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The small molecule inhibitor of the MDM2/p53 interaction Nutlin-3 is a promising anti-cancer agent, which exhibits activity against a variety of cancers, including acute myeloid leukemia (AML). Previous studies have shown that Nutlin-3 variably induces apoptosis and cell cycle arrest in cancer cells while it shows low/absent cytotoxicity in normal cells. However, the reason for the selective pro-apoptotic activity in cancer cells with respect to normal counterparts is incompletely understood. In this study, we have compared the induction of several known target genes of p53 in two p53(wild-type) AML cell lines, OCI-AML3 and MOLM, in comparison with primary normal peripheral blood mononuclear cells (PBMC). Among several p53-target genes activated both in AML cell lines and normal PBMC (BBC3, BAX, MDM2, FAS, CDKN1A, GDF15, GADD45A, TNFRSF10B, TP53I3/PIG3), only TP53I3/PIG3 was selectively activated in MOLM and OCI-AML3, but not in PBMC. The important role of TP53I3/PIG3 in mediating the apoptotic activity of Nutlin-3 was underlined by knock-down experiments with siRNA specific for TP53I3/PIG3, which resulted in a significant decrease in the pro-apoptotic activity of Nutlin-3., (© 2012 Wiley Periodicals, Inc.)

A disseminated model of non-Hodgkin's lymphoma with prevalent liver metastasis was generated by intraperitoneal (i.p.) injection of EBV(+) B lymphoblastoid SKW6.4 in nude-SCID mice. The survival of SKW6.4 xenografts (median survival = 27 days) was significantly improved when hyaluronan scaffolds embedded with mesenchimal stem cells (MSC) were implanted in the abdominal area 4 days after SKW6.4 injection (median survival = 39.5 days). Mice implanted with MSC showed a significant improvement of hepatic functionality in lymphoma xenografts, as demonstrated by measurement of serum ALT/AST levels. Co-culture of MSC with lymphoma cells enhanced the release of hepatocyte growth factor (HGF) by MSC. These data suggest that hyaluronan-embedded MSC exert anti-lymphoma activity by ameliorating hepatic functionality.

The multi-kinase inhibitor dasatinib induced a variable but significant decrease of viability in both p53(wild-type) (EHEB, JVM-2, JVM-3) and p53(mutated) (MEC-1, MEC-2, BJAB) prolymphocytic B leukemic cells, due to a combination of cell cycle block in G1 and apoptosis. Antibody phospho-kinase array analysis revealed that dasatinib inhibited the phosphorylation of various kinases, including ERK1/2 and p38/MAPK as well as of STAT3 transcription factors, in both p53(wild-type) and p53(mutated) cells. Therefore, dasatinib might offer a novel therapeutic strategy not only for p53(wild-type), but also for p53(mutated) B malignancies that have the worst prognosis and urgently need innovative therapeutic approaches.

The soluble member of the TNF-R superfamily osteoprotegerin (OPG) is abundantly released under basal conditions by both mesenchymal stem cells (MSC) and fibroblasts and by endothelial cells upon stimulation with inflammatory cytokines. Since MSC, fibroblasts and endothelial cells represent key elements of the normal and tumor microenvironment and express detectable levels of surface TRAIL receptors, we investigated the effect of TRAIL on OPG release. Unexpectedly, recombinant TRAIL decreased the spontaneous OPG release in all cell types examined. Moreover, TRAIL decreased OPG release also in stromal cells co-cultured with lymphoma cells and counteracted the OPG induction by TN-alpha in HUVEC and MSC. Such down-regulation was not due to a masking effect in the ELISA quantification of the OPG released in the culture supernatants due to binding of OPG to its ligands (TRAIL and RANKL), as demonstrated by competition experiments with recombinant TRAIL and by the lack of RANKL release/induction. In addition, OPG down-regulation was not due to induction of cytotoxic effects by TRAIL, since the degree of apoptosis in response to TRAIL was negligible in all primary cell types. With regards to the possible molecular mechanism accounting for the down-regulation of OPG release by TRAIL, we found that treatment of MSC with TRAIL significantly decreased the phosphorylation levels of p38/MAPK. There is a suggestion that this pathway is involved in the stabilization of OPG mRNA. In this respect, the ability of TRAIL to decrease the release of OPG, in the absence of cell cytotoxicity, was mimicked by the p38/MAPK inhibitor SB203580., (Copyright © 2011 Wiley-Liss, Inc.)

