Daniel Djakiew

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-β) 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 by NSAIDs 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 p75^NTR and Nag-1–specific siRNA shows that NSAID inhibition of cell migration is mediated by Nag-1 and p75^NTR. These results demonstrate a role for Nag-1 in NSAID inhibition of cell migration, but not survival. Mol Cancer Res; 8(12); 1656–64. ©2010 AACR.

Following spinal cord injury (SCI) or peripheral neuropathy, increased levels of the p75 ^NTR death receptor initiate the signal transduction cascade leading to cell death. Investigations of compounds that may ameliorate neuronal cell death have largely used rodent models, which are time consuming, expensive, and cumbersome to perform. Previous studies had demonstrated that steroids, particularly dexamethasone and its analog methylprednisolone sodium succinate, exhibit limited neuroprotective effects against neuronal injury. Significantly, many naturally occurring nonsteroidal plant compounds exhibit structural overlap with steroids. In this report, we present an in vitro cellular screen model to practically examine the efficacy of various phytoestrogens in modulating the ibuprofen-induced expression of p75 ^NTR and reduced cell survival of CCFSTTG1 and U87MG cells in a rescue (postinjury) or prevention (preinjury) regimen. We show that the phytoestrogen, biochanin A, and, to a lesser extent, genistein are more effective than dexamethasone at reducing p75 ^NTR expression and improving the viability of U87MG and CCFSTTG1 before and after p75 ^NTR induction. Furthermore, these studies implicate biochanin A's inactivation of p38-MAPK as a possible contributor to reducing p75 ^NTR with associated increased cell survival. This new in vitro assay facilitates a more time-efficient screening of compounds to suppress p75 ^NTR expression and increase neuronal cell viability prior to their evaluation in animal models of neurological diseases.

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.

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^NTR-associated 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 cause-and-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. Expression of a dominant-negative form of MK2, the kinase downstream of p38 MAPK frequently associated with signaling cascades leading to apoptosis, prevented carprofen induction of the p75^NTR protein. Collectively, we identify carprofen as a highly potent profen capable of inducing p75^NTR-dependent apoptosis via the p38 MAPK pathway in prostate cancer cells. [Mol Cancer Ther 2008;7(11):3539–45]

The p75neurotrophin receptor (p75NTR) has been characterized as a tumor suppressor in human prostate cancer. Ectopic re-expression of this cell surface receptor protein in prostate cancer cell lines has been shown to increase the frequency of apoptosis and concurrently reduce the rate of cellular proliferation. Since it is difficult to maintain a therapeutic level of this membrane receptor protein by systemic administration in the blood, we utilized an alternative gene therapy strategy based upon intra-tumoral injection into xenografts of PC-3 prostate tumor cells of a lipoplex containing the p75NTR gene. Administration of the p75NTR gene into subcutaneous PC-3 xenografts suppressed in a dose-dependent manner the growth of tumors. Within the gene therapy-treated tumors, re-expression of the p75NTR gene product was associated with increased apoptosis and reduced proliferation of tumor cells. These results suggest that the p75NTR may be a suitable candidate for gene therapy of prostate cancer cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used to reduce inflammation and as analgesics by inhibition of cyclooxygenase-2. At higher concentrations, some NSAIDs inhibit proliferation and induce apoptosis of cancer cells. Although several molecular mechanisms have been postulated to explain the anticancer effects of NSAIDs, they do not involve merely the inhibition of cyclooxygenase-2, and a more proximate initiator molecule may be regulated by NSAIDs to inhibit growth. The p75 neurotrophin receptor (p75^NTR) is a proximate cell membrane receptor glycoprotein that has been identified as a tumor and metastasis suppressor. We observed that NSAID treatment of cell lines from bladder and other organs induced expression of the p75^NTR protein. Of the different types of NSAIDs examined, ibuprofen was more efficacious than aspirin and acetaminophen and comparable with (R)-flurbiprofen and indomethacin in induction of p75^NTR protein expression. This rank order NSAID induction of the p75^NTR protein correlated with the ability of these NSAIDs to reduce cancer cell survival. To examine a mechanistic relationship between ibuprofen induction of p75^NTR protein and inhibition of survival, bladder cancer cells were transfected with ponasterone A-inducible vectors that expressed a death domain-deleted (ΔDD) or intracellular domain-deleted (ΔICD) p75^NTR product that acts as a dominant negative antagonist of the intact p75^NTR protein. Expression of ΔDD and ΔICD rescued cells from ibuprofen inhibition of growth. These observations suggest that p75^NTR is an important upstream modulator of the anticancer effects of NSAIDs and that ibuprofen induction of the p75^NTR protein establishes an alternate mechanism by which ibuprofen may exert an anticancer effect.

P75^NTR was identified as a tumor and metastasis suppressor that functions in part via induction of apoptosis in tumor cells. To examine p75^NTR‐dependent apoptosis in tumor cells, we demonstrated that a dose‐dependent increase in p75^NTR expression was associated with a concomitant increase in the mitochondrial proapoptotic effector proteins Bad, Bax and Bik and a decrease in the mitochondrial prosurvival effector proteins phospho‐Bad, Bcl‐2 and Bcl‐x_L. Significantly, p75^NTR‐dependent induction of cytochrome c release from the mitochondria occurred during CHX potentiation of apoptosis. Furthermore, p75^NTR expression largely suppressed expression of IAP‐1 and induced cleavage of procaspase‐9 and procaspase‐7 but not of procaspases 2, 3, 6, 8 and 10. A specific peptide inhibitor of procaspase‐9 cleavage also inhibited cleavage of procaspase‐7, indicating that caspase‐7 is downstream of caspase‐9. As end points of apoptosis, we observed p75^NTR‐dependent annexin V binding to the plasma membrane, an indicator of early apoptotic events, and Hoechst staining of DNA nuclear fragmentation, an indicator of late apoptotic events, whereas control tumor cells that lack expression of the p75^NTR protein did not exhibit either of these apoptotic markers. Together, these results delineate the mitochondria‐mediated apoptotic pathway of the p75^NTR tumor‐suppressor gene product.