The aryl propionic acid R-flurbiprofen selectively induces p75NTR-dependent decreased survival of prostate tumor cells.

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. p75(NTR) 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 R-flurbiprofen 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.

Carprofen induction of p75NTR-dependent apoptosis via the p38 mitogen-activated protein kinase 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.

Attractin is elevated in the cerebrospinal fluid of patients with malignant astrocytoma and mediates glioma cell migration.

PURPOSE: There are a limited number of noninvasive methods available for the monitoring of neoplastic disease in the central nervous system. The goal of our study was to find reliable markers that could be used for disease monitoring as well as to identify new targets for the therapeutic intervention for malignant astrocytoma (WHO grades 3 and 4). EXPERIMENTAL DESIGN: We employed proteomic techniques to identify secreted proteins in the cerebrospinal fluid that were specific to patients with malignant astrocytoma. RESULTS: Among 60 cerebrospinal fluid samples of patients with various central nervous system diseases, attractin was consistently found to be elevated in the samples of patients with malignant astrocytoma. To independently validate these results, we examined attractin expression in a new set of 108 normal and tumoral brain tissue specimens and found elevated expression in 97% of malignant astrocytomas, with the highest levels in grade 4 tumors. Using immunohistochemistry, we further showed that attractin is produced and secreted by the tumor cells. Finally, we showed that cerebrospinal fluid from brain tumor patients induces glioma cell migration and that attractin is largely responsible for this promigratory activity. CONCLUSIONS: Our results find attractin to be a reliable secreted marker for high-grade gliomas. Additionally, our migration studies suggest that it may be an important mediator of tumor invasiveness, and thus, a potential target in future therapies.

Identification of a novel small-molecule inhibitor of the hypoxia-inducible factor 1 pathway.

Hypoxia-inducible factor 1 (HIF-1) is the central mediator of cellular responses to low oxygen and has recently become an important therapeutic target for solid tumor therapy. Inhibition of HIF-1 is expected to result in the attenuation of hypoxia-inducible genes, which are vital to many aspects of tumor biology, including adaptative responses for survival under anaerobic conditions. To identify small molecules inhibiting the HIF-1 pathway, we did a biological screen on a 10,000-membered natural product-like combinatorial library. The compounds of the library, which share a 2,2-dimethylbenzopyran structural motif, were tested for their ability to inhibit the hypoxic activation of an alkaline phosphatase reporter gene under the control of hypoxia-responsive elements in human glioma cells. This effort led to the discovery of 103D5R, a novel small-molecule inhibitor of HIF-1alpha. 103D5R markedly decreased HIF-1alpha protein levels induced by hypoxia or cobaltous ions in a dose- and time-dependent manner, whereas minimally affecting global cellular protein expression levels, including that of control proteins such as HIF-1beta, IkappaBalpha, and beta-actin. The inhibitory activity of 103D5R against HIF-1alpha was clearly shown under normoxia and hypoxia in cells derived from different cancer types, including glioma, prostate, and breast cancers. This inhibition prevented the activation of HIF-1 target genes under hypoxia such as vascular endothelial growth factor (VEGF) and glucose transporter-1 (Glut-1). Investigations into the molecular mechanism showed that 103D5R strongly reduced HIF-1alpha protein synthesis, whereas HIF-1alpha mRNA levels and HIF-1alpha degradation were not affected. 103D5R inhibited the phosphorylation of Akt, Erk1/2, and stress-activated protein kinase/c-jun-NH(2)-kinase, without changing the total levels of these proteins. Further studies on the mechanism of action of 103D5R will likely provide new insights into its validity/applicability for the pharmacologic targeting of HIF-1alpha for therapeutic purposes.

Sodium and Fluid Excretion With Torsemide in Healthy Subjects is Limited by the Short Duration of Diuretic Action.

