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Purpose: To elucidate new molecular mechanisms able to downregulate the mRNA levels of key oncogenes, such as B-Myb and E2F1, in a therapeutic perspective.Experimental Design:B-Myb and E2F1 mRNA levels were evaluated in primary B chronic lymphocytic leukemia (B-CLL, n = 10) and acute myeloid leukemia (AML, n = 5) patient cells, in a variety of p53wild-type and p53mutated/deleted leukemic cell lines, as well as in primary endothelial cells and fibroblasts. Knockdown experiments with siRNA for p53 and E2F1 and overexpression experiments with miR34a were conducted to elucidate the role of these pathways in promoting B-Myb downregulation.Results:In vitro exposure to Nutlin-3, a nongenotoxic activator of p53, variably downregulated the expression of B-Myb in primary leukemic cells and in p53wild-type myeloid (OCI, MOLM) and lymphoblastoid (SKW6.4, EHEB) but not in p53mutated (NB4, BJAB, MAVER) or p53deleted (HL-60) leukemic cell lines. The transcriptional repression of B-Myb was also observed in primary normal endothelial cells and fibroblasts. B-Myb downregulation played a critical role in the cell-cycle block in G1 phase induced by Nutlin-3, as shown by transfection experiments with specific siRNA. Moreover, we have provided experimental evidence suggesting that miR-34a is a central mediator in the repression of B-Myb both directly and through E2F1.Conclusions: Owing to the role of B-Myb and E2F1 transcription factors in controlling cell-cycle progression of leukemic cells, the downregulation of these oncogenes by miR-34a suggests the usefulness of therapeutic approaches aimed to modulate the levels of miR-34a. Clin Cancer Res; 17(9); 2712–24. ©2011 AACR.

Purpose: To analyze the effect of the combination of Dasatinib, a multikinase inhibitor, plus Nutlin-3, a nongenotoxic activator of the p53 pathway, in primary B chronic lymphocytic leukemia (B-CLL) patient samples and B leukemic cell line models.Experimental Design: The induction of cytotoxicity was evaluated in both primary B-CLL cell samples (n = 20) and in p53wild-type (EHEB, JVM-2) and p53deleted/mutated (MEC-2, BJAB) B leukemic cell lines. The role of Akt in modulating leukemic cell survival/apoptosis in response to Dasatinib or Dasatinib + Nutlin-3 was documented by functional experiments carried out using specific pharmacological inhibitors and by overexpression of membrane-targeted constitutively active form of Akt.Results: The combination of Dasatinib + Nutlin-3 exhibited a synergistic cytotoxicity in the majority (19 out of 20) of B-CLL samples, including patients carrying 17p- (n = 4), and in both p53wild-type and p53deleted/mutated B leukemic cell lines. At the molecular level, Dasatinib significantly counteracted the Nutlin-3-mediated induction of the p53 transcriptional targets MDM2 and p21 observed in p53wild-type leukemic cells. Conversely, Nutlin-3 did not interfere with the ability of Dasatinib to decrease the phosphorylation levels of ERK1/2, p38/MAPK, and Akt in both p53wild-type and p53deleted/mutated B leukemic cell lines. A critical role of Akt downregulation in mediating the antileukemic activity of Dasatinib and Dasatinib + Nutlin-3 was demonstrated in experiments carried out by specifically modulating the Akt pathway.Conclusions: These findings suggest that Dasatinib + Nutlin-3 might represent an innovative therapeutic combination for both p53wild-type and p53deleted/mutated B-CLL. Clin Cancer Res; 17(4); 1–9. ©2010 AACR.

Purpose: To investigate the potential link between C-reactive protein (CRP), a known biomarker of acute and chronic inflammation, and TRAIL, a cytokine which plays a key role in the immune-surveillance against tumors.Experimental Design: Primary normal peripheral blood mononuclear cell (PBMC) and CD14+ monocytes were exposed to recombinant CRP (1–10 μmol/L). TRAIL expression was analyzed by ELISA and/or by quantitative real-time PCR (qRT-PCR). In parallel, the potential role of the transcription factor Egr-1 was investigated by analyzing its modulation in response to CRP and by transfection experiments.Results:In vitro CRP exposure induced downregulation of TRAIL expression, both at the mRNA and protein level, in unfractionated PBMC and in purified CD14+ monocytes. TRAIL downregulation was not due to a specific toxicity or to contaminating lipopolysaccharide (LPS), as shown by the lack of induction of monocyte apoptosis and by the inability of the inhibitor of LPS polymyxin B to interfere with CRP activity. Of note, CRP downregulated TRAIL expression/release in CD14+ monocytes also in response to IFN-α, the most potent inducer of TRAIL. At the molecular level, the downmodulation of TRAIL by CRP was accompanied by a significant increase of Egr-1. Consistently, Egr-1 overexpression reduced the baseline levels of TRAIL mRNA, whereas knocking down Egr-1 counteracted the ability of CRP to downregulate TRAIL.Conclusions: Our findings suggest that a chronic elevation of CRP, which occurs during systemic inflammation and often in patients with cancer, might contribute to promote cancer development and/or progression by downregulating TRAIL in immune cells. Clin Cancer Res; 19(8); 1949–59. ©2013 AACR.

Supplementary Table S1.

