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Metformin and the mitochondrial targeting dichloroacetate (DCA) have recently received attention due to their ability to inhibit anaerobic glycolysis, which renders most cancer cells resistant to apoptosis induction. We observed that Metformin alone exhibited a dose-dependent anti-leukemic activity in both B leukemic cell lines and primary B-chronic lymphocytic leukemia (B-CLL) patients' cells and its anti-leukemic activity was enhanced when used in combination with DCA. In order to overcome the problems of poor bioavailability and cellular uptake, which limit DCA efficacy, we have designed and synthetized cocrystals consisting of Metformin and DCA (Met-DCA) at different stoichiometric ratios. Of note, the MetH(2)(++)•2DCA(-) cocrystal exhibited enhanced in vitro anti-leukemic activity, with respect to the treatment with the mix consisting of Metformin plus DCA. In particular, the treatment with the cocrystal MetH(2)(++)•2DCA(-) induced a synergistic apoptotic cell death coupled to a marked down-modulation of the anti-apoptotic Mcl-1 protein. Taken together, our data emphasize that innovative compounds based on Metformin-DCA combination merit to be further evaluated as chemotherapeutic agents for the treatment of B-CLL.

To improve the pharmacokinetic profile of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) an N-terminal specific pegylation was performed to generate pegylated TRAIL (PEG-TRAIL). In in vitro experiments, we found that although PEG-TRAIL was slightly less efficient than recombinant TRAIL in promoting leukemic cell apoptosis, it showed an improved ability to promote migration of bone-marrow mesenchymal stem cells and to elicit the ERK1/2 intracellular signal transduction pathway. Overall, these data suggest that TRAIL pegylation retains, or even enhances, the biological activities of TRAIL relevant for its therapeutic applications.

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-α (TNF-α)–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-α-induced NF-κB 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.

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.

It has been clearly established that receptor activator of nuclear factor kappa B ligand (RANKL) is a key cytokine involved in the differentiation of osteoclastic precursors of the monocytic/macrophagic lineage. However, relatively little information is available on the ability of RANKL to modulate the expression of genes controlling cell survival/apoptosis and proliferation in human osteoclastic cells in comparison to macrophages. For this purpose, CD14+ human peripheral blood mononuclear cells, which express the cognate high affinity receptor activator of nuclear factor kappa B (RANK), were differentiated along the macrophagic or osteoclastic lineage by adding macrophage-colony stimulating factor (M-CSF) or M-CSF plus RANKL in culture for 12 days. RANKL up-regulated the expression of the chemokine MIP1α, which potentiates osteoclastic differentiation and simultaneously activated both anti-apoptotic (Bcl-2) and pro-apoptotic (CIDEB, PYCARD, and BAK-1) genes. Moreover, RANKL markedly up-regulated cylin D2, while it significantly decreased the levels of cyclin A, cyclin-dependent kinase 2, and other cyclin-dependent kinases, in keeping with the notion that end-stage osteoclasts are nondividing cells. Finally, a long-term exposure of RANKL up-regulated the adaptor protein TRAF3 but not TRAF6. Anat Rec, 2007. © 2007 Wiley-Liss, Inc.

Despite improvements in surgical and chemotherapeutic intervention of ovarian cancer over the recent decades, ovarian cancer remains the most lethal cancer in women. Notably, after an initial effective response to chemotherapeutic regimen, therapeutic resistance rises up leading to patient’s death. This scenario highlights the urgent need to develop novel diagnostic and therapeutic strategies. Recently, several efforts to better understand the molecular bases of ovarian cancer using integrated multiplatform molecular profiling have revealed an intrinsic complexity and heterogeneity among ovarian cancers. Concurrently, a growing body of evidences implies fallopian tube epithelium as the likely site of origin of the majority of ovarian cancers. This fallopian tube hypothesis has shifted the attention of ovarian cancer research from the ovarian surface epithelium to the fallopian tube epithelium leading to adjustment of in vitro and in vivo ovarian cancer models. In this review article, we critically summarize recent advances in ovarian cancer preclinical models that have the potential to accelerate and facilitate the discovery of more effective biomarkers and target drugs for personalized cancer therapy.