BACKGROUND: Loop diuretics are highly natriuretic but their short duration of action permits postdiuretic sodium retention, which limits salt loss unless dietary salt is severely restricted. We tested the hypothesis that a more prolonged duration of action would enhance salt loss. METHODS AND RESULTS: Ten healthy participants were crossed over between 20 mg of oral immediate-release or extended-release (ER) torsemide while consuming a fixed diet with 300 mmol·d-1 of Na+. Compared with immediate-release, plasma torsemide after ER was 59% lower at 1 to 3 hours but 97% higher at 8 to 10 hours as a result of a >3-fold prolongation of time to maximal plasma concentrations. The relationship of natriuresis to log torsemide excretion showed marked hysteresis, but participants spent twice as long with effective concentrations of torsemide after ER, thereby enhancing diuretic efficiency. Compared with immediate-release, ER torsemide did not reduce creatinine clearance and increased fluid (1634±385 versus 728±445 mL, P<0.02) and Na+ output (98±15 versus 42±17 mmol, P<0.05) despite an 18% reduction in exposure. Neither formulation increased K+ excretion. CONCLUSIONS: Torsemide ER prolongs urine drug levels, thereby increasing the time spent with effective drug concentrations, reduces postdiuretic Na+ retention, and moderates a fall in glomerular filtration rate. It caused significant Na+ loss even during very high salt intake. Thus, a short duration of action limits salt loss with loop diuretics. These conclusions warrant testing in subjects with edema and heart failure.

Ibuprofen inhibits survival of bladder cancer cells by induced expression of the p75NTR tumor suppressor protein.

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 (p75NTR) 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 p75NTR 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 p75NTR protein expression. This rank order NSAID induction of the p75NTR protein correlated with the ability of these NSAIDs to reduce cancer cell survival. To examine a mechanistic relationship between ibuprofen induction of p75NTR protein and inhibition of survival, bladder cancer cells were transfected with ponasterone A-inducible vectors that expressed a death domain-deleted (DeltaDD) or intracellular domain-deleted (DeltaICD) p75NTR product that acts as a dominant negative antagonist of the intact p75NTR protein. Expression of DeltaDD and DeltaICD rescued cells from ibuprofen inhibition of growth. These observations suggest that p75NTR is an important upstream modulator of the anticancer effects of NSAIDs and that ibuprofen induction of the p75NTR protein establishes an alternate mechanism by which ibuprofen may exert an anticancer effect.

Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis.

Glioblastomas, like other solid tumors, have extensive areas of hypoxia and necrosis. The importance of hypoxia in driving tumor growth is receiving increased attention. Hypoxia-inducible factor 1 (HIF-1) is one of the master regulators that orchestrate the cellular responses to hypoxia. It is a heterodimeric transcription factor composed of alpha and beta subunits. The alpha subunit is stable in hypoxic conditions but is rapidly degraded in normoxia. The function of HIF-1 is also modulated by several molecular mechanisms that regulate its synthesis, degradation, and transcriptional activity. Upon stabilization or activation, HIF-1 translocates to the nucleus and induces transcription of its downstream target genes. Most important to gliomagenesis, HIF-1 is a potent activator of angiogenesis and invasion through its upregulation of target genes critical for these functions. Activation of the HIF-1 pathway is a common feature of gliomas and may explain the intense vascular hyperplasia often seen in glioblastoma multiforme. Activation of HIF results in the activation of vascular endothelial growth factors, vascular endothelial growth factor receptors, matrix metalloproteinases, plasminogen activator inhibitor, transforming growth factors alpha and beta, angiopoietin and Tie receptors, endothelin-1, inducible nitric oxide synthase, adrenomedullin, and erythropoietin, which all affect glioma angiogenesis. In conclusion, HIF is a critical regulatory factor in the tumor microenvironment because of its central role in promoting proangiogenic and invasive properties. While HIF activation strongly promotes angiogenesis, the emerging vasculature is often abnormal, leading to a vicious cycle that causes further hypoxia and HIF upregulation.

Gene therapy of prostate xenograft tumors with a p75NTR lipoplex.

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.

3,3'-diindolylmethane induction of p75NTR-dependent cell death via the p38 mitogen-activated protein kinase pathway in prostate cancer cells.

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.

Prognostic markers of astrocytoma: how to predict the unpredictable?

Astrocytomas are the most frequent tumors originating in the human nervous system. They carry a dismal prognosis as high-grade astroctyoma patients (World Health Organization [WHO] grade III and IV) rarely live beyond 5 years. At present, these tumors are mainly diagnosed through the difficult task of histologic examination of tissue obtained through stereotactic biopsy or tumor resection. In addition to determining the malignancy grade through histologic studies, the only other prognostic factors used in clinical setting are patient age and performance status. To overcome current limitations, research is underway to develop molecular approaches for glioma classification. These include identification, characterization and expansion of clinical (patient characteristics and imaging variables), histologic (WHO classification criteria) and molecular (genetic and proteomic) factors with prognostic potential. In this review the established classification characteristics, along with recent advances that may lead to the addition of new parameters and thus improve patient management and survival, are discussed.

