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.

No abstract

Epidemiologic studies show that patients chronically consuming nonsteroidal anti-inflammatory drugs (NSAID) for arthritis exhibit a reduced incidence of prostate cancer. In addition, some NSAIDs show anticancer activity in vitro. NSAIDs exert their anti-inflammatory effects by inhibiting cyclooxygenase (COX) activity; however, evidence suggests that COX-independent mechanisms mediate decreased prostate cancer cell survival. Hence, we examined the effect of selected aryl propionic acid NSAIDs and structurally related compounds on the decreased survival of prostate cancer cell lines PC-3, DU-145, and LNCaP by induction of the p75 NTR protein. p75NTR has been shown to function as a tumor suppressor in the prostate by virtue of its intracellular death domain that can initiate apoptosis and inhibit growth. The most efficacious compounds for induction of p75 NTR and decreased survival, in rank-order, were R-flurbiprofen, ibuprofen, oxaprozin, fenoprofen, naproxen, and ketoprofen. Because R-flurbiprofen and ibuprofen exhibited the greatest efficacy, we examined their dose-dependent specificity of induction for p75 NTR relative to other members of the death receptor family. Whereas treatment with R-flurbiprofen or ibuprofen resulted in a massive induction of p75 NTR protein levels, the expression of Fas, p55 TNFR , DR3, DR4, DR5, and DR6 remained largely unchanged. Moreover, transfection of either cell line before Rflurbiprofen or ibuprofen treatment with a dominant negative form of p75 NTR to antagonize p75 NTR activity or p75 NTR small interfering RNA to prevent p75 NTR protein expression rescued both cell lines from decreased survival. Hence, R-flurbiprofen and ibuprofen selectively induce p75 NTR -dependent decreased survival of prostate cancer cells independently of COX inhibition. [Cancer Res 2007;67(7):3254-62]

The p75 NTR acts as a tumor suppressor in the prostate, but its expression is lost as prostate cancer progresses and is minimal in established prostate cancer cell lines such as PC-3, DU-145, and LNCaP. Previously, we showed that treatment with R-flurbiprofen or ibuprofen induced p75 NTR expression in PC-3 and DU-145 cells leading to p75 NTR -mediated decreased survival. Here, we investigate the mechanism by which these drugs induce p75 NTR expression. We show that the observed increase in p75NTR protein due to R-flurbiprofen and ibuprofen treatment was accompanied by an increase in p75 NTR mRNA, and this increase in mRNA was the result of increased mRNA stability and not by an up-regulation of transcription. In addition, we show that treatment with R-flurbiprofen or ibuprofen led to sustained activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Furthermore, inhibition of the p38 MAPK pathway with the p38 MAPK-specific inhibitor SB202190 or by small interfering RNA (siRNA) knockdown of p38 MAPK protein prevented induction of p75 NTR by R-flurbiprofen and ibuprofen. We also observed that siRNA knockdown of MAPK-activated protein kinase (MK)-2 and MK3, the kinases downstream of p38 MAPK that are responsible for the mRNA stabilizing effects of the p38 MAPK pathway, also prevented an induction of p75 NTR by R-flurbiprofen and ibuprofen. Finally, we identify the RNA stabilizing protein HuR and the posttranscriptional regulator eukaryotic translation initiation factor 4E as two possible mechanisms by which the p38 MAPK pathway may increase p75 NTR expression. Collectively, the data suggest that R-flurbiprofen and ibuprofen induce p75 NTR expression by increased mRNA stability that is mediated through the p38 MAPK pathway. [Cancer Res 2007;67(23):11402-10]

p75NTR 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, p75NTR 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. NTR is a 75 kDa cell-surface receptor glycoprotein that binds with similar affinity to the neurotrophin family of growth factors. 3 p75 NTR exhibits overlapping sequence identity with several cell-surface proteins, including the tumor necrosis factor receptors (p75 TNFR , p55 TNFR ), Fas, 4,5 and the family of death receptors (DR3, DR4, DR5). Several of these receptor proteins, including p75 NTR , share similar sequence motifs of defined elongation structure, 5 which have been designated "death domains" based on their apoptosis-inducing activity. 6 The expression pattern of p75 NTR is widespread, extending outside of the nervous system to numerous peripheral organs and tissues, including bladder urothelium, 7 where it may negatively regulate cell survival, proliferation and growth. 8 The characterization of p75 NTR as a tumor suppressor 1,2 used 2 distinct human cell lines, the prostate PC-3 line and the TSU-pr1 line, derived from the T24 bladder cancer cell line. 9 Interestingly, even though the gene encoding p75NTR was intact in these cancer cells, expression of p75 NTR protein was suppressed due to loss of mRNA stability. 10 Nevertheless, transgenic reexpression of p75 NTR in these cancer cells by stable and transient transfection showed that p75 NTR inhibits growth of tumor cells in vitro 11 and in vivo 1,2 by induction of apoptosis. Hence, loss of p75 NTR expression appears to eliminate a potential apoptotic pathway in tumor cells, thereby facilitating the immortalization of these epithelia during transformation to a malignant phenotype. 12,13 Considering that p75 NTR is a tumor and metastasis suppressor in humans and that its apoptotic activi...