The role of the tumor necrosis factor (TNF) superfamily member receptor activator of nuclear factor kappa B ligand (RANKL) in promoting the differentiation of osteoclasts has been extensively characterized. In this study, we have investigated the effect of TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of cytokines, in osteoclastogenesis, by using human peripheral blood mononuclear cells and the RAW264.7 murine monocytic cell line. Both cell models differentiate into osteoclast-like cells in presence of RANKL plus macrophage-colony-stimulating factor (M-CSF), as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Unexpectedly, when added in culture in combination with RANKL plus M-CSF, TRAIL inhibited osteoclastic differentiation in both cell models. To investigate the molecular mechanism underlining such inhibitory activity, we analyzed the effect of TRAIL on the mitogen-activated protein kinases (MAPKs) pathways, which play a key role in osteoclastogenesis. Treatment with RANKL plus M-CSF activated both the ERK1/2 and p38/MAPK pathways, which are essential for proliferation and differentiation of preosteoclasts, respectively. Of note, the addition of TRAIL to RANKL plus M-CSF did not affect ERK1/2 but it profoundly inhibited p38/MAPK phosphorylation. Thus, our data demonstrate that TRAIL blocks osteoclastic differentiation and suggest that inhibition of the p38/MAPK pathway by TRAIL likely plays an important role in this process. (Blood. 2004;104:2044-2050)

// Chiara Agnoletto 1,* , Elisabetta Melloni 1,* , Fabio Casciano 1 , Gian Matteo Rigolin 2 , Erika Rimondi 3 , Claudio Celeghini 3 , Laura Brunelli 1 , Antonio Cuneo 2 , Paola Secchiero 1 , Giorgio Zauli 4 1 Department of Morphology, Surgery and Experimental Medicine and LTTA Centre, University of Ferrara, Ferrara, Italy 2 Department of Medical Sciences, University of Ferrara-Arcispedale S.Anna, Ferrara, Italy 3 Department of Life Sciences, University of Trieste, Trieste, Italy 4 Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, Trieste, Italy * These two authors equally contributed to this work Correspondence: Paola Secchiero, email: // Keywords : Sodium dichloroacetate, Nutlin-3, B-CLL, p21 Received : March 21, 2014 Accepted : May 26, 2014 Published : May 26, 2014 Abstract The anti-leukemic activity of the mitochondria-targeting small molecule sodium dichloroacetate (DCA), used alone and in association with the small molecule inhibitor of the p53/MDM2 interaction Nutlin-3, was analyzed in primary B-chronic lymphocytic leukemia (B-CLL) samples (n=22), normal peripheral blood cells (n=10) and in p53 wild-type EHEB, JVM-2, JVM-3 B lymphoblastoid cell lines. DCA exhibited a dose-dependent anti-leukemic activity in both primary B-CLL and B leukemic cell lines with a functional p53 status and showed a synergistic cytotoxic activity when used in combination with Nutlin-3. At the molecular level, DCA positively regulated p53 activity, as documented by post-transcriptional modifications of p53 protein, and synergized with Nutlin-3 in increasing the expression of the p53-target genes MDM2 , PUMA , TIGAR and in particular p21 . The potential role of p21 in mediating the DCA+Nutlin-3 anti-leukemic activity was underscored in knocking-down experiments. Indeed, transfection of leukemic cells with p21 siRNAs significantly decreased the DCA+Nutlin-3-induced cytotoxicity. Taken together, our data emphasize that DCA is a molecule that merits to be further evaluated as a chemotherapeutic agent for B-CLL, likely in combination with other therapeutic compounds.