Hyperglycaemia and increased circulating saturated fatty acids are key metabolic features of type 2 diabetes mellitus (T2DM) that contribute to diabetic retinopathy pathogenesis. Contrarily, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been shown to improve or prevent T2DM. This study aimed at investigating the effect of TRAIL in an in vitro model of human retinal pigment epithelium: the ARPE-19 cell line, treated with palmitic acid (PA) in the presence of high glucose concentration. PA caused a drop in cellular metabolic activity and cell viability as well as an increase in apoptosis rates, which were paralleled by an upregulation of reactive oxygen species (ROS) generation as well as mitochondrial fragmentation. Despite ARPE-19 cells expressing TRAIL-R2 at the cell surface, TRAIL failed to counteract the cytotoxic effects of PA. However, when TRAIL was used alongside PA and then removed or used alone following PA challenge, it partially attenuated PA-induced lipotoxicity. This effect of TRAIL appeared to rely upon the modulation of inflammation and ROS production. Thus, TRAIL exerted a trophic effect on ARPE-19 cells, which became evident only when the lipotoxic insult was removed. Nevertheless, whether recombinant TRAIL might have a therapeutic potential for the treatment of diabetic retinopathy requires further investigation.

In B-chronic lymphocytic leukemia (B-CLL), the interaction between leukemic cells and the microenvironment promotes tumor cell survival. The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib is one of the first-in-class molecules for the treatment of B-CLL patients, however, the emerging mechanisms of resistance to ibrutinib call for new therapeutic strategies. The purpose of the current study was to investigate the ability of ibrutinib plus the MDM2-inhibitor nutlin-3 to counteract the tumor microenvironment protective effect. We observed that primary B-CLL cells cultivated in microenvironment mimicking conditions were protected from apoptosis by the up-regulation of c-MYC and of p53. In the same setting, combined treatments with ibrutinib plus nutlin-3 led to significantly higher levels of apoptosis compared to the single treatments, counteracting the c-MYC up-regulation. Moreover, the combination induced high p53 levels and a significant dissipation of the mitochondrial membrane potential, together with BAX cleavage in the more active p18 form and phospho-BAD down-regulation, that are key components of the mitochondrial apoptotic pathway, enhancing the apoptosis level. Our findings propose a new therapeutic strategy to overcome the tumor microenvironment protection involved in B-CLL resistance to drugs, with possible clinical implications also for other hematologic and solid tumors for which ibrutinib is considered a therapeutic option.

The cholesterol biosynthesis represents a crucial metabolic pathway for cellular homeostasis. The end products of this pathway are sterols, such as cholesterol, which are essential components of cell membranes, precursors of steroid hormones, bile acids, and other molecules such as ubiquinone. Furthermore, some intermediates of this metabolic system perform biological activity in specific cellular compartments, such as isoprenoid molecules that can modulate different signal proteins through the prenylation process. The defects of prenylation represent one of the main causes that promote the activation of inflammation. In particular, this mechanism, in association with oxidative stress, induces a dysfunction of the mitochondrial activity. The purpose of this review is to describe the pleiotropic role of prenylation in neuroinflammation and to highlight the consequence of the defects of prenylation.

Convolvulus pluricaulis (CP) is a common Indian herb, largely employed in Ayurvedic medicine and known for its neuroprotective and neuroinflammatory action. Its effectiveness against several pathologic/sub-pathologic conditions is widely accepted, but it is not yet completely chemically characterized. In recent years, several researchers have pointed out the involvement of CP and other Convolvulaceae in lipidic and glucidic metabolism, particularly in the control of hyperlipidaemia and diabetic conditions. In this scenario, the aim of the study was to chemically characterize the medium polarity part of the CP whole plant and its fractions and to shed light on their biological activity in adipocyte differentiation using the 3T3-L1 cell model. Our results demonstrated that the CP extract and fractions could upregulate the adipocyte differentiation through the modulation of the nuclear receptor PPARγ (Peroxisome Proliferator-Activated Receptor γ), broadly recognized as a key regulator of adipocyte differentiation, and the glucose transporter GLUT-4, which is fundamental for cellular glucose uptake and for metabolism control. CP also showed the ability to exert an anti-inflammatory effect, downregulating cytokines such as Rantes, MCP-1, KC, eotaxin, and GM-CSF, which are deeply involved in insulin resistance and glucose intolerance. Taken together, these data suggest that CP could exert a potential beneficial effect on glycemia and could be employed as an anti-diabetic adjuvant or, in any case, a means to better control glucose homeostasis.

Chenodeoxycholic acid and ursodeoxycholic acid (CDCA and UDCA, respectively) have been conjugated with paclitaxel (PTX) anticancer drugs through a high-yield condensation reaction. Bile acid-PTX hybrids (BA-PTX) have been investigated for their pro-apoptotic activity towards a selection of cancer cell lines as well as healthy fibroblast cells. Chenodeoxycholic-PTX hybrid (CDC-PTX) displayed cytotoxicity and cytoselectivity similar to PTX, whereas ursodeoxycholic-PTX hybrid (UDC-PTX) displayed some anticancer activity only towards HCT116 colon carcinoma cells. Pacific Blue (PB) conjugated derivatives of CDC-PTX and UDC-PTX (CDC-PTX-PB and UDC-PTX-PB, respectively) were also prepared via a multistep synthesis for evaluating their ability to enter tumor cells. CDC-PTX-PB and UDC-PTX-PB flow cytometry clearly showed that both CDCA and UDCA conjugation to PTX improved its incoming into HCT116 cells, allowing the derivatives to enter the cells up to 99.9%, respect to 35% in the case of PTX. Mean fluorescence intensity analysis of cell populations treated with CDC-PTX-PB and UDC-PTX-PB also suggested that CDC-PTX-PB could have a greater ability to pass the plasmatic membrane than UDC-PTX-PB. Both hybrids showed significant lower toxicity with respect to PTX on the NIH-3T3 cell line.