Objective: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibits biological activity on vascular cells in vitro. Rapid variation of circulating TRAIL levels occurs during acute coronary ischemia, suggesting that biological pathways involving TRAIL may be activated during ischemic heart disease. However, whether differential levels of soluble TRAIL in normal individuals are associated with adverse health outcomes has not been investigated. We tested the hypothesis that TRAIL levels predict mortality in a population based sample of community dwelling men and women., Methods: Plasma TRAIL level was measured by ELISA at baseline in 1282 adults (mean age 68 years) enrolled in the InCHIANTI study. Vital status was ascertained over the six-year follow-up., Results: In multivariable Cox regression analysis adjusted for potential confounders including prevalent cardiovascular diseases (CVD), ankle-brachial index, electrocardiogram abnormalities, and inflammatory markers, baseline TRAIL levels were inversely related to all-cause mortality (p=0.008). In stratified analyses, the prognostic effect of TRAIL level was strong and highly significant in participants with prevalent CVD (N=321), (lowest versus highest quartile: HR 3.1; 95% CI 1.5-6.5) while it was negligible in those free of CVD (p value for the interaction term between CVD status and TRAIL levels=0.038). Similar findings were obtained when CVD mortality was considered as the outcome of interest., Conclusions: In older patients with CVD, low levels of TRAIL were associated with increased risk of death over a period of 6 years. Lower concentration of circulating TRAIL may be related to the clinical evolution of older adults with CVD., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Although serum osteoprotegerin (OPG) is significantly increased in diabetic subjects, its potential role in beta cell dysfunction has not been investigated. This study aimed to assess the effect of full-length OPG administered in vivo in mice on pancreatic islet structure and function and its interaction with the renin-angiotensin system (RAS). OPG-treated mice showed increased islet monocyte/macrophage infiltration, fibrosis and apoptosis with reduction of islet function. The remodeling of islet architecture was associated with increased pancreatic expression of components of the RAS, growth factor genes (transforming growth factor β and connective tissue growth factor) and inflammatory molecules (monocyte chemotactic protein-1 and vascular adhesion molecule type 1). Prevention of these changes with improvement of insulin secretion was observed in ramipril treated animals. Our data suggest that OPG might play an important role in promoting beta cell dysfunction and that the upregulation of the local RAS represents one possible mechanism responsible for the OPG-induced beta cell dysfunction., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Objective: To assess the relationship between serum concentrations of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and osteoprotegerin (OPG) and the therapeutic response to disease-modifying antirheumatic drugs (DMARD) in patients with early rheumatoid arthritis (RA)., Methods: Circulating levels of TRAIL and its soluble receptor OPG were measured by ELISA in paired serum samples obtained from 66 patients with early RA at their first visit (baseline) and after 1 year of therapy. Levels of TRAIL and OPG were analyzed in relation to the clinical response, defined by the 28-joint count Disease Activity Score (DAS28)., Results: Both serum TRAIL and OPG increased after DMARD therapy. Baseline levels of TRAIL, but not OPG, were significantly higher (p < 0.05) in the patients that achieved a clinical response by DAS28 after 1 year of therapy, versus patients without clinical response to DMARD. Baseline serum levels of TRAIL were higher (p < 0.01) in rheumatoid factor-negative patients., Conclusion: Our data suggest that the basal level of circulating TRAIL is an important determinant in the therapeutic response to DMARD in patients with early RA.

