Triptolide Inhibits PCa Cells Proliferation cleaved products are indicated. a-tubulin was used as a loading control. (F) and (G) Western blot analysis of PARP and caspase-3 proteins in Triptolide- or Celastrol-treated LNCaP (F) and PC-3 (G) cells. Cells were treated with 1 mM Triptolide or Celastrol for desired times. Uncleaved PARP and caspase-3 and their cleaved products are indicated. a-tubulin was used as a loading control. doi:10.1371/journal.pone.0037693.g002 Triptolide significantly reduced AR mRNA level in dose- dependent and time-dependent manners. We further analyzed the AR protein level in LNCaP cell treated with Triptolide, with Celastrol-treated cells as positive control. As shown in Figure 5C and 5D, Triptolide reduced AR protein expression in the same manners as mRNA level. Since AR functions through binding to androgen-response-elements (AREs) in the promoter region of target genes and regulates these androgen response genes transcription to induce cell proliferation [22], Triptolide-induced suppression of AR expression could inhibit AR-mediated tran- scription. To verify this hypothesis, expression of several AR targets genes in AR-positive LNCaP cells treated with various doses of Triptolide for 24 h was detected by qRT-PCR (Fig. 5E). The expression of PSA, BARD1, Cdk1, Cdk2 and FKBP51, known to be positively regulated by AR, were indeed suppressed. These data suggest that Triptolide inhibited AR transcription activity by suppressing AR expression. In addition to the AR target genes, prostate-specific antigen (PSA) is an important biological marker for the clinical diagnosis of human prostate cancer [23,24]. Therefore, we further detected PSA protein level in LNCaP cell lysate and culture medium by chemiluminescence immunoassay (CLIA). PSA protein level in LNCaP cells and that secreted into medium were decreased following 1 mM Triptolide treatment (Fig. 5F), while the same concentration of Celastrol showed less suppressive effect on PSA. Overall, these results indicate that Triptolide suppresses AR expression and induces AR-mediated transcription inhibition. Triptolide down-regulated c-Jun expression in PCa cells Oncoprotein c-Jun which is often over-expressed in cancer cells is involved in PCa transformation [25]. c-Jun is a major component of the transcription factor AP-1 [26,27]. In addition, c-Jun co-activates AR transcriptional activities, and SENP1 suppresses c-Jun expression [18]. In order to determine whether Triptolide regulates c-Jun expression, we treated PCa cells with various doses Triptolide or Celastrol for 24 h, or with 0.1 mM Triptolide or Celastrol for desired times. As shown in Figure 6A– 6D, c-Jun expression, at mRNA and protein levels, was suppressed by Triptolide in dose-dependent and time-dependent manners. In Figure 3. Triptolide suppressed PC-3 tumor progression in mouse xenograft model. PC-3 cells (26106 per mouse) were injected into 5- week-old nude mice. When solid tumors grew to about 100 mm3, the 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. (A) Effect of Triptolide on xenograft tumor volume. (B) Effect of Triptolide on nude mice body weight. (C) Effect of Triptolide on xenograft tumor weight. (D) Image of xenograft tumor treated with or without Triptolide.* indicates P,0.05 (compared to control). doi:10.1371/journal.pone.0037693.g003 PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e37693