Carprofen induction of p75-dependent apoptosis via the p38 mitogen-activated protein kinase pathway in prostate cancer cells

The p75 neurotrophin receptor (p75) 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 expression in several prostate cancer cell lines leading to p75-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 levels and inhibition of cell survival. Prostate (PC-3 and DU-145) and bladder (T24) cancer cells were more sensitive to carprofen induction of p75associated loss of survival than breast (MCF-7) and fibroblast (3T3) cells. Transfection of prostate cell lines with a dominant-negative form of p75 before carprofen treatment partially rescued cell survival, showing a causeand-effect relationship between carprofen induction of p75 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 by carprofen in both prostate cell lines. Carprofen treatment induced phosphorylation of p38 MAPK as early as within 1 min. Expression of a dominant-negative form ofMK2, the kinase downstream of p38 MAPK frequently associated with signaling cascades leading to apoptosis, prevented carprofen induction of the p75 protein. Collectively, we identify carprofen as a highly potent profen capable of inducing p75-dependent apoptosis via the p38 MAPK pathway in prostate cancer cells. [Mol Cancer Ther 2008;7(11):3539–45] Introduction The p75 neurotrophin receptor (p75) is a 75-kDa cell surface receptor glycoprotein that shares both structural and sequence homology with the tumor necrosis factor receptor superfamily of proteins (1, 2). Some of these proteins (e.g., p75, p55, Fas, DRs3-6, and EDAR) have similar sequence motifs of defined elongated structure (1) designated ‘‘death domains’’ based on their apoptosisinducing function (2). In the human prostate, the p75 protein is progressively lost in pathologic cancer tissues (3). The proportion of epithelial cells that have retained p75 expression in the organ-confined pathologic prostate is inversely associated with increasing Gleason score and preoperative serum prostate-specific antigen concentrations (4). In addition, immunoblot of human prostate epithelial cell lines derived from metastases exhibit a further reduction of p75 expression (5). Significantly, although expression of the p75 protein is suppressed, the gene encoding p75 appears intact in these prostate cancer cells (6). The loss of p75 expression is a result of a loss of mRNA stability (6). Following ectopic reexpression of the p75 in these cancer cells, their rate of apoptosis increased (7). Additionally, the same ectopically expressing p75 cancer cells exhibited a retardation of cell cycle progression characterized by accumulation of cells in G1 phase with a corresponding reduction of cells in the S phase of the cell cycle (7). Consistent with these observations, the p75 has been characterized with both tumor suppressor and metastasis suppressor activity in prostate cancer cells (7, 8). Several studies have shown that nonsteroidal antiinflammatory drugs (NSAID) are effective as anticancer agents for colorectal, breast, pancreatic, squamous cell carcinoma of the head and neck, bladder, ovarian, lung, and prostate cancers (9, 10). With respect to prostate cancer, retrospective studies indicate that there is a significantly reduced risk of prostate cancer associated with regular use of NSAIDs (11–13). In vivo studies using rodents have indicated that NSAIDs can decrease the size of prostate tumors (14, 15) and suppress the metastasis of prostatic cancer (14, 16). There is no common mechanism of action underlying NSAIDs effectiveness against cancer cells. Some Received 6/2/08; revised 8/13/08; accepted 8/24/08. Grant support: NIH grants R01DK52626 and U56CA101429 and Department of Defense Prostate Cancer Research Program grant PC060409. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Daniel Djakiew, Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, 3900 Reservoir Road Northwest, Washington, DC 20057-1436. Phone: 202-687-1203; Fax: 202-687-1823. E-mail: [email protected] Copyright C 2008 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-08-0512 3539 Mol Cancer Ther 2008;7(11). November 2008 on May 29, 2017. © 2008 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst October 30, 2008; DOI: 10.1158/1535-7163.MCT-08-0512