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Lung and liver cancers are among the most deadly types of cancer. Despite improvements in treatment over the past few decades, patient survival remains poor, underlining the need for development of targeted therapies. MicroRNAs represent a class of small RNAs, frequently deregulated in human malignancies. We now report that miR221&222 are over-expressed in aggressive non small cell lung cancer and hepatocarcinoma cells, as compared with less invasive and/or normal lung and liver cells. We show that miR-221&222, by targeting PTEN and TIMP3 tumor suppressors, induce TRAIL resistance and enhance cellular migration through the activation of the AKT pathway and metallopeptidases. Finally, we demonstrate that the MET oncogene is involved in miR-221&222 activation, through the c-Jun transcription factor.

L'opera, basata sul Prometheus di M. Schünke, E. Schulte e U. Schumacher con illustrazioni di M. Voll e K. Wesker, include tre volumi: I, Basi Anatomiche per la Semeiotica; II, Basi Anatomiche per la Fisiopatologia; III: Basi Anatomiche per le Neuroscienze. Il trattato è impostato in maniera moderna, rendendo complementari la trattazione regionalistica e quella sistematica, prevedendone e consentendone il pieno utilizzo virtualmente in tutte le sedi accademiche, a prescindere dal numero di crediti e dall’organizzazione del corso. Ne risulta uno strumento didattico che consentirà non solo la personalizzazione dello studio, ma anche il successivo approfondimento dei contenuti per tutta la vita professionale del medico.

Patients with systemic lupus erythematosus (SLE) have a significant increase in cardiovascular risk although they display a preserved number of circulating angiogenic CD3+CD31+CXCR4+ T cells (Tang), a subpopulation of T cells which promotes repair of damaged endothelium. This happens due to the concomitant expansion of a Tang subset with immunosenescent features, including the loss of CD28. Therefore, the aim of this study was to elucidate the interplay between Tang subpopulations and endothelial cells in a group of young SLE patients without previous cardiovascular events. Twenty SLE female patients aged less than 40 years and 10 healthy controls (HCs) were recruited. Serum levels of interleukin (IL) -6, -8, matrix metalloproteinase (MMP) -9 and interferon (IFN) -γ were measured and flow cytometric analysis of endothelial progenitor cells (EPCs) and Tang subsets were performed. Human umbilical vein endothelial cells (HUVECs) proliferation and pro-inflammatory phenotype in response to subjects’ serum stimulation were also evaluated. Results showed that the percentage of Tang and EPCs subsets was reduced in SLE patients compared with HCs, with a marked increase of senescent CD28null cells among Tang subset. Furthermore, IL-8 serum levels were directly correlated with the percentage of Tang and inversely related to the CD28null Tang subsets, SLE disease activity index -2000 (SLEDAI-2K) was inversed related to Tang cells percentage. We indirectly evaluated the role of the Tang subset on the endothelium upon stimulation with serum from subjects with a low percentage of Tang CD3+ cells in HUVECs. HUVECs displayed pro-inflammatory phenotype with up-regulation of mRNA for IL-6, intercellular adhesion molecule (ICAM) -1, endothelial leukocyte adhesion molecule (ELAM) -1; cell proliferation rate was directly related to IL-8 serum levels and EPCs percentage. In highly selected young SLE patients without previous CV events, we found that the deterioration of Tang compartment is an early event in disease course, preceding the development of an overt cardiovascular disease and potentially mediated by SLE-specific mechanisms. The overcome of the CD28null subset exerts detrimental role over the Tang phenotype, where Tang could exert an anti-inflammatory effect on endothelial cells and might orchestrate via IL-8 the function of EPCs, ultimately modulating endothelial proliferation rate.

Physical exercise is considered to be one of the beneficial factors of a proper lifestyle and is nowadays seen as an indispensable element for good health, able to lower the risk of disorders of the cardiovascular, endocrine and osteomuscular apparatus, immune system diseases and the onset of potential neoplasms. A moderate and programmed physical exercise has often been reported to be therapeutic both in the adulthood and in aging, since capable to promote fitness. Regular exercise alleviates the negative effects caused by free radicals and offers many health benefits, including reduced risk of all-cause mortality, sarcopenia in the skeletal muscle, chronic disease, and premature death in elderly people. However, physical performance is also known to induce oxidative stress, inflammation, and muscle fatigue. Many efforts have been carried out to identify micronutrients and natural compounds, also known as nutraceuticals, able to prevent or attenuate the exercise-induced oxidative stress and inflammation. The aim of this review is to discuss the benefits deriving from a constant physical activity and by the intake of antioxidant compounds to protect the body from oxidative stress. The attention will be focused mainly on three natural antioxidants, which are quercetin, resveratrol and curcumin. Their properties and activity will be described, as well as their benefits on physical activity and on aging, which is expected to increase through the years and can get favorable benefits from a constant exercise activity.