Background: This study was designed to evaluate the potential role of metalloproteinase 2 (MMP2) in promoting the cleavage of TNF-related apoptosis inducing ligand (TRAIL), whose circulating levels are decreased after acute myocardial infarction (AMI)., Methods: The levels of MMP2 and of tissue inhibitor of metalloprotease 2 (TIMP2), as well as of TRAIL, were measured by ELISA in the serum of AMI patients and in the culture supernatant of endothelial cells., Results: In AMI patients the serum levels of TRAIL showed a significant inverse correlation with the MMP2/TIMP2 ratio. In vitro MMP2 induced the cleavage of recombinant TRAIL and inactivated its ability of inducing apoptosis. Moreover, exposure of endothelial cells to TNF-alpha increased the MMP2/TIMP2 ratio in the culture supernatant., Conclusions: An impaired MMP2/TIMP2 ratio might be involved in the decreased levels of circulating TRAIL observed after AMI., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: Although multimodality treatment can induce high rate of remission in many subtypes of non-Hodgkin's lymphoma (NHL), significant proportions of patients relapse with incurable disease. The effect of human bone marrow (BM) mesenchymal stem cells (MSC) on tumor cell growth is controversial, and no specific information is available on the effect of BM-MSC on NHL., Methodology/principal Findings: The effect of BM-MSC was analyzed in two in vivo models of disseminated non-Hodgkin's lymphomas with an indolent (EBV(-) Burkitt-type BJAB, median survival = 46 days) and an aggressive (EBV(+) B lymphoblastoid SKW6.4, median survival = 27 days) behavior in nude-SCID mice. Intra-peritoneal (i.p.) injection of MSC (4 days after i.p. injection of lymphoma cells) significantly increased the overall survival at an optimal MSC:lymphoma ratio of 1:10 in both xenograft models (BJAB+MSC, median survival = 58.5 days; SKW6.4+MSC, median survival = 40 days). Upon MSC injection, i.p. tumor masses developed more slowly and, at the histopathological observation, exhibited a massive stromal infiltration coupled to extensive intra-tumor necrosis. In in vitro experiments, we found that: i) MSC/lymphoma co-cultures modestly affected lymphoma cell survival and were characterized by increased release of pro-angiogenic cytokines with respect to the MSC, or lymphoma, cultures; ii) MSC induce the migration of endothelial cells in transwell assays, but promoted endothelial cell apoptosis in direct MSC/endothelial cell co-cultures., Conclusions/significance: Our data demonstrate that BM-MSC exhibit anti-lymphoma activity in two distinct xenograft SCID mouse models of disseminated NHL.

Background: This study was designed to evaluate the biological significance of simultaneous changes in the circulating levels of osteoprotegerin (OPG) and TNF-related apoptosis inducing ligand (TRAIL) in patients with coronary artery disease (CAD), and, in particular, with acute myocardial infarction (AMI)., Methods: Total levels of OPG and TRAIL were measured by ELISA in patients with AMI (n=113), unstable angina (UA, n=21) and healthy controls (n=120)., Results: Since OPG was elevated during the acute phase (first 12-24-48h) after AMI and in patients with UA with respect to healthy controls, while TRAIL was decreased in acute AMI patients, CAD patients were characterized by an increased OPG/TRAIL ratio. Moreover, the OPG/TRAIL ratio was significantly (p<0.05) higher in the acute AMI patients who developed heart failure (HF) than in those who did not develop HF in the follow-up., Conclusions: An impaired OPG/TRAIL ratio after AMI is related to a higher risk of HF., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

The number of circulating mesenchymal stem cells (MSC), analyzed after acute myocardial infarction (AMI), was lower in AMI patients who developed heart failure (HF) in the follow-up. Conversely, the circulating levels of tumor necrosis factor (TNF)-alpha, and osteoprotegerin (OPG) were higher in AMI patients who developed HF with respect to the patients who did not develop HF. In vitro exposure to TNF-alpha enhanced the migration of MSC in response to TNF-related apoptosis-inducing ligand (TRAIL) and significantly increased the release of OPG by endothelial cells. On the contrary, OPG dose-dependently neutralized the in vitro pro-migratory activity of TRAIL. Thus, TNF-alpha exhibits opposite effects on MSC migration driven by TRAIL: it is capable of potentiating MSC migration as well as of inhibiting MSC migration as an indirect consequence of OPG induction, which might result in a suboptimal recruitment of circulating MSC after AMI in those patients who develop HF in the follow-up.