Dear Editor: It has been extensively documented that osteoprotegerin (OPG), a soluble member of the TNF receptor superfamily, inhibits osteoclastogenesis by binding to receptor activator of NF-kB ligand (RANKL) and preventing interaction with its cognate transmembrane receptor RANK [reviewed in 1]. However, OPG can also interact with, and neutralize, TNF-related apoptosis inducing ligand (TRAIL), whose extracellular domain shares a 30% homology with the extracellular domain of RANKL [reviewed in 2]. Although some inconsistencies on the differential binding affinity of OPG for RANKL versus TRAIL were present in initial studies [2, 3], it has been recently demonstrated that the affinity of native OPG for native TRAIL is comparable to that for RANKL (45 nM versus 23 nM, respectively) at 37°C, as determined by plasmon surface resonance analysis [4]. Consistently with this biochemical study, OPG has been shown to act in a paracrine and autocrine manner by binding TRAIL and promoting the survival of multiple myeloma [5], prostate cancer [6], ameloblastoma cells [7] and synovial fibroblasts [8]. Interestingly, previous data from different groups have shown that recombinant TRAIL modulates the differentiation of erythroid and myeloid precursors [9–10], while inhibits both human and mouse osteoclastogenesis when added to pre-osteoclast cultures induced to differentiate with recombinant macrophage colony-stimulating factor (M-CSF) +RANKL as well as to mature osteoclasts [11–14]. On the other hand, a couple of studies suggested that recombinant soluble TRAIL might promote osteoclastogenesis [4, 15], and the proposed molecular mechanism to explain such observation was a competition between TRAIL and RANKL for OPG binding. However, it should be noticed that Vitovsky et al. used much higher concentrations of TRAIL (500 ng/ml) than RANKL (30 ng/ml) or OPG (50 ng/ml) and more importantly used mouse bone marrow pre-osteoclasts [4]. In this respect, it has been clearly shown that mouse pre-osteoclasts only express TRAIL-R2 [2], while human peripheral blood-derived pre-osteoclasts express both death receptors TRAIL-R1 and TRAIL-R2 as well as TRAIL-R4 [11–14]. Therefore, to further elucidate the important issue of the interplay between RANKL, TRAIL and OPG in human osteoclastogenesis, we have cultured adherent PBMC with M-CSF+RANKL for 14 days in the absence or presence of recombinant OPG and recombinant TRAIL, prepared as previously described [16]. TRAIL and OPG were added alone or in combination. Importantly, all cytokines were used at the same concentration (50 ng/ml). As expected [11–14], the addition of TRAIL to M-CSF+RANKL significantly (P < 0.05) inhibited osteoclast formation (Fig. 1A and B). Of note, recombinant OPG completely abrograted (P < 0.05) osteoclast formation irrespectively of the presence of recombinant TRAIL in culture (Fig. 1A and B). The anti-osteoclastic activity of OPG could not be ascribed to a low affinity of OPG for TRAIL since OPG (50 ng/ml) efficiently inhibited the apoptosis induced by TRAIL (50 ng/ml) in HL-60 leukemic cells (data not shown). 1 Effect of combined treatment of RANKL, TRAIL and OPG on osteoclastic differentiation. Adherent PBMC were cultured with M-CSF alone for 6 days. Then cells were cultured for additional 14 days with the addition of RANKL in the absence or presence of TRAIL ... Our current observations on one hand confirm that TRAIL has anti-osteoclastic activity and on the other hand indicate that it does not affect the potent anti-osteoclastic activity of OPG at least in the simplified model of osteoclastogenesis represented by human PBMC induced to differentiate by M-CSF+RANKL. Taken together with previous studies [4, 11–15], these data also suggest that the relative concentrations of TRAIL, RANKL and OPG in the local microenvironment are likely key determinant for the regulation of osteoclastogenesis.