OBJECTIVE Epidemiological evidence indicates that excess fat may be beneficial for bone health, offering protective effects against the onset of postmenopausal osteoporosis. Experimental data suggest that this link might be due to the direct effect of adipokines on bone tissue. Confirmatory evidence of this association, however, remains limited. METHODS The levels of a panel of selected adipokines including interleukin (IL)-6, -8, -1β, adipsin, lipocalin-2/neutrophil gelatinase-associated ipocalin, tumor necrosis factor alpha, monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, hepatocyte growth factor, resistin, leptin, and adiponectin in a group of osteopenic and osteoporotic postmenopausal women were compared with those of unaffected women (n = 127). RESULTS Univariate analysis revealed that leptin and adiponectin were significantly correlated with bone mineral density (BMD). In particular, leptin was positively associated with BMD of the spine (r = 0.22, P

The TNF-related apoptosis inducing ligand TRAIL is a member of the TNF superfamily that has been firstly studied and evaluated for its anti-cancer activity, and the insights into its biology have already led to the identification of several TRAIL-based anticancer strategies with strong clinical therapeutic potentials. Nonetheless, the TRAIL system is far more complex and it can lead to a wider range of biological effects other than the ability of inducing apoptosis in cancer cells. By virtue of the different receptors and the different signalling pathways involved, TRAIL plays indeed a role in the regulation of different processes of the innate and adaptive immune system and this feature makes it an intriguing molecule under consideration in the development/progression/treatment of several immunological disorders. In this context, central nervous system represents a peculiar anatomic site where, despite its "status" of immune-privileged site, both innate and adaptive inflammatory responses occur and are involved in several pathological conditions. A number of studies have evaluated the role of TRAIL and of TRAIL-related pathways as pro-inflammatory or protective stimuli, depending on the specific pathological condition, confirming a twofold nature of this molecule. In this light, the aim of this review is to summarize the main preclinical evidences of the potential/involvement of TRAIL molecule and TRAIL pathways for the treatment of central nervous system disorders and the key suggestions coming from their assessment in preclinical models as proof of concept for future clinical studies.

Inflammation plays an important role in pregnancy, and cytokine and matrix metalloproteases (MMPs) imbalance has been associated with premature rupture of membranes and increased risk of preterm delivery. Previous studies have demonstrated that lactoferrin (LF), an iron-binding protein with anti-inflammatory properties, is able to decrease amniotic fluid (AF) levels of IL-6. Therefore, we aimed to evaluate the effect of vaginal LF administration on amniotic fluid PGE2level and MMP-TIMP system in women undergoing genetic amniocentesis. One hundred and eleven women were randomly divided into controls (n=57) or treated with LF 4 hours before amniocentesis (n=54). Amniotic fluid PGE2, active MMP-9 and MMP-2, and TIMP-1 and TIMP-2 concentrations were determined by commercially available assays and the values were normalized by AF creatinine concentration. PGE2, active MMP-9, and its inhibitor TIMP-1 were lower in LF-treated group than in controls (p<0.01,p<0.005, andp<0.001, resp.). Conversely, active MMP-2 (p<0.0001) and MMP-2/TIMP-2 molar ratio (p<0.001) were increased, whilst TIMP-2 was unchanged. Our data suggest that LF administration is able to modulate the inflammatory response following amniocentesis, which may counteract cytokine and prostanoid imbalance that leads to abortion. This trial is registered with Clinical Trial numberNCT02695563.

