Triptolide Inhibits PCa Cells Proliferation in a time-dependent manner. Cells were treated with 0.1 mM Triptolide or Celastrol for indicated times, SENP1 and b-actin mRNA levels were determined by qRT-PCR. (C) Effect of Triptolide on SENP1 mRNA level in PCa cells. IC50 was shown as calculated by Prism 5.04. (D) and (E) Triptolide decreased SENP1 protein level in LNCaP (D) and PC-3 (E) in a dose-dependent manner. Cells were treated with indicated doses of Triptolide or Celastrol for 24 h before Western blot analysis. a-tubulin was used as a loading control. (F) Triptolide decreased SENP1 protein level in PCa cells in a time-dependent manner. PC-3 cells were treated with 1 mM Triptolide for indicated time before Western blot analysis. b-actin was used as a loading control. (G) Triptolide enhanced cellular SUMOylation in PC-3 cells. PC-3 cells were treated with 1 mM Triptolide or Celastrol for 24 h before Western blot analysis using SUMO-1 antibody. NEM was used as a positive control for a SUMO protease inhibitor. doi:10.1371/journal.pone.0037693.g004 contrast, Celastrol had no effect on c-Jun expression. To further study the effect of Triptolide on activity of c-Jun, we analyzed the c-Jun target genes expression by qRT-PCR. c-Jun target genes Cyclin A2, Cyclin D1, ETV1 and p21, which are known to be up- regulated by c-Jun were suppressed following Triptolide treatment (Fig. 6F). These data indicate that Triptolide suppresses c-Jun expression at mRNA and protein level and resultes in c-Jun mediated transcription inhibition. Down-regulation or over-expression of SENP1, c-Jun or AR inhibited Triptolide anti-PCa efficacy Since we observed that Triptolide suppressed SENP1 expres- sion, enhanced cellular SUMOylation, inhibited AR and c-Jun expression and suppressed AR/c-Jun-mediated transcription, we asked whether Triptolide anti-PCa efficacy relies on SENP1, c-Jun or AR expression in LNCaP and PC-3 cells. To answer this question, firstly we knocked down SENP1, c-Jun or AR expression by specific siRNA in LNCaP (with siRNA of SENP1, c-Jun or AR) and PC-3 (with siRNA of SENP1 or c-Jun) cells. Twenty-four hours after siRNA transfection, cells were treated with 100 nM Triptolide or vehicle control for 48 h, the viable cells number was counted then. Efficiency of siRNA againt SENP1, c-Jun and AR were evaluated by Western blot (Fig. 7C). Interestingly, in the absence of Triptolide treatment, silencing of SENP1, c-Jun or AR in LNCaP cells reduced cellular viability (Fig. 7A). Similarly, silencing of SENP1 or c-Jun in PC-3 cells also decreased cellular viability (Fig. 7B). This observation may indicate that cytotoxicity of Triptolide in PCa cells could result from Triptolide-induced downregulation of SENP1, AR or c-Jun. In order to reveal the effect of Triptolide on the SENP1, AR or c-Jun silenced cells, we introduced the value of ‘the viability ratio’ which is obtained by dividing the viable cell number of the Triptolide-treated group by that of the control group without Triptolide-treatment. Interest- ingly, knockdown of SENP1, c-Jun or AR appeared to increase cells viability ratio under Triptolide treatment (Fig. 7A, 7B), suggesting higher Triptolide resistance. We further checked the effect of SENP1, c-Jun or AR over-expression on Triptolide anti- PCa efficacy. PCa cells were transfected or co-transfected with SENP1, c-Jun and AR expression plasmids (Fig. 7F). An irrelevant protein EGFP was used as a negative control and a Triptolide binding protein XPB was used as a positive control. 48 h after transfection, cells were treated with Triptolide for another 48 h and viable cells number was counted. Ectopic expression of these proteins were evaluated by Western blot (Fig. 7F). As shown in Fig. 7D–7E, ectopic expression of SENP1, c-Jun or AR significantly increased PCa cells viability ratio under Triptolide treatment, indicating that rescuing these three Triptolie down- regulating proteins expression could inhibit Triptolide cell toxicity. Furthermore, co-expression of these proteins conferred higher cells viability ratio than respective individual expression, suggesting that all these proteins are involved in Triptolide cytotoxity. In contrast, over-expression of an irrelevant protein EGFP did not affect the toxicity of Triptolide, which showed similar cells viability ratio with empty vector control, suggesting that the effect of SENP1, c- Jun or AR over-expression on Triptolide toxicity were indeed resulted from their molecular function. Interestingly, ectopic expression of SENP1, c-Jun or AR showed similar effect under Triptolide treatment as XPB over-expression did. It was reported that Triptolide binds XPB to induce RNA polymerase II– mediated transcription inhibition, suggesting that down-regulation of SENP1, c-Jun and AR expression are important for Triptolide effect. We noticed that ectopic expression of these proteins including EGFP mildly decreased cells number, especially in PC-3 cell line. This may be due to the robust expression of these proteins affect other protein expression. Nevertheless, this effect did not influence Triptolide activity since ectopic expression of EGFP showed similar cell viability ratio with vector control under Triptolide treatment, and ectopic expression or co-expression of SENP1, c-Jun or AR induced cell number increase in Triptolide treatment groups in LNCaP cells comparing with vector control. Taken together, down-regulation or over-expression of SENP1, c- Jun or AR appears to significantly inhibit Triptolide anti-PCa toxicity, indicating that down-regulation of SENP1, c-Jun or AR by Tritpolide is important for Tritplode activity, suggesting these proteins are key targets of Triptolide for its’ anti-PCa effect. Discussion The Chinese herb Tripterygium wilfordii Hook F has been used for centuries in traditional Chinese medicine for treating fever, chills, edema and carbuncle. Recently, this herb has attracted intensive attention from researcher worldwide for its potent efficacy on many diseases. More than 100 small active compounds have been extracted from this herb. The diterpenoid epoxide Triptolide and the quinine triterpene Celastrol, two main bioactive components of Tripterygium wilfordii Hook F, exhibit anti-tumor activity. They have been shown to suppress cell proliferation and induce apoptosis in many types of cancer including breast cancer [3,28], pancreas cancer [4,29], prostate cancer [1,5,30], either in cell culture assay or in xenografted tumor assay. Celastrol has been identified as a natural 26 s proteasomal inhibitor that has great potential for PCa therapy [1]. But the efficacy of Triptolide on human PCa has been less studied. In this study, we demonstrated that Triptolide suppressed proliferation and induced apoptosis in two PCa cell lines: the androgen-dependent LNCap cells and androgen-independent PC-3 cells. This is in consistent with the findings of Li et al [31]. Furthermore, Triptolide suppressed xenografted PC-3 tumor progression in nude mice. Based on cell viability assay and IC50 value, Triptolide exhibited more potent effect than Celastrol on PCa cells and had antiproliferation activity at a nanomolar grade. It was reported that Triptolide inhibits proliferation of all 60 cancer cell lines in US National Cancer Institute with average IC50 value at 12 nM [32]. Triptolide also reveals a antiproliferative activity on a panel of 12 different cell lines with IC50 values in the 3–70 nM range [33]. All these data suggest that Triptolide is a potent natural anti-tumor compound and has potential for PCa therapy. Triptolide has been shown to induce apoptosis in many cancer cells in vitro or in vivo. In this study, we demonstrated that both PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e37693