Janus kinases (JAK) are a family of tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) that transduce cytokine-mediated signals through the JAK–STAT metabolic pathway. These kinases act by regulating the transcription of specific genes capable of inducing biological responses in several immune cell subsets. Inhibition of Janus kinases interferes with the JAK–STAT signaling pathway. Besides being used in the treatment of cancer and inflammatory diseases, in recent years, they have also been used to treat inflammatory conditions, such as graft-versus-host disease (GVHD) and cytokine release syndrome as complications of allogeneic hematopoietic stem cell transplantation and cell therapy. Recently, the FDA approved the use of ruxolitinib, a JAK1/2 inhibitor, in the treatment of acute steroid-refractory GVHD (SR-aGVHD), highlighting the role of JAK inhibition in this immune deregulation. Ruxolitinib was initially used to treat myelofibrosis and true polycythemia in a high-dose treatment and caused hematological toxicity. Since a lower dosage often could not be effective, the use of ruxolitinib was suspended. Subsequently, ruxolitinib was evaluated in adult patients with SR-aGVHD and was found to achieve a rapid and effective response. In addition, its early low-dose use in pediatric patients affected by GVHD has proved effective, safe, and reasonably preventive. The review aims to describe the potential properties of ruxolitinib to identify new therapeutic strategies.

Innate and adaptive immune responses have a well-known link and represent the distinctive origins of several diseases, many of which may be the consequence of the loss of balance between these two responses. Indeed, autoinflammation and autoimmunity represent the two extremes of a continuous spectrum of pathologic conditions with numerous overlaps in different pathologies. A common characteristic of these dysregulations is represented by hyperinflammation, which is an exaggerated response of the immune system, especially involving white blood cells, macrophages, and inflammasome activation with the hyperproduction of cytokines in response to various triggering stimuli. Moreover, hyperinflammation is of great interest, as it is one of the main manifestations of COVID-19 infection, and the cytokine storm and its most important components are the targets of the pharmacological treatments used to combat COVID-19 damage. In this context, the purpose of our review is to provide a focus on the pathogenesis of autoinflammation and, in particular, of hyperinflammation in order to generate insights for the identification of new therapeutic targets and strategies.

Ligands and receptors of the tumor necrosis factor (TNF) superfamily regulate immune responses and homeostatic functions with potential diagnostic and therapeutic implications. Kidney disease represents a global public health problem, whose prevalence is rising worldwide, due to the aging of the population and the increasing prevalence of diabetes, hypertension, obesity, and immune disorders. In addition, chronic kidney disease is an independent risk factor for the development of cardiovascular disease, which further increases kidney-related morbidity and mortality. Recently, it has been shown that some TNF superfamily members are actively implicated in renal pathophysiology. These members include TNF-related apoptosis-inducing ligand (TRAIL), its decoy receptor osteoprotegerin (OPG), and TNF-like weaker inducer of apoptosis (TWEAK). All of them have shown the ability to activate crucial pathways involved in kidney disease development and progression (e.g. canonical and non-canonical pathways of the transcription factor nuclear factor-kappa B), as well as the ability to regulate cell proliferation, differentiation, apoptosis, necrosis, inflammation, angiogenesis, and fibrosis with double-edged effects depending on the type and stage of kidney injury. Here we will review the actions of TRAIL, OPG, and TWEAK on diabetic and non-diabetic kidney disease, in order to provide insights into their full clinical potential as biomarkers and/or therapeutic options against kidney disease.

Mitoquinone (MitoQ) is a mitochondrial reactive oxygen species scavenger that is characterized by high bioavailability. Prior studies have demonstrated its neuroprotective potential. Indeed, the release of reactive oxygen species due to damage to mitochondrial components plays a pivotal role in the pathogenesis of several neurodegenerative diseases. The present study aimed to examine the impact of the inflammation platform activation on the neuronal cell line (DAOY) treated with specific inflammatory stimuli and whether MitoQ addition can modulate these deregulations. DAOY cells were pre-treated with MitoQ and then stimulated by a blockade of the cholesterol pathway, also called mevalonate pathway, using a statin, mimicking cholesterol deregulation, a common parameter present in some neurodegenerative and autoinflammatory diseases. To verify the role played by MitoQ, we examined the expression of genes involved in the inflammation mechanism and the mitochondrial activity at different time points. In this experimental design, MitoQ showed a protective effect against the blockade of the mevalonate pathway in a short period (12 h) but did not persist for a long time (24 and 48 h). The results obtained highlight the anti-inflammatory properties of MitoQ and open the question about its application as an effective adjuvant for the treatment of the autoinflammatory disease characterized by a cholesterol deregulation pathway that involves mitochondrial homeostasis.

Rehabilitative exercise outcomes and plasma concentrations of soluble adhesion molecules (sEndoglin, sE-Selectin, sL-Selectin, sICAM-1, sNCAM, sNCAM-1, sVCAM-1, sPECAM-1, sVAP-1) were evaluated in 60 severely disabled progressive multiple sclerosis (MS) patients at 4-time points. Changes of sE-Selectin, sL-Selectin, and sPECAM-1 concentrations were observed over time, and their variations were significantly correlated with rehabilitative outcome variations. Baseline sVAP-1 concentrations were able to predict functional mobility recovery. Our data suggest that the evaluation of adhesion molecules in plasma provides useful information to interpret rehabilitative exercise processes and to identify potential predictors of the rehabilitation-induced changes in mobility outcomes in MS patients.