In the last two decades, new insights have been gained regarding sex/gender-related differences in cardiovascular disease (CVD). CVD represents the leading cause of death worldwide in both men and women, accounting for at least one-third of all deaths in women and half of deaths in women over 50 years in developing countries. Important sex-related differences in prevalence, presentation, management, and outcomes of different CVDs have been recently discovered, demonstrating sex/gender-specific pathophysiologic features in the presentation and prognosis of CVD in men and women. A large amount of evidence has highlighted the role of sex hormones in protecting women from CVDs, providing an advantage over men that is lost when women reach the menopause stage. This hormonal-dependent shift of sex-related CVD risk consequently affects the overall CVD epidemiology, particularly in light of the increasing trend of population aging. The benefits of physical activity have been recognized for a long time as a powerful preventive approach for both CVD prevention and aging-related morbidity control. Exercise training is indeed a potent physiological stimulus, which reduces primary and secondary cardiovascular events. However, the underlying mechanisms of these positive effects, including from a sex/gender perspective, still need to be fully elucidated. The aim of this work is to provide a review of the evidence linking sex/gender-related differences in CVD, including sex/gender-specific molecular mediators, to explore whether sex- and gender-tailored physical activity may be used as an effective tool to prevent CVD and improve clinical outcomes in women.

Abdominal adhesions (AA) account for the most common complication of peritoneal surgery with bowel obstruction being the severest problem in the absence of effective predicting biomarkers. Anti-AA-barriers or adhesiolysis did not completely prevent bowel obstruction, although there is evidence they might reduce related complications requiring reoperation. In addition, gender-related predispositions have not been adequately investigated. We explored the role of coagulation Factor XIII (F13A1 and F13B subunit-genes) in patients following laparotomy, mostly median/lower median incision line. Globally, 426 patients (54%,♀), were PCR-SNP-genotyped for FXIIIA V34L (rs5985), FXIIIA P564L (rs5982), FXIIIA Y204F (rs3024477) and FXIIIB H95R (rs6003). Patients’ clinical phenotypes were: Group-A (n = 212), those who developed AA, and 55.2% of them developed bowel obstruction (subgroup-A1), the remaining were subgroup-A2; Group B (n = 214) were those who did not develop AA (subgroup-B1; 53.3%) or symptoms/complications (subgroup-B2). Among different laparotomy, colon surgery associated with AA at a major extent (OR = 5.1; 3.24–7.8; P

In chronic diseases, hypoxia and physical inactivity are associated with atherosclerosis progression. In contrast, a lower mortality from coronary artery disease and stroke is observed in healthy humans residing at high altitude in hypoxic environments. Eleven young, male volunteers completed the following 10-day campaigns in a randomized order: hypoxic ambulatory, hypoxic bed rest and normoxic bed rest. Before intervention, subjects were evaluated in normoxic ambulatory condition. Normobaric hypoxia was achieved in a hypoxic facility simulating 4000 m of altitude. Following hypoxia, either in bed rest or ambulatory condition, markers of cardiometabolic risk shifted toward a more atherogenic pattern consisting of: (a) lower levels of total HDL cholesterol and HDL2 sub-fraction and decreased hepatic lipase; (b) activation of systemic inflammation, as determined by C-reactive protein and serum amyloid A; (c) increased plasma homocysteine; (d) decreased delta-5 desaturase index in cell membrane fatty acids, a marker of insulin sensitivity. Bed rest and hypoxia additively decreased total HDL and delta-5 desaturase index. In parallel to the pro-atherogenic effects, hypoxia activated selected anti-atherogenic pathways, consisting of increased circulating TNF-related apoptosis-inducing ligand (TRAIL), a protective factor against atherosclerosis, membrane omega-3 index and erythrocyte glutathione availability. Hypoxia mediated changes in TRAIL concentrations and redox glutathione capacity (i.e., GSH/GSSG ratio) were greater in ambulatory conditions (+34 ± 6% and +87 ± 31%, respectively) than in bed rest (+17 ± 7% and +2 ± 27% respectively). Hypoxia-induced cardiometabolic risk is blunted by moderate level of physical activity as compared to bed rest. TRAIL and glutathione redox capacity may contribute to the positive interaction between physical activity and hypoxia. Highlights: - Hypoxia and bed rest activate metabolic and inflammatory markers of atherogenesis. - Hypoxia and physical activity activate selected anti-atherogenic pathways. - Hypoxia and physical activity positive interaction involves TRAIL and glutathione.

TNF-related apoptosis inducing ligand (TRAIL) is an intensively studied cytokine, in particular for its anticancer activity. The discovery that conjunctival sac fluid contains extremely high levels of soluble TRAIL as compared to other body fluids suggested important implications in the context of the immunological surveillance of the eye, in particular of the anterior surface. In this review, we discuss the potential physiopathologic and therapeutic role of the TRAIL/TRAIL receptor system in a variety of ocular cancers. Moreover, since an increasing amount of data has indicated the important biological activities of the TRAIL/TRAIL receptor systems also in a completely different pathologic context such as diabetes mellitus, in the second part of this review we summarize the currently available data on the involvement of TRAIL in the ocular complications of diabetes mellitus as modulator of the inflammatory and angiogenic response in the eye.