Background: TNF-related apoptosis inducing ligand (TRAIL) is a pleiotropic cytokine, which plays a key role in the immune system as well as in controlling the balance of apoptosis and proliferation in various organs and tissues. Objective: To investigate the presence and levels of soluble TRAIL in human colostrum and milk. Methods: The levels of soluble human TRAIL were measured in human colostrum (day 2 after delivery) and breast milk (day 5 after delivery). The presence of TRAIL was also measured in infant formula. Results: Levels of soluble TRAIL in the colostrum and mature human milk were, respectively, at least 400 and 100 fold higher than those detected in human serum. No TRAIL was detected in formula. Conclusion: Human soluble TRAIL is present at extremely high levels in human colostrum and human milk and might have a significant role in mediating the anti-cancer activity of human milk.

Twenty patients affected by chronic venous disease (CVD) in tertiary venous network and/or saphenous vein were analyzed before surgical ablation by echo-color-doppler for the hemodynamic parameters reflux time (RT) and resistance index (RI), a negative and a positive prognostic factor, respectively. RT and RI were next correlated with relevantin vitroparameters of venous endothelial cells (VEC) obtained from surgical specimens, such as cell migration in response to serum gradient, proliferation index, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression, as well as cytokines release. Of interest, ICAM-1 expression in patient-derived VEC cultures correlated positively with RT and negatively with RI. Moreover, RT showed a positive correlation with the baseline osteoprotegerin (OPG) expression by VEC and an inverse correlation with VEC proliferation index. On the other hand, RI correlated positively with TNF-related apoptosis inducing ligand (TRAIL) expression. Among the cytokines released by VEC, GM-CSF showed a positive correlation with VEC proliferation and TRAIL expression and a negative correlation with OPG, ICAM-1 and VCAM-1 expression. Sincein vitrorecombinant GM-CSF induced VEC proliferation and counteracted the induction of ICAM-1, VCAM-1 and OPG upon exposure to TNF-α, our data suggest an anti-inflammatory activity of GM-CSF on venous endothelial cells.

Background: The immune modulator TNF-related apoptosis-inducing ligand (TRAIL) has been found at extremely high levels in human milk of women with normal gestation at day 5 after delivery. Objective: To investigate the presence and the levels of soluble TRAIL in human milk of women with preterm delivery at different time points post-partum (32, 34, and 36 weeks from conception). Methods: The levels of soluble TRAIL were analyzed by ELISA in the breast milk of a group of 25 women with preterm delivery at different gestational ages. Results: Soluble TRAIL was present at high levels in human milk since early post-conceptional ages (32 weeks). No significant differences in TRAIL levels were noticed with respect to different gestational ages, or with respect to time of collection when comparing, in a selected group of patients, samples obtained between 15 and 26 days with those obtained 27 and 40 days after birth. Conclusion: Due to the key immunoregulatory role of human soluble TRAIL, the presence of high levels of TRAIL in the milk of women with preterm delivery and its maintenance at high levels up to 72 days after birth support the importance of breastfeeding the preterm newborn.

Osteoprotegerin (OPG) is a soluble member of the tumor necrosis factor (TNF) receptor family, initially characterized for its ability to inhibit the receptor activator of NFkB ligand (RANKL)-stimulated formation of osteoclasts [1]. OPG also interacts with TNF-related apoptosis inducing ligand (TRAIL) [2], a different member of the TNF super-family whose extracellular domain shares a 35% homology with RANKL [3]. Several studies have demonstrated that OPG is elevated in the serum/plasma of patients affected by different types of cancer (reviewed in [4]), but the potential role of OPG with respect to cancer development/progression is unknown. Among different potential cellular sources of circulating OPG, endothelial cells represent a major source of OPG under basal conditions as well as in response to inflammatory stimuli [5]. Hydrogen sulfide (H2S)—known for decades as a toxic gas—is endogenously generated from cysteine, in reactions catalyzed by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) [6–8]. Mounting data on endogenously generated H2S have now included this gas in the family of gasotransmitters, together with nitric oxide (NO) and carbon monoxide (CO), and its effects are intensively investigated both at the cellular and molecular level [7]. On these bases, the aim of the present study was to investigate in vitro the effect of H2S on the expression and release of OPG by human vascular endothelial cells in the absence or presence of the pro-inflammatory cytokine TNF-α.