Eosinophils are major effector cells against parasites, fungi, bacteria, and viruses. However, these cells also take part in local and systemic inflammation, which are central to eczema, atopy, rhinitis, asthma, and autoimmune diseases. A role for eosinophils has been also shown in vascular thrombotic disorders and in cancer. Many, if not all, above-mentioned conditions involve the release of intracellular nucleotides (ATP, ADP, UTP, etc.) and nucleosides (adenosine) in the extracellular environment. Simultaneously, eosinophils further release ATP, which in autocrine and paracrine manners, stimulates P2 receptors. Purinergic signaling in eosinophils mediates a variety of responses including CD11b induction, ROI production, release of granule contents and enzymes, as well as cytokines. Exposure to extracellular ATP also modulates the expression of endothelial adhesion molecules, thereby favoring eosinophil extravasation and accumulation. In addition, eosinophils express the immunosuppressive adenosine P1 receptors, which regulate degranulation and migration. However, pro-inflammatory responses induced by extracellular ATP predominate. Due to their important role in innate immunity and tissue damage, pharmacological targeting of nucleotide- and nucleoside-mediated signaling in eosinophils could represent a novel approach to alleviate eosinophilic acute and chronic inflammatory diseases. These innovative approaches might also have salutary effects, particularly in host defense against parasites and in cancer.

Background and Aims Cardiovascular disease is the main cause of death worldwide, but the collective efforts to prevent this pathological condition are directed exclusively to individuals at higher risk due to hypercholesterolemia, hypertension, obesity, diabetes. Recently, vitamin D deficiency was identified as a risk factor for cardiovascular disease in healthy people, as it predisposes to different vascular dysfunctions that can result in plaque development and fragility. In this scenario, the fundamental aim of the study was to reproduce a disease model inducing vitamin D deficiency and atheromatosis in ApoE-/- mice and then to evaluate the impact of this vitamin D status on the onset/progression of atheromatosis, focusing on plaque formation and instability. Methods and Results In our murine disease model, vitamin D deficiency was achieved by 3 weeks of vitamin D deficient diet along with intraperitoneal paricalcitol injections, while atheromatosis by western-type diet. Under these experimental conditions, vitamin D deficient mice developed more unstable atheromatous plaques with reduced or absent fibrotic cap. Since calcium and phosphorus metabolism and also cholesterol and triglycerides systemic concentration were not affected by vitamin D level, our results highlighted the role of vitamin D deficiency in the formation/instability of atheromatous plaque and, although further studies are needed, suggested a possible intervention with vitamin D to prevent or delay the atheromatous disease. Conclusions The data obtained open the question about the potential role of the vitamins in the pharmacological treatments of cardiovascular disorders as coadjutant of the primary drugs used for these pathologies., Graphical abstract Image 1, Highlights • The vitamin D deficiency represents a risk factor for cardiovascular disease. • The vitamin D deficiency has been correlated to atheromatosis progression. • A model of vitamin D deficiency was fundamental to evaluate the role of the vitamin D the onset/progression of atheromatosis. • The vitamin D deficiency was clearly involved in an early formation of atherosclerotic lesions. • The vitamins play a potential role in the pharmacological treatments of cardiovascular disorders.

Despite the great progress in screening techniques and medical treatments, colorectal cancer remains one of the most widespread cancers in both sexes, with a high death rate. In this work, the volatile compounds released from human colon cancer tissues were detected by a set of four different chemoresistive sensors, made with a nanostructured powder of metal-oxide materials, inserted into an innovative patented device. The sensor responses to the exhalation of a primary cancer sample and of a healthy sample (both of the same weight, collected during colorectal surgery from the intestine of the same patient) were statistically analyzed. The sensors gave reversible, reproducible, and fast responses for at least one year of continuous use, making them quite superior in respect to the existing diagnostic methods. Preliminary results obtained using principal component analysis of the sensor responses to samples removed from 13 patients indicate that the nanostructured sensors employed in this study were able to distinguish between healthy and tumor tissue samples with coherent responses (the discrimination power of the most sensitive sensor was about 17%), highlighting a strong potential for clinical practice.

Early post-transplant is the critical phase for the success of hematopoietic stem cell transplantation (HSCT). New viral infections and the reactivations associated with complete ablation of the recipient’s T-cell immunity and inefficient reconstitution of the donor-derived system represent the main risks of HSCT. To date, the pharmacological treatments for post-HSCT viral infection-related complications have many limitations. Adoptive cell therapy (ACT) represents a new pharmacological strategy, allowing us to reconstitute the immune response to infectious agents in the post-HSC period. To demonstrate the potential advantage of this novel immunotherapy strategy, we report three cases of pediatric patients and the respective central nervous system complications after donor lymphocyte infusion.

Mevalonate Kinase Deficiency (MKD) is a rare inborn disease belonging to the family of periodic fever syndromes. The MKD phenotype is characterized by systemic inflammation involving multiple organs, including the nervous system. Current anti-inflammatory approaches to MKD are only partially effective and do not act specifically on neural inflammation. According to the new emerging pharmacology trends, the repositioning of drugs from the indication for which they were originally intended to another one can make mechanistic-based medications easily available to treat rare diseases. According to this perspective, the squalene synthase inhibitor Lapaquistat (TAK-475), originally developed as a cholesterol-lowering drug, might find a new indication in MKD, by modulating the mevalonate cholesterol pathway, increasing the availability of anti-inflammatory isoprenoid intermediates. Using an in vitro model for MKD, we mimicked the blockade of the cholesterol pathway and evaluated the potential anti-inflammatory effect of Lapaquistat. The results obtained showed anti-inflammatory effects of Lapaquistat in association with a low blockade of the metabolic pathway, while this effect did not remain with a tighter blockade. On these bases, Lapaquistat could be configured as an effective treatment for MKD’s mild forms, in which the residual enzymatic activity is only reduced and not almost completely absent as in the severe forms.