// Ayman A.M. Alameen 1, 2, * , Carolina Simioni 1, * , Alberto M. Martelli 3 , Giorgio Zauli 1 , Simona Ultimo 1 , James A. McCubrey 4 , Arianna Gonelli 1 , Giorgia Marisi 5 , Paola Ulivi 5 , Silvano Capitani 1, 6 , Luca M. Neri 1 1 Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy 2 Department of Chemical Pathology, Faculty of Medical Laboratory Sciences, University of Khartoum, Khartoum, Sudan 3 Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy 4 Department of Microbiology & Immunology, Brody School of Medicine, East Carolina University, Greenville, NC, USA 5 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e Cura dei Tumori (IRST) IRCCS, Meldola, Italy 6 LTTA Center, University of Ferrara, Ferrara, Italy * These authors have contributed equally to this work Correspondence to: Luca M. Neri, email: luca.neri@unife.it Silvano Capitani, email: silvano.capitani@unife.it Keywords: T lymphocytes, PI3K/Akt/mTOR signaling, T-acute lymphoblastic leukemia, targeted therapies, autophagy Received: May 05, 2016 Accepted: July 11, 2016 Published: August 1, 2016 ABSTRACT An attractive molecular target for novel anti-cancer therapies is the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway which is commonly deregulated in many types of cancer. Nevertheless, the effects of PI3K/Akt/mTOR inhibitors on T lymphocytes, a key component of immune responses, have been seldom explored. In this study we investigated the effects on human CD4 + T-cells of a panel of PI3K/Akt/mTOR inhibitors: BGT226, Torin-2, MK-2206, and ZSTK474. We also assessed their efficacy against two acute leukemia T cell lines. T lymphocytes were stimulated with phytohemagglutinin. Inhibitor effects on cell cycle and apoptosis were analyzed by flow cytometry, while cytotoxicity was assessed by MTT assays. In addition, the activation status of the pathway as well as induction of autophagy were analyzed by Western blotting. Quiescent healthy T lymphocytes were unaffected by the drugs whereas mitogen-stimulated lymphocytes as well as leukemic cell lines displayed a cell cycle block, caspase-dependent apoptosis, and dephosphorylation of key components of the signaling pathway. Autophagy was also induced in proliferating lymphocytes and in JURKAT and MOLT-4 cell lines. When autophagy was inhibited by 3-methyladenine or Bafilomycin A1, drug cytotoxicity was increased, indicating that autophagy is a protective mechanism. Therefore, our findings suggest that PI3K/Akt/mTOR inhibitors preserve lymphocyte viability. This is a valuable result to be taken into account when selecting drugs for targeted cancer therapy in order to minimize detrimental effects on immune function.

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.