The p38 MAPK pathway mediates aryl propionic acid induced messenger rna stability of p75 NTR in prostate cancer cells.

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 p75(NTR) 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.

Proteomic identification of biomarkers in the cerebrospinal fluid (CSF) of astrocytoma patients.

The monitoring of changes in the protein composition of the cerebrospinal fluid (CSF) can be used as a sensitive indicator of central nervous system (CNS) pathology, yet its systematic application to analysis of CNS neoplasia has been limited. There is a pressing need for both a better understanding of gliomagenesis and the development of reliable biomarkers of the disease. In this report, we used two proteomic techniques, two-dimensional gel electrophoresis (2-DE), and cleavable Isotope-Coded Affinity Tag (cICAT) to compare CSF proteomes to identify tumor- and grade-specific biomarkers in patients bearing brain tumors of differing histologies and grades. Retrospective analyses were performed on 60 samples derived from astrocytomas WHO grade II, III, and IV, schwannomas, metastastic brain tumors, inflammatory samples, and non-neoplastic controls. We identified 103 potential tumor-specific markers of which 20 were high-grade astrocytoma-specific. These investigations allowed us to identify a spectrum of signature proteins that could be used to distinguish CSF derived from control patients versus those with low- (AII) or high-grade (AIV) astrocytoma. These proteins may represent new diagnostic, prognostic, and disease follow-up markers when used alone or in combination. These candidate biomarkers may also have functional properties that play a critical role in the development and malignant progression of human astrocytomas, thus possibly representing novel therapeutic targets for this highly lethal disease.

The p75(NTR) tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells.

The p75 neurotrophin receptor (p75(NTR)) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75(NTR) retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted (DeltaDD) dominant-negative antagonist of p75(NTR) showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75(NTR)-dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75(NTR) expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75(NTR) rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75(NTR) was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75(NTR)-dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75(NTR) expressing prostate cancer cells.

Proteomic analysis of cerebrospinal fluid discriminates malignant and nonmalignant disease of the central nervous system and identifies specific protein markers.

CNS diseases are often accompanied by changes in the protein composition of cerebrospinal fluid (CSF). SELDI-TOF-MS provides an approach for identifying specific protein markers of disease in biological fluids. We compared the CSF proteomes from patients with neoplastic and reactive/inflammatory CNS diseases to identify potential biomarkers. SELDI-TOF-MS was performed on CSF derived from lumbar puncture of 32 patients, including 10 with CNS malignancies, 12 with inflammatory or reactive conditions, and 10 with unknown CNS disease. Using the SAX-2 (strong anionic exchange) chip, we uncovered three conserved protein peak ranges within each disease category. For neoplastic diseases, we identified conserved peaks at 7.5-8.0 kDa (9/10 samples), 15.1-15.9 kDa (8/10 samples), and 30.0-32.0 kDa (5/10 samples). In reactive/inflammatory diseases, conserved peaks were found at 6.7-7.1 kDa (10/12 samples), 11.5-11.9 kDa (12/12 samples), and 13.3-13.7 kDa (9/12 samples). A protein from the 30.0 to 32.0 kDa peak range found in neoplastic CSF was identified by MALDI analysis as carbonic anhydrase, a protein overexpressed in many malignancies including high-grade gliomas. Similarly, cystatin C was identified in the 13.3-13.7 kDa peak range in non-neoplastic CSF and was most prominent in inflammatory conditions. Our approach provides a rational basis for identifying biomarkers that could be used for detection, diagnosis, and monitoring of CNS diseases.

Regulation of Bim by TCR signals in CD4/CD8 double-positive thymocytes.