Background/aims: Since elevated plasma levels of osteoprotegerin (OPG) represent a risk factor for death and heart failure in patients affected by diabetes mellitus and coronary artery disease, this study aimed to elucidate potential roles of OPG in the pathogenesis of atherosclerosis., Methods and Results: Recombinant human full-length OPG, used at concentrations comparable to the elevated levels found in the serum of diabetic patients, significantly increased the proliferation rate of rodent vascular smooth muscle cells (VSMC). To mimic the moderate chronic elevation of OPG observed in diabetic patients, low doses (1 microg/mouse) of full-length human OPG were injected intraperitoneally every 3 weeks in diabetic apolipoprotein E (apoE)-null mice. The group of animals treated for 12 weeks with recombinant OPG showed a small increase in the total aortic plaque area at necropsy in comparison to vehicle-treated animals. Importantly, while no differences in the amount of interstitial collagen or the degree of macrophage infiltration were observed between OPG-treated and vehicle-treated apoE-null diabetic animals, a significant increase in the number of alpha-actin-positive smooth muscle cells was observed in the plaques of OPG-treated mice., Conclusions: Our data suggest that OPG promotes VSMC proliferation and might be directly involved in pathogenetic aspects of atherosclerosis., (Copyright 2009 S. Karger AG, Basel.)

The small molecule inhibitor of the MDM2/p53 interaction Nutlin-3 significantly up-regulated the steady-state mRNA and protein levels of Notch1 in TP53(wild-type) (OCI, SKW6.4) but not in TP53(deleted) (HL-60) or TP53(mutated) (BJAB) leukemic cell lines. A direct demonstration that NOTCH1 was a transcriptional target of p53 in leukemic cells was obtained in experiments carried out with siRNA for p53. Moreover, inhibition of Notch1 expression using Notch1-specific siRNA significantly increased cytotoxicity in TP53(wild-type) leukemic cells. Of note, Nutlin-3 up-regulated Notch1 expression also in primary TP53(wild-type) B-chronic lymphocytic leukemia (B-CLL) cells and the combined use of Nutlin-3 plus pharmacological gamma-secretase inhibitors of the Notch signaling showed a synergistic cytotoxicity in both TP53(wild-type) leukemic cell lines and primary B-CLL cells. A potential drawback of gamma-secretase inhibitors was their ability to enhance osteoclastic maturation of normal circulating preosteoclasts induced by RANKL + M-CSF. Notwithstanding, Nutlin-3 completely suppressed osteoclastogenesis irrespective of the presence of gamma-secretase inhibitors. Taken together, these data indicate that the p53-dependent up-regulation of Notch1 in response to Nutlin-3 represents an antiapoptotic feedback mechanism able to restrain the potential therapeutic efficacy of Nutlin-3 in hematologic malignancies. Therefore, therapeutic combinations of Nutlin-3 + gamma-secretase inhibitors might potentiate the cytotoxicity of Nutlin-3 in p53(wild-type) leukemic cells.

Objective: Complement activation products contribute to a large number of inflammatory diseases, including RA. We have investigated whether osteoprotegerin (OPG) may concur with the soluble terminal complement complex (SC5b-9) to the inflammatory cascade characterizing RA., Methods: Levels of SC5b-9 and OPG in the plasma and SF of patients with active RA were determined by ELISA. The presence of SC5b-9 and OPG in RA synovial lesions was analysed by immunohistochemistry. Cultured endothelial cells were used for in vitro leucocyte/endothelial cell adhesion assays. In addition, endothelial cells were exposed to SC5b-9 in order to evaluate the effects on the production of OPG protein, as well as the activation of the OPG promoter., Results: Patients affected by active RA are characterized by elevated levels of both SC5b-9 and OPG in plasma and/or SF. Of note, we have observed a co-localization of SC5b-9 and OPG in endothelial cells of post-capillary venules of RA synovial lesions. Data on endothelial cell cultures showed that exposure to SC5b-9 induced the up-regulation of OPG expression/release, stimulating the transcriptional activity of the OPG promoter, and synergized with TNF-alpha in up-regulating OPG production., Conclusions: Our findings demonstrate that SC5b-9 induces OPG production by endothelial cells and we propose that the SC5b-9-mediated up-regulation of OPG may be an important mechanism whereby complement contributes in promoting and/or enhancing the inflammation in RA.