Among 14 peripheral blood samples obtained from patients affected by B chronic lymphocytic leukemia (B-CLL) at initial stages (Rai 0–1) of the disease, 6 showed intermediate/high levels of Zap-70 while 8 displayed low/absent levels of Zap-70. Although Zap-70high and Zap-70low B-CLL samples displayed similar levels of surface death receptor TRAIL-R2, recombinant TRAIL induced cytotoxicity only in a subset of Zap-70low B-CLL samples while Zap-70high were completely resistant to TRAIL. The gene expression profiling was next analyzed in all B-CLL samples treated with either chlorambucil or recombinant TRAIL. While chlorambucil up-regulated the steady-state mRNA levels of known p53 target genes, such as PUMA, Fas/CD95 and MDM2 in all B-CLL samples examined, it significantly down-regulated survivin in Zap-70low but not in Zap-70high. On the other hand, recombinant TRAIL up-regulated the expression of several cytokines (IL-1β, IL-1α, IL-8), which have been involved in promoting B-CLL cell survival. In particular, TRAIL selectively up-regulated IL-1β in Zap-70low B-CLL samples, while it markedly and selectively up-regulated its own mRNA and that of cyclooxigenase-2 (COX-2) in Zap-70high. Taken together, our findings suggest that a significant expression of Zap-70 modulate the response of B-CLL to TRAIL, which might represents an initial step in the pathogenesis of B-CLL. J. Cell. Physiol. 213: 229–236, 2007. © 2007 Wiley-Liss, Inc.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF gene superfamily, which induces apoptosis through engagement of death receptors. TRAIL is unusual as compared to the other cytokines of this family, as it interacts with a complex system of receptors consisting of two pro-apoptotic death receptors (TRAIL-R1 and TRAIL-R2) and three decoy receptors (TRAIL-R3, TRAIL-R4 and osteoprotegerin). Moreover, with respect to other members of the TNF superfamily, such as CD95L and TNF-alpha, TRAIL has generated great interest as a potential tumor-specific cancer therapeutic because as a stable soluble trimer it selectively induces apoptosis in many transformed cells but not in normal cells. Of note, TRAIL cytotoxicity is at least partially independent of the major systems involved in resistance to chemotherapy, such as p53 wild-type function and multidrug resistance (MDR) genes. Since one fundamental problem of most cancers is the development of multiple mechanisms of resistance, which progressively reduce or suppress the therapeutic efficacy of conventional chemotherapy, new therapeutic approaches that either restore the pro-apoptotic activity of chemotherapeutic drugs or by-pass the mechanisms of resistance are highly desirable. This review will focus on the potential of TRAIL for its application in the therapy of hematological malignancies, used either alone or in combination with chemotherapy. The scenario emerging from the literature is that the treatment and management of hematological malignancies will require the rational combination of TRAIL plus conventional or new drugs in a regimen that would optimize the anti-neoplastic activity in malignant cells resistant to chemotherapy through restoration of the pro-apoptotic activity of TRAIL.

OBJECTIVE: It has been shown that SOCS-1 plays an important role in the proper control of cytokine/growth factor responses and acts as a tumor suppressor in acute myeloid leukemias. Therefore, the objective of the present study was to evaluate the in vitro effect of treatment with Nutlin-3, a small molecule inhibitor of the MDM2/p53 interaction, on the expression of the suppressor of cytokine signaling 1 in primary acute myeloid leukemia cells and in myeloid cell lines with differential p53 status. METHOD: The expression of the suppressor of cytokine signaling 1 was quantitatively analyzed by real-time PCR in myeloid p53wild-type (OCI and MOLM) and p53null HL-60, leukemic cell lines, in patient-derived acute myeloid leukemia blasts, and in primary normal cell types, such as macrophages, endothelial cells, and bone marrow mesenchymal stem cells. The p53-dependence of the suppressor of cytokine signaling 1 upregulation that is induced by Nutlin-3 was analyzed in experiments performed using siRNA for p53, while the functional upregulation of the suppressor of cytokine signaling 1 was analyzed by assessing the levels of phosphorylated STAT-3. RESULTS: Nutlin-3 significantly upregulated the transcription of the suppressor of cytokine signaling 1 in p53wild-type OCI and MOLM but not in p53deleted p53null HL60, myeloid leukemic cell lines, as well as in primary acute myeloid leukemia blasts. Conversely, and somewhat unexpectedly, Nutlin-3 did not modulate the suppressor of cytokine signaling 1 expression in primary normal macrophages, endothelial cells, and bone marrow mesenchymal stem cells. The p53-dependent upregulation of the suppressor of cytokine signaling 1 by Nutlin-3 was associated with the downregulation of phosphorylated STAT-3, a major molecular target of the suppressor of cytokine signaling 1. CONCLUSION: Overall, our data suggest a potential role for the suppressor of cytokine signaling 1 as a therapeutic target of Nutlin-3 in p53 wild-type acute myeloid leukemias.

Stromal-derived factor-1α (SDF-1α), the high-affinity ligand of CXC-chemokine receptor 4 (CXCR4), induced a progressive increase of apoptosis when added to the Jurkat CD4+/CXCR4+ T cell line. The SDF-1α-mediated Jurkat cell apoptosis was observed in serum-free or serum-containing cultures, peaked at SDF-1α concentrations of 10–100 ng/ml, required 3 days to take place, and was completely blocked by the z-VAD-fmk tripeptide caspase inhibitor. Although SDF-1α did not modify the expression of TNF-α or that of TNF-RI and TNF-RII, it increased the expression of surface Fas/APO-1 (CD95) and intracellular Fas ligand (CD95L) significantly. Moreover, the ability of SDF-1α to induce apoptosis was inhibited by an anti-CD95 Fab′ neutralizing antibody. These findings suggest a role for SDF-1α in the homeostatic control of CD4+ T-cell survival/apoptosis mediated by the CD95-CD95L pathway.