// Rebecca Voltan 1, * , Erika Rimondi 2, * , Elisabetta Melloni 1 , Gian Matteo Rigolin 3 , Fabio Casciano 1 , Maria Vittoria Arcidiacono 1 , Claudio Celeghini 2 , Antonio Cuneo 3 , Giorgio Zauli 1 , Paola Secchiero 1 1 Department of Morphology, Surgery and Experimental Medicine and LTTA Centre, University of Ferrara, Ferrara, Italy 2 Department of Life Sciences, University of Trieste, Trieste, Italy 3 Department of Medical Sciences, Section of Hematology, University of Ferrara, Ferrara, Italy * These authors have contributed equally to this work Correspondence to: Paola Secchiero, email: paola.secchiero@unife.it Keywords: B leukemic cells, Ibrutinib, MDM-2 inhibitors, apoptosis, combination therapy Received: July 20, 2016 Accepted: September 09, 2016 Published: September 20, 2016 ABSTRACT Objective: The aim of this study was to investigate the anti-leukemic activity of the Bruton tyrosine kinase inhibitor Ibrutinib in combination with the small molecule MDM-2 inhibitor Nutlin-3 in preclinical models. Methods: The potential efficacy of the Ibrutinib/Nutlin-3 combination was evaluated in vitro in a panel of B leukemic cell lines (EHEB, JVM-2, JVM-3, MEC-1, MEC-2) and in primary B-chronic lymphocytic leukemia (B-CLL) patient samples, by assessing cell viability, cell cycle profile, apoptosis and intracellular pathway modulations. Validation of the combination therapy was assessed in a B leukemic xenograft mouse model. Results: Ibrutinib exhibited variable anti-leukemic activity in vitro and the combination with Nutlin-3 synergistically enhanced the induction of apoptosis independently from the p53 status. Indeed, the Ibrutinib/Nutlin-3 combination was effective in promoting cytotoxicity also in primary B-CLL samples carrying 17p13 deletion and/or TP53 mutations, already in therapy with Ibrutinib. Molecular analyses performed on both B-leukemic cell lines as well as on primary B-CLL samples, while confirming the switch-off of the MAPK and PI3K pro-survival pathways by Ibrutinib, indicated that the synergism of action with Nutlin-3 was independent by p53 pathway and was accompanied by the activation of the DNA damage cascade signaling through the phosphorylation of the histone protein H2A.X. This observation was confirmed also in the JVM-2 B leukemic xenograft mouse model. Conclusions: Taken together, our data emphasize that the Ibrutinib/Nutlin-3 combination merits to be further evaluated as a therapeutic option for B-CLL.

Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated protein that endows its carrier with (lipo-)lactonase-dependent antioxidative features. Low levels of PON1 activity have been observed in association with obesity, a major risk factor for cardiovascular disease (CVD). Considering the well-recognized atheroprotective role of PON1, exogenous/endogenous factors that might modulate its levels/activity are raising great interest. Since adipokines represent a molecular link between obesity and CVD, we here explored the possible impact of these substances on PON1 activity/expression. The levels of interleukin (IL)-6, IL-8, tumor necrosis factor alpha, monocyte chemoattractant protein-1, hepatocyte growth factor, resistin, leptin, and adiponectin were measured along with arylesterase, paraoxonase, and lactonase activities of PON1 in 107 postmenopausal women. Moreover, the direct effect of resistin on PON1 expression was evaluated in vitro. Multivariate analysis revealed that only resistin was significantly and inversely correlated with PON1-lactonase activities (r = &minus, 0.346, p &lt, 0.001) regardless of confounding factors such as age or HDL-cholesterol. It is worth noting that no statistical link was found between adipokine and arylesterase or paraoxonase, the two promiscuous activities of PON1. Notably, resistin down-regulated PON1 expression occurred in hepatocellular carcinoma cultures. Our study suggests that resistin might be a negative modulator of PON1 expression and anti-oxidative activity.

Objective. “Oxinflammation” is a recently coined term that defines the deleterious crosstalk between inflammatory and redox systemic processes, which underlie several diseases. Oxinflammation could be latently responsible for the predisposition of certain healthy individuals to disease development. The oxinflammatory pathway has been recently suggested to play a crucial role in regulating the activity of TNF-related apoptosis-inducing ligand (TRAIL), a TNF superfamily member that can mediate multiple signals in physiological and pathological processes. Therefore, we investigated the associations between TRAIL and key players of vascular redox homeostasis. Methods. We measured circulating TRAIL levels relative to praoxonas-1, lipoprotein phospholipase-A2, and ceruloplasmin levels in a cohort of healthy subjects (n=209). Results. Multivariate analysis revealed that ceruloplasmin levels were significantly inversely associated with TRAIL levels (r=−0.431, p<0.001). The observed association retained statistical significance after adjustment for additional confounding factors. After stratification for high-sensitivity C-reactive protein levels, the inverse association between TRAIL and ceruloplasmin levels remained strong and significant (r=−0.508, p<0.001, R2=0.260) only in the presence of inflammation, confirming the role of inflammation as emerged in in vitro experiments where recombinant TRAIL decreased ceruloplasmin expression levels in TNF-treated PBMC cultures. Conclusion. The results indicated that in an inflammatory milieu, TRAIL downregulates ceruloplasmin expression, highlighting a signaling axis involving TRAIL and ceruloplasmin that are linked via inflammation and providing important insights with potential clinical implications.