Little is known on the ability of different epithelia to release soluble TNF-related apoptosis-inducing ligand (TRAIL) and the relevance of TRAIL secretion by epithelial cells is still incompletely understood. On these bases, we have measured the concentration of soluble TRAIL by ELISA in the conjunctival sac fluid. It was the highest ever detected in a biological fluid (mean value of 26,800 pg/ml), being approximately 20-fold greater than that found in human saliva and >200-fold greater than that detected in human serum. On the other hand, osteoprotegerin, the soluble decoy receptor of TRAIL, was almost undetectable in the conjunctival sac fluid. Of note, the levels of soluble TRAIL measured in conjunctival sac fluid were in the range able to induce in vitro apoptosis of lymphoma cells. By in situ immunohistochemistry, TRAIL protein expression was predominantly detected in the corneal epithelium and, to a less extent, in the conjunctival epithelium. By flow cytometry analysis, membrane-associated TRAIL was documented in isolated corneal epithelial cells obtained from patients undergoing photorefractive keratectomy (PRK). The key contribution provided by corneal epithelium to the production of soluble TRAIL was underscored in time-course experiments, in which a marked decrease of the levels of soluble TRAIL in the conjunctival sac fluid was demonstrated 1 day after PRK followed by a progressive recovery at days 5-30 after PRK. Taken together, our findings strongly support a major role of soluble TRAIL in protecting cornea and conjunctiva from tumor formation and/or invasion., ((c) 2008 Wiley-Liss, Inc.)

Background: Since soluble TRAIL exhibits anti-inflammatory and anti-atherosclerotic activities both in vitro and in animal models, this study was designed to assess the relationship between the serum levels of TRAIL and clinical outcomes in patients with acute myocardial infarction (AMI)., Methodology/principal Findings: Levels of TRAIL were measured by ELISA in serial serum samples obtained from 60 patients admitted for AMI, both during hospitalization and in a follow-up of 12 months, as well as in 60 healthy control subjects. Serum levels of TRAIL were significantly decreased in patients with AMI at baseline (within 24 hours from admission), compared with healthy controls, and showed a significant inverse correlation with a series of negative prognostic markers, such as CK, CK-MB and BNP. TRAIL serum levels progressively increased at discharge, but normalized only at 6-12 months after AMI. Of note, low TRAIL levels at the patient discharge were associated with increased incidence of cardiac death and heart failure in the 12-month follow-up, even after adjustment for demographic and clinical risk parameters (hazard ratio [HR] of 0.93 [95% CI, 0.89 to 0.97]; p = 0.001)., Conclusions/significance: Although the number of patients studied was limited, our findings indicate for the first time that circulating TRAIL might represent an important predictor of cardiovascular events, independent of conventional risk markers.

Adult multipotent stromal cells (MSCs), also known as mesenchymal stem cells, represent an important source of cells for the repair of a number of damaged tissues. Both bone marrow (BM)-derived and amniotic MSCs expressed detectable surface levels of two (tumor necrosis factor-related apoptosis-inducing ligand receptor 2 [TRAIL-R2] and TRAIL-R4) of four transmembrane TRAIL receptors. Although the best-characterized activity of TRAIL-R2 is the transduction of apoptotic signals, neither recombinant TRAIL (rTRAIL) nor infection with an adenovirus-expressing TRAIL induced cytotoxic effects on MSCs. Moreover, whereas rTRAIL did not affect proliferation or differentiation of MSCs along the osteogenic and adipogenic lineages, it significantly promoted the migration of human MSCs in range of concentrations comparable to that of soluble TRAIL in human plasma (100 pg/ml). Since rTRAIL induced the rapid phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in MSC cultures and pretreatment with pharmacological inhibitors of the ERK1/2 pathway efficiently counteracted the rTRAIL-induced human MSC migration, these data indicate that ERK1/2 is involved in mediating the ability of rTRAIL to stimulate MSC migration. Taking into consideration that the soluble factors able to induce MSC migration have not been extensively characterized, our current data indicate that the TRAIL/TRAIL-R system might play an important role in the biology of MSCs. Disclosure of potential conflicts of interest is found at the end of this article.