Chronic gastritis is associated frequently with persistent infection by Helicobacter pylori. However, not all patients with chronic gastritis have evidence of H. pylori infection, suggesting that other factors might contribute to the development of gastritis. The present study was undertaken to evaluate a possible etiologic role of human herpesvirus 7 (HHV-7). HHV-7 DNA was detected in about 80% of gastric biopsies, both in healthy mucosa from individuals without evidence of inflammation and in biopsies from patients with histologically confirmed chronic gastric inflammation. HHV-7 was present also in H. pylori negative samples, was associated specifically with gastric tissue and not with residual blood within the mucosa, and was present with high viral loads. HHV-7 DNA persisted in several patients also after remission of gastric inflammation and the viral presence did not correlate with specific symptoms. Analysis by RT-PCR showed that HHV-7 is transcriptionally inactive in chronic gastritis lesions. These observations show that gastric tissue represents a site of HHV-7 latent infection and a potential reservoir for viral reactivation.

Transcription of human herpesvirus-7 (HHV-7) in cultures of productively infected T-cells was studied. Transcription of HHV-7 was regulated by the typical herpesvirus cascade in which alpha, beta and gamma genes are sequentially transcribed. Transcripts of U10, U14, U18, U31, U39, U41, U42, U53, U73 and U89/90 were detected 3 h after infection and were not inhibited by the absence of protein synthesis and therefore were alpha functions. U19 and U18/20 were beta genes; their transcription was inhibited by cycloheximide but not by phosphonoacetate, an inhibitor of DNA synthesis. U60/66 and U98/100 were gamma genes since their spliced transcripts were not detected in cells treated with phosphonoacetate. HHV-7 transcription was regulated by complex mechanisms, which involve the temporal coordinated activation of specific viral promoters and post-transcriptional processing. Splice mechanisms were also temporally regulated. Transcription of U89/90 pre-mRNA and splice took place simultaneously in the immediate-early phase. On the other hand, U16/17 pre-mRNA was synthesized with typical alpha kinetics, but the spliced product was regulated as a beta function. Likewise, the primary transcripts of U60/66 and U98/100 were alpha and beta, respectively, but both spliced products were synthesized in the late phase of virus replication. Finally, HHV-7 supported a bona fide latent infection in the adult population, since viral transcripts were not detected in peripheral blood mononuclear cells of healthy donors infected with HHV-7.

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.

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-α 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.

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.

Although the role of serine/threonine protein kinase C (PKC) in malignant transformation is known from decades, an anti-PKC based approach in cancer therapy was hampered for the difficulties in developing pharmacological compounds able to selectively inhibit specific PKC isoforms. In this review, the role of PKC-epsilon and PKC-delta in promoting and counteracting tumor progression in different types of cancer, respectively, will be discussed in relationship with promising therapeutic perspectives based either on small molecule inhibitors or on natural compounds. Among a myriad of molecules able to modulate PKC activity, we will focus on the role of the enzastaurin and briostatin-1, which already entered clinical trials for several human cancers.

Although several studies have shown that the serum levels of osteoprotegerin (OPG) are significantly elevated in patients affected with atherosclerotic lesions in coronary and peripheral arteries, the cellular source and the role of OPG in the physiopathology of atherosclerosis are not completely defined. Therefore, we aimed to investigate the potential contribution of mesenchymal stem cells in the production/release of OPG. OPG was detectable by immunohistochemistry in aortic and coronary atherosclerotic plaques, within or in proximity of intimal vascular smooth muscle cells (SMC). In addition, bone marrow mesenchymal stem cell (MSC)-derived vascular SMC as well as primary aortic SMC released in the culture supernatant significantly higher levels of OPG with respect to MSC-derived endothelial cells (EC) or primary aortic EC. On the other hand, in vitro exposure to full-length human recombinant OPG significantly increased the proliferation rate of aortic SMC cultures, as monitored by bromodeoxyuridine incorporation. Taken together, these data suggest that OPG acts as an autocrine/paracrine growth factor for vascular SMC, which might contribute to the progression of atherosclerotic lesions.

