The nonsteroidal anti-inflammatory drugs (NSAID) R-flurbiprofen and ibuprofen have been shown to induce expression of p75 NTR (neurotrophin receptor) in prostate cancer cell lines. p75 NTR , a tumor necrosis factor receptor superfamily member, is a proapoptotic protein that functions as a tumor suppressor in the human prostate. Expression of p75 NTR is lost as prostate cancer progresses and is minimal in several metastatic prostate cancer cell lines. NSAIDs induce p75 NTR through activation of the p38 mitogen-activated protein kinase (MAPK) pathway, with a concomitant decrease in cell survival. Here, we show that treatment with R-flurbiprofen and ibuprofen induces expression of the NSAID-activated gene-1 (Nag-1) protein, a divergent member of the TGF beta (TGF-b) family, in PC-3 cells. Using the selective pharmacologic inhibitor of p38 MAPK, SB202190, and p38 MAPK-specific siRNA (small interfering RNA), we show that Nag-1 induction following NSAID treatment is mediated by the p38 MAPK pathway. p75 NTR -specific siRNA pretreatment shows that Nag-1 induction byNSAIDs is downstream of p75 NTR induction. Decreased survival of NSAID-treated cells is rescued by p75 NTR -specific siRNA but not by Nag-1 siRNA. Transwell chamber and in vitro wound healing assays demonstrate decreased cell migration upon NSAID treatment. Pretreatment of PC-3 cells with p75NTR and Nag-1-specific siRNA shows that NSAID inhibition of cell migration is mediated by Nag-1 and p75NTR . These results demonstrate a role for Nag-1 in NSAID inhibition of cell migration, but not survival.

The p75 neurotrophin receptor (p75 NTR ) functions as a tumor suppressor in prostate epithelial cells, where its expression declines with progression to malignant cancer. Previously, we showed that treatment with R-flurbiprofen or ibuprofen induced p75 NTR expression in several prostate cancer cell lines leading to p75 NTR -mediated decreased survival. Using the 2-phenyl propionic acid moiety of these profens as a pharmacophore, we screened an in silico database of 30 million compounds and identified carprofen as having an order of magnitude greater activity for induction of p75 NTR levels and inhibition of cell survival. Prostate (PC-3 and DU-145) and bladder (T24) cancer cells were more sensitive to carprofen induction of p75 NTRassociated loss of survival than breast (MCF-7) and fibroblast (3T3) cells. Transfection of prostate cell lines with a dominant-negative form of p75 NTR before carprofen treatment partially rescued cell survival, showing a causeand-effect relationship between carprofen induction of p75 NTR levels and inhibition of survival. Carprofen induced apoptotic nuclear fragmentation in prostate but not in MCF-7 and 3T3 cells. Furthermore, small interfering RNA knockdown of the p38 mitogen-activated protein kinase (MAPK) protein prevented induction of p75 NTR by carprofen in both prostate cell lines. Carprofen treatment induced phosphorylation of p38 MAPK as early as within 1 min.

The p75 NTR functions as a tumor suppressor in prostate epithelial cells, where its expression declines with progression to malignant cancer. Previously, we showed that treatment with the nonsteroidal anti-inflammatory drug, indomethacin, induced p75 NTR expression in the T24 cancer cell line leading to p75 NTR -mediated decreased survival. Utilizing the indole moiety of indomethacin as a pharmacophore, we identified in rank-order with least efficacy, ketorolac, etodolac, indomethacin, 5-methylindole-3-acetic acid, indole-3-carbinol, and 3,3′-diindolylmethane (DIM) exhibiting greatest activity for induction of p75 NTR levels and inhibition of cell survival. Prostate (PC-3, DU-145) and bladder (T24) cancer cells were more sensitive to DIM induction of p75 NTR -associated loss of survival than breast (MCF7) and fibroblast (3T3) cells. Transfection of the PC-3 prostate cell line with a dominant-negative form of p75 NTR before DIM treatment significantly rescued cell survival demonstrating a cause and effect relationship between DIM induction of p75 NTR levels and inhibition of survival. Furthermore, siRNA knockdown of the p38 mitogen-activated protein kinase (MAPK) protein prevented induction of p75 NTR by DIM in the PC-3 prostate cell line. DIM treatment induced phosphorylation of p38 MAPK as early as within 1 minute. Collectively, we identify DIM as an indole capable of inducing p75 NTR -dependent apoptosis via the p38 MAPK pathway in prostate cancer cells.

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