We have compared the cytotoxic/cytostatic responses of the SKW6.4 lymphoblastoid B-cells to the alkylating agent chlorambucil, the purine analog fludarabine, the non-genotoxic activator of the p53 pathway, Nutlin-3, used alone or in association with the death-inducing ligand recombinant TRAIL. Exposure to chlorambucil, fludarabine, and Nutlin-3 induced p53 accumulation and variably affected cell cycle progression in SKW6.4 lymphoblastoid cells. In particular, chlorambucil induced cell cycle accumulation at the G2/M checkpoint; Nutlin-3 induced early cell cycle arrest at the G1/S checkpoint, while fludarabine showed an intermediate behavior. On the other hand, recombinant TRAIL alone did not affect cell cycle progression but induced a rapid increase of apoptosis. Analysis of the gene expression profile of the p53-transcriptional targets showed distinct features between chlorambucil, Nutlin-3 and fludarabine, which likely account for their differential effect on cell cycle in SKW6.4 cells. In particular, chlorambucil upregulated the steady-state mRNA expression of SFN/14-3-3sigma, a gene involved in G2/M cell cycle arrest. Of note, all agonists upregulated TRAIL-R2 expression in SKW6.4 cells both at the mRNA and protein levels. Consistently, pretreatment with chlorambucil, fludarabine and Nutlin-3 enhanced SKW6.4 sensitivity to TRAIL-mediated apoptosis., (Copyright 2007 Wiley-Liss, Inc.)

It has been shown that the expression of osteoprotegerin (OPG) is up-regulated in tumor-associated endothelial cells as well as in the sera of patients affected by both solid tumors and hematologic malignancies. We now report that sera of p53(-/-) mice contain higher levels of OPG with respect to p53(+/+) mice and that endothelial cells, in which p53 was knocked down by siRNA, release increased levels of OPG with respect to mock-transfected cells. Conversely, activation of the p53 pathway by the MDM2 small molecule antagonist Nutlin-3 significantly attenuated both spontaneous and tumor necrosis factor-alpha (TNF-alpha)-induced OPG mRNA and protein release in endothelial cell cultures. OPG promoter functional assays and chromatin immunoprecipitation experiments revealed inhibitory effects of Nutlin-3 on the TNF-alpha-induced NF-kappaB DNA binding activity to the OPG promoter. Because OPG inhibits the pro-tumoricidal activity of TNF-related apoptosis-inducing ligand, our findings suggest that, besides its well-documented functions within the malignant cancer cells, the ability of p53 to down-modulate OPG production by endothelial cells may be an additional important mechanism whereby it exerts non-cell-autonomous tumor suppression function.

Experimental evidences indicate that the TNF family member TNF-related apoptosis inducing ligand (TRAIL) might be involved in modulating osteoclastic differentiation. The ability of recombinant soluble TRAIL to affect bone density in vivo was evaluated by using 4-week-old mice subcutaneously (s.c.) injected with TRAIL for 8 days. TRAIL injection induced a significant increase of tibia trabecular thickness and total bone mass in 4-week-old mice, accompanied by a significant decrease of TRAP serum levels, without modulation of osteocalcin and osteoprotegerin (OPG). Parallel experiments performed in vitro showed that inhibition of osteoclastic differentiation, induced by treatment of human peripheral blood osteoclast precursors with TRAIL, was associated to inhibition of receptor activator of nuclear factor kappa B ligand (RANKL)-induced accumulation of p27(Kip1). The potential role of p27(Kip1) pathway in mediating the anti-osteoclastic activity of TRAIL was further suggested by in vitro gene knock-down experiments performed in osteoclast precursor cultures. Taken together, our data strongly suggest that recombinant TRAIL inhibits osteoclastogenesis by inducing the ubiquitin-mediated degradation of p27(Kip1)., ((c) 2007 Wiley-Liss, Inc.)