SummaryLung and liver cancers are among the most deadly types of cancer. Despite improvements in treatment over the past few decades, patient survival remains poor, underlining the need for development of targeted therapies. MicroRNAs represent a class of small RNAs frequently deregulated in human malignancies. We now report that miR-221&222 are overexpressed in aggressive non-small cell lung cancer and hepatocarcinoma cells, as compared with less invasive and/or normal lung and liver cells. We show that miR-221&222, by targeting PTEN and TIMP3 tumor suppressors, induce TRAIL resistance and enhance cellular migration through the activation of the AKT pathway and metallopeptidases. Finally, we demonstrate that the MET oncogene is involved in miR-221&222 activation through the c-Jun transcription factor.

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-α 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. © The Author 2009. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved.

At variance to solid tumors, which show percentage of p53 deletions and/or mutations close to 50%, more than 80% of haematological malignancies express wild-type p53 at diagnosis. Therefore, activation of the p53 pathway by antagonizing its negative regulator murine double minute 2 (MDM2) might offer a new therapeutic strategy for the great majority of haematological malignancies. Recently, potent and selective small-molecule MDM2 inhibitors, the Nutlins, have been identified. Studies with these compounds have strengthened the concept that selective, non-genotoxic p53 activation might represent an alternative to the current cytotoxic chemotherapy. Interestingly, Nutlins not only are able to induce apoptotic cell death when added to primary leukemic cell cultures, but also show a synergistic effect when used in combination with the chemotherapeutic drugs commonly used for the treatment of haematological malignancies. Of interest, Nutlins also display non-cell autonomous biological activities, such as inhibition of vascular endothelial growth factor, stromal derived factor-1/CXCL12 and osteprotegerin expression and/or release by primary fibroblasts and endothelial cells. Moreover, Nutlins have a direct anti-angiogenic and anti-osteoclastic activity. Thus, Nutlins might have therapeutic effects by two distinct mechanisms: a direct cytotoxic effect on leukemic cells and an indirect non-cell autonomous effect on tumor stromal and vascular cells, and this latter effect might be therapeutically relevant also for treatment of haematological malignancies carrying p53 mutations.

We have investigated the effect of combined treatment with CpG-oligodeoxynucleotide (CpG-ODN) plus Nutlin-3, a small molecule inhibitor of the murine double minute 2/p53 interaction, on the immune activation, cell cycle progression, and apoptosis of peripheral blood B chronic lymphocytic leukemia (B-CLL) cells. CpG-ODN induced a robust up-regulation of immune activation markers (CD54, CD69, CD80, CD86, MHC-II) in Zap70high and Zap70low B-CLL samples. Although cotreatment of B-CLL cells with CpG-ODN + Nutlin-3 did not interfere with such immune activation, CpG-ODN potentiated the Nutlin-3-mediated induction of the death receptors CD95 and TRAIL receptor 2. Importantly, treatment with CpG-ODN did not interfere with the ability of Nutlin-3 to inhibit cell cycle progression and to induce apoptosis. Thus, a therapeutic regimen including CpG-ODN plus Nutlin-3 might have the advantage to preserve the immune activation of B-CLL cells while restraining the prosurvival/proliferative potential of CpG-ODN treatment.

Nutlin-3, a nongenotoxic activator of the p53 pathway, dose-dependently (range 0.1 to 10 μmol/L) inhibited the formation of capillaries in an in vivo matrigel assay, as well as the formation of capillary-like structures in an in vitro coculture system composed of endothelial cells surrounded by fibroblasts. In contrast to the chemotherapeutic agent doxorubicin, nutlin-3 showed no induction of apoptosis in vitro either in the cocultures or in isolated vascular endothelial cells, even when used at the highest concentration (10 μmol/L). However, treatment with pharmacological inhibitors of the nuclear factor κB and phosphatidylinositol 3-kinase/Akt pathways sensitized endothelial cells to nutlin-3–induced apoptosis. Although nutlin-3 and doxorubicin induced a comparable p53 accumulation in endothelial cells, nutlin-3 was significantly more efficient than doxorubicin in upregulating the p53 target genes CDKN1A/p21, MDM2, and GDF-15, as well as in inhibiting cell cycle progression. However, the predominant in vitro effect of nutlin-3 was its strong antimigratory activity observed at concentrations significantly lower (0.1 μmol/L) than those required to inhibit endothelial cell cycle progression. Taken together, our data suggest that the antiangiogenic activity of nutlin-3 observed in vivo was mainly attributable to inhibition of endothelial cell migration, to some extent attributable to cell cycle arrest, and to a lesser extent attributable to induction of apoptosis.

