Triptolide Inhibits PCa Cells Proliferation Figure 7. Down-regulation or over-expression of SENP1, c-Jun or AR inhibited Triptolide anti-PCa efficacy. (A) and (B) Down-regulation of SENP1, c-Jun or AR expression increased cell viability upon Triptolide treatment. LNCaP and PC-3 cells were transfected with specific siRNA against SENP1, c-Jun and AR. 24 h after transfection, the transfected cells were treated with 100 nM triptolide or vehicle control for 48 h, then the viable cell number was counted in each treatment. (A) Effect of knockdown SENP1, c-Jun and AR on Triptolide efficacy in LNCaP cell. Left chart shows the viable cell number in each treatment. Right chart shows the viability ratio. (B) Effect of knockdown SENP1 and c-Jun on Triptolide efficacy in PC-3 cell. * indicates P,0.05 (compared to control). (C) Down-regulation of SENP1, c-Jun and AR protein levels by siRNA in (A) and (B). (D) and (E) Over- expression of SENP1, c-Jun and AR rescued cell viability upon Triptolide treatment. PCa cells were transfected or cotransfected with 800 ng SENP1, c- Jun and AR expression plasmids DNA as indicated. EGFP protein was used as a negative control and the Triptolide binding protein XPB was used as a positive control. Empty vector was used as blank control and to keep the total amount of plasmids DNA equal in each well. 48 h after transfection, cells were treated with Triptolide for another 48 h and viable cell number were counted. (D) Effect of ectopic expression or coexpression of SENP1, c- Jun and AR on Triptolide efficacy in LNCaP cells. (E) Effect of ectopic expression or coexpression of SENP1 and c-Jun on Triptolide efficacy in PC-3 cells. * indicates P,0.05 (compared to control). (F) Ectopic expression or coexpression of SENP1, c-Jun and AR in PCa cells evaluated by Western blot using anti-Flag and anti-HA antibodies. doi:10.1371/journal.pone.0037693.g007 Triptolide and Celastrol significantly induced apoptosis in the two PCa cell lines tested associated with caspase-3 activation and PARP cleavage. The AR-positive cell line LNCaP seems more sensitive to these two compounds than AR-negative cell line PC-3. Wang et al [34] reported that Triptolide induces apoptosis in cervical adenocarcinoma cells HeLa and pancreatic carcinoma cells PANC-1, associated with activation of caspase-8/9/3 and cleavage of PARP and Bid. Wan et al [35] found that Triptolide induces apoptosis in promyelocytic leukemia cells HL-60 with concomitant DNA fragmentation, S phase cell cycle arrest, PLoS ONE | www.plosone.org 11 May 2012 | Volume 7 | Issue 5 | e37693