Doi:10.1371/journal.pone.0065889.gResults Piperine inhibits proliferation and induces death in both androgen Contributes to cancer pathogenesis in adult animals [1]. Once transcription has been dependent (AD) LNCaP and androgen independent (AI) DU145, 22RV1, PC-3 cells 10781694 in vitroWe first determined the anti-proliferative effects of piperine on human Title Loaded From File Prostate carcinoma cells including androgen sensitive (LNCaP) (Figure 1A) and androgen insensitive (PC-3, 22Rv1, DU145 cells: Figure 1B?D). The cells were treated with (5?00 mM) piperine for 24, 48, 72 hours. The treatment of LNCaP (AD) and PC-3 (AI) cells with piperine resulted in significant reduction of proliferation or viability in a dose dependent manner with an IC50 values of 60 mM and 75 mM respectively, as assessed by MTT. In case of 22Rv1 and DU-145 prostate cancer cells, piperine treatment exhibited higher IC-50 values of 110 mM and 160 mM respectively. Thus, piperine seems to be capable of exerting a differential level of cytotoxic effects depending on the type of prostate cancer cells with androgen dependent prostate cancer cells (LNCaP) being the most sensitive one. The IC50 values obtained from this cell viability assay results were used to evaluateWestern blotting analysis: Piperine treatment activates the expression of caspase-3 and cleaves PARP-The activation of executioner caspases, i.e caspase-3, results in the cleavage of a broad spectrum of 16985061 cellular target proteins, including poly (ADP-Ribose) polymerase-1 (PARP-1), leading to the cell death [13]. Therefore, we determined the effect of piperine on the activation of caspase-3 and Poly (ADP) Ribose Polymerase. Immunoblot analysis of LNCaP (Figure 5A and Figure 5C) and DU-145, PC-3 cells (Figure 5B) treated with piperine resulted in an increase in the cleavage of caspase-3 and PARP-1 when compared with cells treated with DMSO alone. These results wereAnti Prostate Cancer Effects of PiperineFigure 4. Piperine induced caspase activation in prostate cancer cell lines. A NIR-FLIVO 747-conjugated poly-caspase probe, which is a cellpermeable fluorescent detector of active caspases, was employed to determine whether piperine activates caspase in prostate cancer cells. The LNCaP and PC-3 cell lines were treated with piperine and then tested for caspase activation. Results showed that piperine induced global caspase activation in both LNCaP (A) and PC-3 cells (B) as early as 24 hours. Experiments were repeated four times and obtained similar results as shown in this representative figure. doi:10.1371/journal.pone.0065889.gAnti Prostate Cancer Effects of PiperineFigure 5. Piperine activates apoptotic markers in androgen dependent and androgen independent prostate cancer cells by targeting AR, NF-kB and STAT-3 transcription factors. (a) Western blot analysis showed that 60 mM piperine inhibits the expression of AR, STAT-3 and NF-kB transcription factors in LNCaP cells while simultaneously activating apoptotic signals (Caspase-3 and PARP-1 activation). (b). Western blot analysis showed that 160 mM and 75 mM piperine treatment inhibits the expression of STAT-3 and NF-kB transcription factors in DU-145 and PC-3 cells by activating apoptotic markers (caspase-3 and PARP-1 activation). C) Immunoblot results showed thatpiperine at lower dose of 25 mM also inhibits the expression of AR, STAT-3 and NF-kB transcription factors in LNCaP cells in addition to downregulating PSA expression. Changes in the expression of proteins are indicated by + or 2 sign. LNCaP, DU-145 and PC-3 cells treated with 0.1 DMSO alone served as controls.Doi:10.1371/journal.pone.0065889.gResults Piperine inhibits proliferation and induces death in both androgen dependent (AD) LNCaP and androgen independent (AI) DU145, 22RV1, PC-3 cells 10781694 in vitroWe first determined the anti-proliferative effects of piperine on human prostate carcinoma cells including androgen sensitive (LNCaP) (Figure 1A) and androgen insensitive (PC-3, 22Rv1, DU145 cells: Figure 1B?D). The cells were treated with (5?00 mM) piperine for 24, 48, 72 hours. The treatment of LNCaP (AD) and PC-3 (AI) cells with piperine resulted in significant reduction of proliferation or viability in a dose dependent manner with an IC50 values of 60 mM and 75 mM respectively, as assessed by MTT. In case of 22Rv1 and DU-145 prostate cancer cells, piperine treatment exhibited higher IC-50 values of 110 mM and 160 mM respectively. Thus, piperine seems to be capable of exerting a differential level of cytotoxic effects depending on the type of prostate cancer cells with androgen dependent prostate cancer cells (LNCaP) being the most sensitive one. The IC50 values obtained from this cell viability assay results were used to evaluateWestern blotting analysis: Piperine treatment activates the expression of caspase-3 and cleaves PARP-The activation of executioner caspases, i.e caspase-3, results in the cleavage of a broad spectrum of 16985061 cellular target proteins, including poly (ADP-Ribose) polymerase-1 (PARP-1), leading to the cell death [13]. Therefore, we determined the effect of piperine on the activation of caspase-3 and Poly (ADP) Ribose Polymerase. Immunoblot analysis of LNCaP (Figure 5A and Figure 5C) and DU-145, PC-3 cells (Figure 5B) treated with piperine resulted in an increase in the cleavage of caspase-3 and PARP-1 when compared with cells treated with DMSO alone. These results wereAnti Prostate Cancer Effects of PiperineFigure 4. Piperine induced caspase activation in prostate cancer cell lines. A NIR-FLIVO 747-conjugated poly-caspase probe, which is a cellpermeable fluorescent detector of active caspases, was employed to determine whether piperine activates caspase in prostate cancer cells. The LNCaP and PC-3 cell lines were treated with piperine and then tested for caspase activation. Results showed that piperine induced global caspase activation in both LNCaP (A) and PC-3 cells (B) as early as 24 hours. Experiments were repeated four times and obtained similar results as shown in this representative figure. doi:10.1371/journal.pone.0065889.gAnti Prostate Cancer Effects of PiperineFigure 5. Piperine activates apoptotic markers in androgen dependent and androgen independent prostate cancer cells by targeting AR, NF-kB and STAT-3 transcription factors. (a) Western blot analysis showed that 60 mM piperine inhibits the expression of AR, STAT-3 and NF-kB transcription factors in LNCaP cells while simultaneously activating apoptotic signals (Caspase-3 and PARP-1 activation). (b). Western blot analysis showed that 160 mM and 75 mM piperine treatment inhibits the expression of STAT-3 and NF-kB transcription factors in DU-145 and PC-3 cells by activating apoptotic markers (caspase-3 and PARP-1 activation). C) Immunoblot results showed thatpiperine at lower dose of 25 mM also inhibits the expression of AR, STAT-3 and NF-kB transcription factors in LNCaP cells in addition to downregulating PSA expression. Changes in the expression of proteins are indicated by + or 2 sign. LNCaP, DU-145 and PC-3 cells treated with 0.1 DMSO alone served as controls.