Deletions and/or mutations of p53 are relatively rare and late events in the natural history of B-cell chronic lymphocytic leukemia (B-CLL). However, it is unknown whether p53 signaling is functional in B-CLL and if targeted nongenotoxic activation of the p53 pathway by using nutlin-3, a small molecule inhibitor of the p53/MDM2 interaction, is sufficient to kill B-CLL cells. In vitro treatment with nutlin-3 induced a significant cytotoxicity on primary CD19+ B-CLL cells, but not on normal CD19+ B lymphocytes, peripheral-blood mononuclear cells, or bone marrow hematopoietic progenitors. Among 29 B-CLL samples examined, only one was resistant to nutlin-3–mediated cytotoxicity. The induction of p53 by nutlin-3 in B-CLL samples was accompanied by alterations of the mitochondrial potential and activation of the caspase-dependent apoptotic pathway. Among several genes related to the p53 pathway, nutlin-3 up-regulated the steady-state mRNA levels of PCNA, CDKN1A/p21, GDF15, TNFRSF10B/TRAIL-R2, TP53I3/PIG3, and GADD45. This profile of gene activation showed a partial overlapping with that induced by the genotoxic drug fludarabine. Moreover, nutlin-3 synergized with both fludarabine and chlorambucil in inducing B-CLL apoptosis. Our data strongly suggest that nutlin-3 should be further investigated for clinical applications in the treatment of B-CLL.

Evidence suggests that cyclooxygenase-2 (COX-2) increases tumorigenic potential by promoting resistance to apoptosis. Because B chronic lymphoid leukemia (B-CLL) cells exhibit a defective apoptotic response, we analyzed CD19 + B lymphocytes purified from the peripheral blood of B-CLL patients. Microarray analysis showed a variable (up to 38-fold) increase in the steady-state mRNA levels of COX-2 in B-CLL lymphocytes compared with normal CD19 + B lymphocytes. The up-regulation of COX-2 in B-CLL cells was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analyses. Moreover, immunohistochemical analysis of B-CLL bone marrow infiltrates confirmed clear expression of COX-2 in leukemic cells. Ex vivo treatment with the COX-2 inhibitor NS-398 significantly decreased the survival of leukemic cells by increasing the rate of spontaneous apoptosis in 13 of 16 B-CLL samples examined, but it did not affect the survival of normal lymphocytes. Pretreatment with NS-398 significantly potentiated the cytotoxicity induced by chlorambucil in 8 of 16 B-CLL samples examined. Moreover, although recombinant tumor necrosis factor-related apoptosis inducing ligand (TRAIL)/Apo2L showed little cytotoxic effect in most B-CLL samples examined, pretreatment with NS-398 sensitized 8 of 16 B-CLL samples to TRAIL-induced apoptosis. Taken together, our data indicate that COX-2 overexpression likely represents an additional mechanism of resistance to apoptosis in B-CLL and that pharmacological suppression of COX-2 might enhance chemotherapy-mediated apoptosis.

Exposure of endothelial cells to recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induced a modest (2-fold) increase of HL-60 cell adhesion as compared to TNF-α (40-fold) or interleukin 1β (IL-1β; 20-fold). However, pretreatment of endothelial cultures with TRAIL determined a significant reduction of the proadhesive activity induced by both TNF-α and IL-1β. Unexpectedly, the antiadhesive activity of TRAIL was not due to interference with the nuclear factor κB (NF-κB)-mediated up-regulation of surface intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin adhesion molecules in response to inflammatory cytokines. In searching for the molecular mechanism underlying this biologic activity of TRAIL, a cDNA microarray analysis was performed. TRAIL pretreatment variably down-modulated the mRNA steady-state levels of several TNF-α-induced chemokines, and, in particular, it abrogated the TNF-α-mediated up-regulation of CCL8 and CXCL10. Of note, the addition of optimal concentrations of recombinant CCL8 plus CXCL10 to endothelial cultures completely restored the proadhesive activity of TNF-α. Moreover, experiments performed with agonistic anti-TRAIL receptor antibodies demonstrated that both TRAIL-R1 and TRAIL-R2 contributed, although at different levels, to TRAIL-induced chemokine modulation. Taken together, our data suggest that TRAIL might play an important role in modulating leukocyte/endothelial cell adhesion by selectively down-regulating CCL8 and CXCL10 chemokines.

Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development. Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches. Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3-4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Gly(bright)) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Gly(bright)) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation. These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro.