0.05 (compared to control). (C) Effect of Triptolide on LNCaP cells. (D) Effect of Celastrol on LNCaP cells. (E) Effect of Triptolide on PC-3 cells. (F) Effect of Celastrol on PC-3 cells. (G) and (H) PCa Cells were treated with indicated concentrations of Triptolide or Celastrol and cells viability was determined by MTT assay. All assays were performed in triplicate, and data shown are mean 6 SD of four independent experiments. (G) Effect of Triptolide and Celastrol on LNCaP cells. (H) Effect of Triptolide and Celastrol on PC-3 cells. doi:10.1371/journal.pone.0037693.g001 treated PC-3 cells. We then followed the status of nuclear enzyme poly (ADP-ribose) polymerase (PARP) that is one of the main cleavage targets of Caspase-3. PARP cleavage was enhanced in Triptolide-treated LNCaP cells, and to a less degree, in Celastrol- treated LNCaP cells (Fig. 2F). Again, lower levels of PARP fragments were detectable in Celastrol or Triptolide-treated PC-3 cells (Fig. 2G). Altogether, these data demonstrated that Triptolide, and to a less degree, Celastrol, induce apoptosis in PCa cells associated with activation of caspase-3 and cleavage of PARP. Triptolide suppressed xenografted PC-3 tumor growth in vivo We observed that Triptolide can inhibit PCa cell proliferation and induce cell apoptosis in vitro. To determine the efficacy of Triptolide in vivo, we carried out xenograft study. PC-3 cells were implanted subcutaneously in nude mice. When the tumors became 3 palpable (,100 mm ), mice were treated intraperitonneally with either a vehicle control or Triptolide at 0.4 mg/kg daily for 15 days. Tumor sizes and body weight were measured every three days. As shown in Figure 3, the volume (Fig. 3A) and weight (Fig. 3C) of xenograft tumors treated with Triptolide were significantly smaller than those in the control group (Fig. 3D), indicating that Triptolide has potent anti-tumor effect in vivo. Meanwhile, there was no significant difference in body weight between the treated and the control group (Fig. 3B), suggesting that administered dose of Triptolide has no significant toxicity to mice. Triptolide down-regulated SENP1 expression in PCa cells Since Triptolide inhibited PCa cell proliferation and induced cells apoptosis in vitro, and suppressed xenografted tumor growth in vivo, we were interested in the mechanisms underlying these anti- tumor effects. Previous studies showed that SENP1 is over- expressed in PCa cells and tumors samples [15]. SENP1 over- expression in transgenic mice promotes the development of malignant lesions in the prostate gland [15]. These data suggest that over-expression of SENP1 mediates PCa development and SENP1 could be a potential target for PCa therapy. To investigate whether Triptolide regulates SENP1 expression, firstly we analyzed SENP1 mRNA level by real-time PCR in LNCaP and PC-3 cells treated with various doses of Triptolide or Celastrol for 24 h, or treated with 0.1 mM Triptolide or Celastrol for desired times. As shown in Figure 4A and 4B, Triptolide significantly decreased SENP1 mRNA levels in both LNCaP and PC-3 cells in dose-dependent and time-dependent manners. Table 1. IC50 values. Name Celastrol Triptolide Celastrol Triptolide Time (h) 48 48 48 48 IC50 (mM) 0.4881 0.0203 1.195 0.009754 Cell PC-3 PC-3 LNCaP LNCaP doi:10.1371/journal.pone.0037693.t001 Meanwhile, Celastrol also reduced SENP1 mRNA level following a brief treatment at low doses. In contrast, at high doses and with longer treatment time, Celastrol enhanced SENP1 mRNA level in both treated PCa cell lines. To evaluate the efficacy of Triptolide- induced down-regulation of SENP1 expression, we determined the dose of Triptolide required to induce 50% decrease in SENP1 mRNA (IC50). Both LNCaP and PC-3 cells were treated with more than 6 doses Triptolide for 24 h, as shown in Figure 4C, Triptolide efficiently induced the decrease in SENP1 mRNA level with IC50 value 0.07579 mM and 0.07071 mM for LNCaP and PC-3 cells respectively. We further measured the SENP1 protein level in PCa cells treated with Triptolide or Celastrol. Triptolide was able to decrease the SENP1 protein level in both dose- dependent (Fig. 4D, 4E) and time-dependent (Fig. 4F) manners. In contrast, Celastrol did not significantly reduce SENP1 protein level in treated PCa cells. These results are consistent with our qRT-PCR results and indicate that Triptolide suppresses SENP1 expression in PCa cells at both mRNA and protein levels. SENP1 plays an important role in SUMOylation processes. As an isopeptidase, SENP1 deconjugates SUMO from SUMOylated protein. A reduced SENP1 expression in Triptolide-treated PCa cells would result in an enhanced cellular SUMOylation. To verify this expectation, the cellular SUMOylation level in PC-3 cells treated with 1 mM Triptolide or Celastrol for 24 h was detected by Western blot using SUMO-1 antibody. Cells without treatment but lysed in lysis buffer in the presence of 20 mM N- ethylmaleimide (NEM), a potent inhibitor of deubiquitinating enzymes and of SUMO hydrolase activity, was used a positive control [20]. As shown in Figure 4G, compare to control, Triptolide treatment significantly enhanced total SUMOylation level. Interestingly, Celastrol also enhanced total SUMOylation level, but with less efficiency compared to Triptolide, which could result from its proteasome suppression activity. It was reported that SUMOylation of some proteins, such as HIF1 [12] and PML [21], promotes these proteins ubiquitin-dependent degradation. As a proteasome inhibitor, Celastrol could suppress degradation of these SUMOylated proteins and induce their accumulation. To confirm this result, we performed similar experiments using another SUMO-1 monoclonal antibody. The western blot results showed that the levels of SUMO-1 heterodimers were increased in Triptolide treated samples (Fig. S2A, S2B). We also found that the level of SUMO-1 monomer was reduced in Triptolide treated samples (Fig. S2C). This could result from either down-regulated SENP1 expression, or the increase of SUMO-1 heterodimers. Overall, Triptolide is able to negatively regulate SENP1 expres- sion at both mRNA and protein levels, resulting in an enhanced cellular SUMOylation level in PCa cells. Triptolide suppressed androgen receptor (AR) expression in LNCaP cells AR plays an important role in normal prostate development and maintenance, and in prostate cancer progression. Celastrol was reported to suppress AR expression in LNCaP cells [1]. We raised the question whether Triptolide inhibits AR expression and AR-mediated transcription in PCa cells. We analyzed AR mRNA level in LNCaP cells treated with various doses of Triptolide and Celastrol for 24 h or with 0.1 mM Triptolide and Celastrol for desired times by real-time PCR. As shown in Figure 5A and 5B, PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e37693