A range of cell types of mesenchymal origin express α-smooth muscle actin (α-SMA), a protein that plays a key role in controlling cell motility and differentiation along the fibrocyte and myofibroblast lineages. Although α-SMA is often expressed in stromal cells associated to a variety of cancers including hematological malignancies, up to now the role of anti-cancer drugs on α-SMA has not been deeply investigated. In this study, we demonstrated that Nutlin-3, the small molecule inhibitor of the MDM2/p53 interactions, significantly up-regulated the mRNA and protein levels of α-SMA in normal macrophages as well as in p53(wild-type) but not in p53(mutated/null) myeloid leukemic cells. The p53-dependence of α-SMA up-regulation induced by Nutlin-3 was demonstrated in experiments performed with siRNA for p53. Of note, Nutlin-3 mediated up-regulation of α-SMA in OCI leukemic cells was accompanied by cell adhesion to plastic substrate and by reduced cell migratory response in transwell assays. Notably, the role of α-SMA induction in the modulation of myeloid cell migration was clearly documented in α-SMA gene knockdown experiments. In addition, Nutlin-3 significantly up-regulated α-SMA expression in primary endothelial cells, but not in fibroblasts and mesenchymal stem cells (MSC). Conversely, transforming growth factor-β1 up-regulated α-SMA in fibroblasts and MSC, but not in macrophages and endothelial cells. Taken together, these data indicate that Nutlin-3 is a potent inducer of α-SMA in both normal and leukemic myeloid cells as well as in endothelial cells.

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.

BackgroundSince circulating levels of TNF-related apoptosis inducing ligand (TRAIL) may be important in the physiopathology of pregnancy, we tested the hypothesis that TRAIL levels change at delivery in response to stressful conditions.Methods/principal findingsWe conducted a longitudinal study in a cohort of 73 women examined at week 12, week 16, delivery and in the corresponding cord blood (CB). Serum TRAIL was assessed in relationship with maternal characteristics and to biochemical parameters. TRAIL did not vary between 12 (67.6±27.6 pg/ml, means±SD) and 16 (64.0±16.2 pg/ml) weeks' gestation, while displaying a significant decline after partum (49.3±26.4 pg/ml). Using a cut-off decline >20 pg/ml between week 12 and delivery, the subset of women with the higher decline of circulating TRAIL (41.7%) showed the following characteristics: i) nullipara, ii) higher age, iii) operational vaginal delivery or urgent CS, iv) did not receive analgesia during labor, v) induced labor. CB TRAIL was significantly higher (131.6±52 pg/ml) with respect to the corresponding maternal TRAIL, and the variables significantly associated with the first quartile of CB TRAIL (ConclusionsStressful partum conditions and elevated CRP levels are associated with a decrease of circulating TRAIL.

The effect of the single-chain alkylphospholipid perifosine was analyzed in p53(wild-type) (SKW6.4, OCI and MOLM), p53(mutated) (BJAB, MAVER) and p53(null) (HL-60) leukemic cell lines. Perifosine promoted cytotoxicity with a combination of apoptosis induction in all cell lines and cell cycle block at the G(2)M checkpoint, which was selectively observed in p53(mutated) BJAB and MAVER cell lines. At the molecular level, perifosine induced hypophosphorylation of retinoblastoma protein and the degradation of E2F1 protein in p53(mutated) but not in p53(wild-type) cells. These data indicate that perifosine potentially represents an innovative therapeutic approach for p53(mutated) hematological malignancies.

We have analyzed the effect of the synthetic glucocorticoid dexamethasone, used alone or in combination with recombinant TRAIL, on in vitro osteoclastic differentiation of peripheral blood-derived macrophages cultured in the presence of macrophage-colony stimulating factor (M-CSF) + RANKL for 12-14 days. Dexamethasone exhibited different effects based on the concentration used. Indeed, while at 10(-7) M dexamethasone reduced the number of mature osteoclasts, at 10(-8) M showed no significant effects and at 10(-9) M significantly increased the number of mature osteoclasts, with respect to cells cultured with only M-CSF + RANKL. On the other hand, the addition in culture of recombinant TRAIL inhibited the output of mature osteoclasts induced by M-CSF + RANKL. However, the presence of dexamethasone (10(-8) or 10(-9) M) into the culture medium significantly counteracted the anti-osteoclastic activity of TRAIL. In order to ascertain whether dexamethasone, might also interfere with the anti-leukemic activity of TRAIL, the degree of apoptosis induced by TRAIL was evaluated in several myeloid (OCI, MOLM, HL-60) and lymphoid (SKW6.4, MAVER, BJAB) leukemic cell lines. The levels of TRAIL-triggered apoptosis were not significantly different between leukemic cells cultured in the absence or presence of dexamethasone. Concerning the molecular mechanism mediating the dexamethasone-suppression of the TRAIL activity in pre-osteoclasts, but not in leukemic cells, we found that dexamethasone induced a significant down-regulation of the surface levels of TRAIL-R2 in cells of the osteoclastic lineage but not in leukemic cells. The ability of dexamethasone to counteract the TRAIL pathway envisions a novel mechanism mediating the pro-osteoclastic activity of dexamethasone in vivo.

Protein kinase C (PKC)-epsilon, a component of the serine/threonine PKC family, has been shown to influence the survival and differentiation pathways of normal hematopoietic cells. Here, we have modulated the activity of PKC-epsilon with specific small molecule activator or inhibitor peptides. PKC-epsilon inhibitor and activator peptides showed modest effects on HL-60 maturation when added alone, but PKC-epsilon activator peptide significantly counteracted the pro-maturative activity of tumor necrosis factor (TNF)-alpha towards the monocytic/macrophagic lineage, as evaluated in terms of CD14 surface expression and morphological analyses. Moreover, while PKC-epsilon inhibitor peptide showed a reproducible increase of TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis, PKC-epsilon activator peptide potently counteracted the pro-apoptotic activity of TRAIL. Taken together, the anti-maturative and anti-apoptotic activities of PKC-epsilon envision a potentially important proleukemic role of this PKC family member.

The small molecule inhibitor of the MDM2/p53 interaction Nutlin-3 significantly up-regulated the steady-state mRNA and protein levels of Notch1 in TP53wild-type (OCI, SKW6.4) but not in TP53deleted (HL-60) or TP53mutated (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 TP53wild-type leukemic cells. Of note, Nutlin-3 up-regulated Notch1 expression also in primary TP53wild-type B-chronic lymphocytic leukemia (B-CLL) cells and the combined use of Nutlin-3 plus pharmacological γ-secretase inhibitors of the Notch signaling showed a synergistic cytotoxicity in both TP53wild-type leukemic cell lines and primary B-CLL cells. A potential drawback of γ-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 γ-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 + γ-secretase inhibitors might potentiate the cytotoxicity of Nutlin-3 in p53wild-type leukemic cells.

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).

Exposure of human pre-osteoclasts to the MDM2 antagonist Nutlin-3 activated the p53 pathway and significantly decreased the entry of pre-osteoclasts in the S phase in response to RANKL. Moreover, repeated exposure to Nutlin-3 suppressed osteoclastic differentiation, without affecting cell survival at any culture time. Introduction: The p53 oncosuppressor coordinates an intracellular network involved in protection from malignant transformation and cell cycle control; its activation is tightly regulated by the murine double minute 2 (MDM2) gene and p53-MDM 2 interaction can be disrupted by selective small molecule inhibitors, the Nutlins. Although the ability of Nutlins to suppress the growth of wildtype p53 tumors has been clearly established, their biological activity in normal cells and tissues has not been extensively studied. Materials and Methods: Peripheral blood mononuclear cell pre-osteoclasts were cultured with macrophage-colony stimulating factor (M-CSF ) + RANKL or co-cultured with SaOS-2 osteosarcoma cells in the presence of IL-1β to induce osteoclastic differentiation. Cell cycle was analyzed by BrdU incorporation. The degree of osteoclastic differentiation was monitored at different culture times by TRACP and DAPI staining, as well as by TRACP-5b ELISA. Finally, the role of p53 in mediating the biological activity of Nutlin-3 was studied using specific siRNA. Results: Exposure of human pre-osteoclasts to RANKL induced an early (24 h) increase in the percentage of cells in the S phase, followed by the exit from the cell cycle at later time-points. The simultaneous addition of Nutlin-3 and RANKL dose-dependently decreased the percentage of pre-osteoclasts in the S phase and induced a rapid accumulation of p53 protein coupled with the induction of p53 target genes. Unexpectedly, the administration of Nutlin-3 to pre-osteoclasts at early culture times significantly suppressed the final output of osteoclasts at day 14 of culture. The role of p53 in mediating this biological activity of Nutlin-3 was underscored by gene knockdown experiments, in which the anti-osteoclastic activity of Nutlin-3 was significantly counteracted by siRNA specific for p53. Nutlin-3 also significantly decreased the formation of osteoclasts in a co-culture system of SaOS-2 osteosarcoma and pre-osteoclastic cells. Conclusions: These findings indicate that Nutlin-3 abrogates both pre-osteoclastic proliferation and differentiation through a p53-dependent pathway and may have therapeutic implications for those neoplastic diseases characterized by an abnormal osteoclastic activity.

Background— Although in vitro studies have suggested that tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) might be involved in vascular biology, its potential role in the pathogenesis and/or treatment of atherosclerosis has not been investigated. Methods and Results— Both recombinant human TRAIL and an adeno-associated virus vector expressing human TRAIL were used to deliver TRAIL in apolipoprotein E (apoE)–null mice in which diabetes mellitus was induced by destruction of islet cells with streptozotocin. Diabetes in apoE-null mice was associated with a significant increase in atherosclerotic plaque area and complexity in the aorta as assessed by a marked increase in interstitial collagen, cellular proliferation, and macrophage infiltration and a focal loss of endothelial coverage. Repeated intraperitoneal injections of recombinant human TRAIL and a single intravenous injection of adeno-associated virus–human TRAIL significantly attenuated the development of atherosclerotic plaques in apoE-null animals. TRAIL also markedly affected the cellular composition of plaque lesions by inducing apoptosis of infiltrating macrophages and increasing the vascular smooth muscle cell content. Moreover, TRAIL promoted the in vitro migration of cultured human aortic vascular smooth muscle cells but not of monocytes or macrophages. Conversely, TRAIL selectively induced apoptosis of human cultured macrophages but not of vascular smooth muscle cells. Conclusions— Overall, data from the present study indicate that atherosclerosis in diabetic apoE-null mice is ameliorated by systemic TRAIL administration and that adeno-associated virus–mediated TRAIL gene delivery might represent an innovative method for the therapy of diabetic vascular diseases.

Despite the fact that tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and its receptors (TRAIL-Rs) are expressed in intestinal mucosa, little is known about the biological role of this system in intestinal cell physiology. The expression of surface TRAIL and TRAIL-R1, -R2, -R3, -R4 were examined by flow cytometry in the immortalized human cell line tsFHI under culture conditions promoting growth or growth arrest and expression of differentiated traits. A progressive increase of surface TRAIL expression paralleled tsFHI differentiation, consistently with immunohistochemistry analysis showing an increase of TRAIL immunostaining along the crypt-villus axis in normal jejuneal mucosa. In spite of the presence of TRAIL-R1 and TRAIL-R2 "death receptors," recombinant TRAIL was not cytotoxic for tsFHI cells. Exposure of tsFHI to recombinant TRAIL rather increased/anticipated the expression levels of the cyclin-dependent kinase inhibitors p21 and p27, which mediate the induction of growth arrest and the stabilization of differentiated traits, respectively, as well as of the canonical differentiation marker DPPIV. The differentiation inducing activity of TRAIL was abolished by pre-incubation with a Fc-TRAIL-R2 chimera. On the other hand, TRAIL did not significantly modulate the levels of osteoprotegerin (OPG), CXCL8/IL-8, CXCL9/MIG, and CXCL10/IP10 spontaneously released or induced by inflammatory cytokines. Taken together, these data suggest that TRAIL might act as a paracrine trophic cytokine on intestinal epithelium, promoting intestinal cell differentiation.