Bim, a BH3-only Bcl-2 family member, is required for apoptosis of thymocytes in response to negative selection signals. Regulation of the apoptotic activity of Bim during negative selection is not understood. In this study we demonstrate that in murine thymocytes undergoing apoptosis in response to anti-CD3epsilon injection, levels of Bim protein expression do not change. In immature thymocytes, Bim is associated with mitochondria before stimulation and is not regulated by a change in subcellular localization during apoptosis. We also show that Bim(EL) is rapidly phosphorylated in thymocytes in response to CD3epsilon cross-linking both in vivo and in vitro, and that phosphorylation is sustained for at least 24 h. Analysis of MHC-deficient mice shows that phosphorylation of Bim occurs in CD4/CD8 double-positive thymocytes and does not depend on activation of mature T cells. We also find that TCR cross-linking on thymocytes induces an increase in the proportion of Bcl-x(L) bound to Bim at late time points. Our results favor a model in which strong TCR signals regulate the apoptotic activity of Bim by phosphorylation and subsequent changes in binding to Bcl-x(L) in immature thymocytes.

The p75NTR mediates a bifurcated signal transduction cascade through the NF kappa B and JNK pathways to inhibit cell survival.

p75NTR is most abundantly expressed in the nervous system, but is also widely expressed in many other organs and tissues where it primarily functions as a negative regulator of cell survival. In the prostate, p75NTR functions as an inhibitory protein capable of slowing proliferation and inducing apoptosis. It has been shown that p75NTR is expressed in the normal prostate, progressively lost from malignant tumor cells in vivo, and largely absent from prostate cancer cell lines derived from metastases. Although the role of p75NTR in prostate cancer has been well established, the signal transduction pathway that mediates its inhibitory activity has only been partially elucidated. This study demonstrates that exogenous expression of p75NTR down-regulates, in a dose-dependent manner, a bifurcated signaling cascade that results in reduced expression of potent transcription effectors. This two-arm signal transduction cascade was directly linked to the upstream receptor by using dominant-negative deletion constructs of p75NTR that rescued tumor cells from p75NTR-induced loss of survival and promotion of apoptosis. Furthermore, the dominant negatives rescued alterations in the levels of signal transduction intermediates. Conversely, the use of kinase-inactive intermediates that are downstream of the receptor further reduced expression of involved transcription effectors and reduced survival of the cells. These results provide a definitive link between the proximate p75NTR and signal transduction intermediates leading to the transcription effectors NF kappa B and JNK, with associated growth suppression and induction of apoptosis.

Inhibition of cell-cycle effectors of proliferation in bladder tumor epithelial cells by the p75NTR tumor suppressor.

The neurotrophin (NTR) receptor (p75(NTR)) is a cell-surface glycoprotein that binds to the neurotrophin family of growth factors, of which the prototypic member is nerve growth factor (NGF). This receptor was previously shown to retard cell-cycle progression by inducing accumulation of cells in G(1) with a concomitant reduction of cells in the S phase of the cell cycle. Furthermore, p75(NTR) was shown to be an effective tumor suppressor of bladder cancer cell growth in vivo. In order to investigate the mechanism of p75(NTR)-dependent suppression of cell-cycle progression, we utilized transgenic clones of bladder tumor cells that express p75(NTR) in increasing concentrations to demonstrate an effect of p75(NTR) on the levels of cell-cycle regulatory proteins that modulate proliferation of tumor cells. A rank-order (dose-dependent) increase in p75(NTR) protein expression was associated with a decrease in cell proliferation. This p75(NTR)-dependent suppression of proliferation was rescued with NGF. In the absence of ligand, a dose-dependent increase in p75(NTR) protein expression was associated with reduced expression of cyclin D1, cyclin E, and cyclin-dependent kinase 2 (cdk2) as well as decreased cdk2 activity. There was also a decrease in the expression of hyper-phosphorylated retinoblastoma protein, the transcription factor E2F1, and proliferating cell nuclear antigen, and there was an increase in expression of hypophosphorylated Rb and the cdk inhibitor p16(Ink4a) with increasing p75(NTR) expression. Treatment of tumor cells with NGF ameliorated these p75(NTR)-dependent changes in the levels of cell-cycle regulatory proteins and rescued the tumor cells from p75(NTR)-dependent inhibition of proliferation. Hence, it can be concluded that p75(NTR) inhibits proliferation by altering the expression of cell-cycle regulatory proteins and that NGF ameliorates this effect.

The p75(NTR) tumor suppressor induces caspase-mediated apoptosis in bladder tumor cells.

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.