Human and rat primary sub-cultured vascular smooth muscle cells (VSMCs) showed clear expression of the death receptors TRAIL-R1 and TRAIL-R2; however, recombinant soluble TRAIL did not induce cell death when added to these cells. TRAIL tended to protect rat VSMCs from apoptosis induced either by inflammatory cytokines tumor necrosis factor-alpha + interleukin-1beta + interferon-gamma or by prolonged serum withdrawal, and promoted a significant increase in VSMC proliferation and migration. Of note, all the biological effects induced by TRAIL were significantly inhibited by pharmacological inhibitors of the ERK pathway. Western blot analysis consistently showed that TRAIL induced a significant activation of ERK1/2, and a much weaker phosphorylation of Akt, while it did not affect the p38/MAPK pathway. Taken together, these data strengthen the notion that the TRAIL/TRAIL-R system likely plays a role in the biology of the vascular system by affecting the survival, migration and proliferation of VSMCs.

Increasing experimental evidence suggests that both tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its soluble decoy receptor osteoprotegerin (OPG) are involved in vascular biology. In particular, emerging data indicate that recombinant soluble TRAIL may act as a molecule with potential anti-inflammatory activity in vascular physiopathology. Conversely, the presence of leukocytes expressing membrane-bound TRAIL in atherosclerotic lesions might be involved in the destabilization of atherosclerotic plaques by inducing apoptotic cell death of vascular smooth muscle cells in an inflammatory milieu. Also OPG seems to be involved in vascular homeostasis, by acting in a paracrine or autocrine manner as a survival factor for endothelial cells. However, an increased production of OPG may have a role in the development of vascular dysfunction likely by multiple potential mechanisms, not only related to its ability to neutralize TRAIL-activity but also mediated by its heparin-binding domain. In this review we have summarized and discussed both in vitro and in vivo data that suggest potential roles of TRAIL and OPG in vascular physiopathology. Further studies are needed to address how the TRAIL/OPG interaction, their reciprocal balance and/or interplay affect vascular biology in order to design innovative therapeutic strategies in vascular diseases.

Most neuroblastoma cell lines do not express apical caspases 8 and 10, which play a key role in mediating tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity in a variety of malignant cell types. In this study, we demonstrated that TRAIL induced a moderate but significant increase of apoptosis in the caspase 8/10-deficient SK-N-SH neuroblastoma cell line, through activation of a novel caspase 9/7 pathway. Concomitant to the induction of apoptosis, TRAIL also promoted a significant increase of prostaglandin E2 (PGE2) release by SKN-SH cells. Moreover, coadministration of TRAIL plus indomethacin, a pharmacological inhibitor of cyclooxygenase (COX), showed an additive effect on SKN-SH cell death. In spite of the ability of TRAIL to promote the phosphorylation of both ERKi/2 and p38/MAPK, which have been involved in the control of COX expression/activity, neither PD98059 nor SB203580, pharmacological inhibitors of the ERKi/2 and p38/MAPK pathways, respectively, affected either PGE2 production or apoptosis induced by TRAIL. Finally, both induction of apoptosis and PGE2 release were completely abrogated by the broad caspase inhibitor z-VAD4mk, suggesting that both biologic end points were regulated in SK-N-SH cells through a caspase 9/7-dependent pathway.