Background: Chronic kidney disease (CKD) patients experience a high risk of cardiovascular disease (CV); however, the factors involved in CV-related morbidity and mortality in these patients have not been fully defined. Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a cytokine, which exhibits pleiotropic activities on endothelial, vascular smooth muscle and inflammatory cells, with relevant effects on atheromatous plaque formation. On this basis, the present study aims to investigate the role of TRAIL in atheromatosis progression in CKD patients. Methods: Circulating TRAIL levels were measured in 378 CKD patients belonging to the Spanish National Observatory of Atherosclerosis in Nephrology (NEFRONA) study. All patients were free of previous CV events. Carotid and femoral B-mode ultrasound was performed to detect the presence of plaque at baseline and after 24 months of follow-up. Results: The lowest levels of TRAIL at baseline were significantly (p

Photorefractive keratectomy (PRK) represents a therapeutic option to remodel corneal stroma and to compensate refractive errors, which involves inflammatory and/or regenerative processes. In this context, the modulation of cytokines/chemokines in the conjunctival sac fluid and their role in the maintenance of the corneal microenvironment during the healing process upon refractive procedures has not been deeply investigated. In this study, serial samples of conjunctival sac fluid of patients (n=25) undergoing PRK were harvested before and at different time points after surgery. The levels of 29 cytokines/chemokines/growth factors involved in inflammatory/immune processes were measured with a multiplex array system. The results have firstly highlighted the different pattern of cytokine expression between the microenvironment at the anterior surface of the eye and the systemic circulation. More importantly, the kinetic of modulation of cytokines/chemokines at the conjunctival level following PRK revealed that while the majority of cytokines/chemokines showed a significant decrease, MCP-1 emerged in light of its pronounced and significant increase soon after PRK and during the follow-up. This methodological approach has highlighted the role of MCP-1 in the healing process following PRK and has shown a potential for the identification of expression/modulation of soluble factors for biomarker profiling in ocular surface diseases.

// Paola Secchiero 1, * , Rebecca Voltan 1, * , Erika Rimondi 1 , Elisabetta Melloni 1 , Emmanouil Athanasakis 2 , Veronica Tisato 1 , Stefania Gallo 1 , Gian Matteo Rigolin 3 and Giorgio Zauli 1 1 Department of Morphology, Surgery and Experimental Medicine and LTTA Centre, University of Ferrara, Ferrara, Italy 2 Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, Trieste, Italy 3 Department of Medical Sciences, Section of Hematology, University of Ferrara, Ferrara, Italy * These authors have contributed equally to this work Correspondence to: Giorgio Zauli, email: giorgio.zauli@unife.it Paola Secchiero, email: paola.secchiero@unife.it Keywords: B-leukemic cells, Ibrutinib, γ-secretase inhibitors, NOTCH1, combination therapy Received: May 05, 2017 Accepted: June 20, 2017 Published: July 22, 2017 ABSTRACT Ibrutinib blocks B-cell receptor signaling and interferes with leukemic cell-to-microenvironment interactions. Ibrutinib plays a key role in the management of B-CLL and is recommended for first line treatment of high-risk CLL patients with 17p deletion. Therefore, elucidating the factors governing sensitivity/resistance to Ibrutinib represents a relevant issue. For this purpose, in 3 B-CLL patient samples harboring functional TP53 mutations, the frequency of the mutated clones was monitored during in vivo Ibrutinib therapy, revealing a progressive decline of the frequency of TP53 mut clones during 12 months of treatment. In parallel, the anti-leukemic activity of Ibrutinib was assessed in vitro on B-CLL patient cell cultures in combination with γ-secretase inhibitors (GSI). In the in vitro assays, the combination of Ibrutinib+GSI exhibited enhanced cytotoxicity on B-CLL cells also in the presence of stroma and it was coupled to the down-regulation of the stroma-activated NOTCH1 and c-MYC pathways. Moreover, the combined treatment was effective in reducing CXCR4 expression and functions. Therefore, the ability of GSI to enhance the Ibrutinib anti-leukemic activity in B-CLL cells, by down-regulating the NOTCH1 and c-MYC pathways, warrants further experimentation for its potential therapeutic applications.

The effect of the multi-kinase inhibitor Sorafenib was investigated in an in vitro model of human osteoclastogenesis, represented by peripheral blood mononuclear cells (PBMCs) induced to differentiate into osteoclast-like cells in presence of receptor activator of nuclear factor kappa B ligand (RANKL) plus macrophage-colony stimulating factor (M-CSF). Sorafenib significantly inhibited osteoclastic formation at clinically achievable concentrations (1-3 μM) and promoted autophagia with minimal induction of apoptosis. At the molecular levels, the M-CSF + RANKL combination increased the expression level of the Bcl-2 family member Mcl-1 protein, which is known to play a key role in the control of both cell survival and autophagia. The simultaneous treatment with Sorafenib significantly down-regulated endogenous Mcl-1 expression. Conversely, over-expression of Mcl-1 in primary human macrophages significantly counteracted the anti-osteoclastic activity of Sorafenib, strongly suggesting that Mcl-1 down-regulation played a major role in mediating the inhibitory activity of Sorafenib in cells of the osteoclastic lineage.

The basal SOCS1 mRNA levels were significantly lower in p53(mutated) BJAB and MAVER leukemic cell lines with respect to p53(wild-type) SKW6.4 and JVM-2 leukemic cell lines, p53(wild-type) primary B chronic lymphocytic leukemia (B-CLL) cells and primary normal peripheral blood mononuclear cells (PBMC). Moreover, the MDM2 small molecule inhibitor Nutlin-3 significantly increased the levels of SOCS1 mRNA in both primary p53(wild-type) B-CLL cells as well as in p53(wild-type) B leukemic cell lines, but not in p53(mutated) B leukemic cell lines nor in primary PBMC. Of note, a significant inverse correlation was observed between SOCS1 mRNA and miR-155 levels in Nutlin-3-treated primary B-CLL cells and PBMC, suggesting that the miRNA-155/SOCS1 axis represents a potentially important therapeutic target of Nutlin-3 in B-CLL.

Acute Lymphoblastic Leukemia (ALL) is an aggressive hematologic disorder and constitutes approximately 25% of cancer diagnoses among children and teenagers. Pediatric patients have a favourable prognosis, with 5-years overall survival rates near 90%, while adult ALL still correlates with poorer survival. However, during the past few decades, the therapeutic outcome of adult ALL was significantly ameliorated, mainly due to intensive pediatric-based protocols of chemotherapy. Mammalian (or mechanistic) target of rapamycin (mTOR) is a conserved serine/threonine kinase belonging to the phosphatidylinositol 3-kinase (PI3K)-related kinase family (PIKK) and resides in two distinct signalling complexes named mTORC1, involved in mRNA translation and protein synthesis and mTORC2 that controls cell survival and migration. Moreover, both complexes are remarkably involved in metabolism regulation. Growing evidence reports that mTOR dysregulation is related to metastatic potential, cell proliferation and angiogenesis and given that PI3K/Akt/mTOR network activation is often associated with poor prognosis and chemoresistance in ALL, there is a constant need to discover novel inhibitors for ALL treatment. Here, the current knowledge of mTOR signalling and the development of anti-mTOR compounds are documented, reporting the most relevant results from both preclinical and clinical studies in ALL that have contributed significantly into their efficacy or failure.

Purpose: To analyze the effect of the combination of Dasatinib, a multikinase inhibitor, plus Nutlin-3, a nongenotoxic activator of the p53 pathway, in primary B chronic lymphocytic leukemia (B-CLL) patient samples and B leukemic cell line models. Experimental Design: The induction of cytotoxicity was evaluated in both primary B-CLL cell samples (n = 20) and in p53wild-type (EHEB, JVM-2) and p53deleted/mutated (MEC-2, BJAB) B leukemic cell lines. The role of Akt in modulating leukemic cell survival/apoptosis in response to Dasatinib or Dasatinib + Nutlin-3 was documented by functional experiments carried out using specific pharmacological inhibitors and by overexpression of membrane-targeted constitutively active form of Akt. Results: The combination of Dasatinib + Nutlin-3 exhibited a synergistic cytotoxicity in the majority (19 out of 20) of B-CLL samples, including patients carrying 17p- (n = 4), and in both p53wild-type and p53deleted/mutated B leukemic cell lines. At the molecular level, Dasatinib significantly counteracted the Nutlin-3-mediated induction of the p53 transcriptional targets MDM2 and p21 observed in p53wild-type leukemic cells. Conversely, Nutlin-3 did not interfere with the ability of Dasatinib to decrease the phosphorylation levels of ERK1/2, p38/MAPK, and Akt in both p53wild-type and p53deleted/mutated B leukemic cell lines. A critical role of Akt downregulation in mediating the antileukemic activity of Dasatinib and Dasatinib + Nutlin-3 was demonstrated in experiments carried out by specifically modulating the Akt pathway. Conclusions: These findings suggest that Dasatinib + Nutlin-3 might represent an innovative therapeutic combination for both p53wild-type and p53deleted/mutated B-CLL. Clin Cancer Res; 17(4); 1–9. ©2010 AACR.

Purpose: To characterize the role of the oncogene DEK in modulating the response to either Nutlin-3, a small-molecule inhibitor of the MDM2/p53 interaction, or chlorambucil in primary B-chronic lymphocytic leukemia (B-CLL) cells. Experimental Design: DEK mRNA and protein levels were evaluated in primary B-CLL samples (n = 21), p53wild-type SKW6.4, p53mutated BJAB lymphoblastoid cell lines, and normal CD19+ B lymphocytes–treated Nutlin-3 or chlorambucil (10 μmol/L, each). Knocking down experiments with either p53 or DEK small interfering RNA (siRNA) were done to investigate the potential role of p53 in controlling the expression of DEK and the role of DEK in leukemic cell survival/apoptosis. Results: Both Nutlin-3 and chlorambucil downregulated DEK in primary B-CLL samples (n = 21) and SKW6.4 but not in BJAB cells. Knocking down p53 attenuated the effect of Nutlin-3 on DEK expression, whereas knocking down DEK significantly increased both spontaneous and Nutlin-3–induced apoptosis. Conversely, counteracting DEK downmodulation by using p53 small interfering RNA reduced Nutlin-3–mediated apoptosis. On the other hand, Nutlin-3 potently induced p53 accumulation, but it did not affect DEK levels in normal CD19+ B lymphocytes. Conclusions: These data show that the downregulation of DEK in response to either Nutlin-3 or chlorambucil represents an important molecular determinant in the cytotoxic response of leukemic cells, and suggest that strategies aimed to downregulate DEK might improve the therapeutic potential of these drugs. Clin Cancer Res; 16(6); 1824–33

Osteoprotegerin (OPG) is a soluble tumor necrosis factor receptor family member, which potently inhibits RANKL-mediated osteoclastogenesis. Numerous constructs have been created for therapeutic purposes in which the heparin-binding and death homology domains of OPG were removed and the remaining peptide (amino acids 22-194) was fused to the Fc domain of human IgG1 (OPG-Fc). The administration of OPG-Fc efficiently counteracted bone loss in a variety of preclinical models of cancers. However, several in vitro studies have shown that native or recombinant full-length OPG not only neuralizes RANKL, but also the death-inducing ligand TRAIL, suggesting that OPG might potentially counteract the anti-tumor activity of TRAIL. Additional evidence suggests that full-length OPG possesses RANKL- and TRAIL-independent biological properties, mainly related to the promotion of endothelial cell survival and angiogenesis. Finally, breast tumor cells overexpressing OPG have shown increased bone metastatic potential in vivo. The relevance of these apparently conflicting findings in tumor cell biology is highlighted.

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.

The mitochondrial kinase inhibitor dichloroacetate (DCA) has recently received attention in oncology due to its ability to target glycolysis. However, DCA molecule exhibits poor bioavailability and cellular uptake with limited ability to reach its target mitochondria. To overcome these biases, we have synthesized novel DCA-loaded compounds. The selection of the most promising therapeutic molecule was evaluated by combining in vitro assays, to test the antitumoral potential on leukemic cells, and a preliminary characterization of the molecule stability in vivo, in mice. Among the newly synthesized compounds, we have selected the multiple DCA-loaded compound 10, characterized by a tertiary amine scaffold, because it exhibited enhanced (>30-fold) in vitro antitumor activity with respect to DCA and increased in vivo stability. On the basis of these results, we believe that compound 10 should be considered for further preclinical evaluations for the treatment of cancers and/or other diseases characterized by altered metabolic origin.

The small-molecule inhibitor of murine double minute (MDM-2), Nutlin-3, induced variable apoptosis in primary acute myeloid leukemia (AML) blasts and promoted myeloid maturation of surviving cells, as demonstrated by analysis of CD11b and CD14 surface antigens and by morphologic examination. Although the best-characterized activity of Nutlin-3 is activation of the p53 pathway, Nutlin-3 induced maturation also in one AML sample characterized by p53 deletion, as well as in the p53(-/-) human myeloblastic HL-60 cell line. At the molecular level, the maturational activity of Nutlin-3 in HL-60 cells was accompanied by the induction of E2F1 transcription factor, and it was significantly counteracted by specific gene knockdown with small interfering RNA for E2F1. Moreover, Nutlin-3, as well as tumor necrosis factor (TNF) alpha, potentiated the maturational activity of recombinant TNF-related apoptosis-inducing ligand (TRAIL) in HL-60 cells. However, although TNF-alpha significantly counteracted the proapoptotic activity of TRAIL, Nutlin-3 did not interfere with the proapoptotic activity of TRAIL. Taken together, these data disclose a novel, potentially relevant therapeutic role for Nutlin-3 in the treatment of both p53 wild-type and p53(-/-) AML, possibly in association with recombinant TRAIL.

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.

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.

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)

In order to investigate the biologic activity of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) on human erythropoiesis, glycophorin A (GPA)+ erythroid cells were generated in serum-free liquid phase from human cord blood (CB) CD34+ progenitor cells. The surface expression of TRAIL-R1 was weakly detectable in the early-intermediate phase of erythroid differentiation (days 4-6; dim-intermediate GPA expression), whereas a clear-cut expression of TRAIL-R2 was observed through the entire course of erythroid differentiation (up to days 12-14; bright GPA expression). On the other hand, surface TRAIL-R3 and -R4 were not detected at any culture time. Besides inducing a rapid but small increase of apoptotic cell death, which was abrogated by the pan-caspase inhibitor z-VAD-fmk, the addition of recombinant TRAIL at day 6 of culture inhibited the generation of morphologically mature erythroblasts. Among the intracellular pathways investigated, TRAIL significantly stimulated the extracellular signal-regulated kinase 1/2 (ERK1/2) but not the p38/mitogen-activated protein kinase (MAPK) or the c-Jun NH2-terminal kinase (JNK) pathway. Consistently with a key role of ERK1/2 in mediating the negative effects of TRAIL on erythroid maturation, PD98059, a pharmacologic inhibitor of the ERK pathway, but not z-VAD-fmk or SB203580, a pharmacologic inhibitor of p38/MAPK, reverted the antidifferentiative effect of TRAIL on CB-derived erythroblasts.

Cyclooxygenase (COX)-1 or -2 and prostaglandin (PG) synthases catalyze the formation of various PGs and thromboxane (TX) A 2 . We have investigated the expression and activity of COX-1 and -2 during human megakaryocytopoiesis. We analyzed megakaryocytes from bone marrow biopsies and derived from thrombopoietin-treated CD34 + hemopoietic progenitor cells in culture. Platelets were obtained from healthy donors and patients with high platelet regeneration because of immune thrombocytopenia or peripheral blood stem cell transplantation. By immunocytochemistry, COX-1 was observed in CD34 + cells and in megakaryocytes at each stage of maturation, whereas COX-2 was induced after 6 days of culture, and remained detectable in mature megakaryocytes. CD34 + cells synthesized more PGE 2 than TXB 2 (214 ± 50 vs. 30 ± 10 pg/10 6 cells), whereas the reverse was true in mature megakaryocytes (TXB 2 8,440 ± 2,500 vs. PGE 2 906 ± 161 pg/10 6 cells). By immunostaining, COX-2 was observed in 2 and TXB 2 to a significantly greater extent in patients than in healthy subjects. Finally, we found that COX-2 and the inducible PGE-synthase were coexpressed in mature megakaryocytes and in platelets. We conclude that both COX-isoforms contribute to prostanoid formation during human megakaryocytopoiesis and that COX-2-derived PGE 2 and TXA 2 may play an unrecognized role in inflammatory and hemostatic responses in clinical syndromes associated with high platelet turnover.

The regulatory human immunodeficiency virus-1 (HIV-1) Tat protein shows pleiotropic effects on the survival and growth of both HIV-1-infected and uninfected CD4+ T lymphocytes. In this study, we have demonstrated that low concentrations (10 ng/ml) of extracellular Tat protein induce the expression of both c-fos mRNA and protein in serum-starved Jurkat CD4+ lymphoblastoid T cells. Using deletion mutants, we demonstrates that the SRE, CRE and, to a lesser extent, also the SIE domains (all placed in the first 356 bp of c-fos promoter) play a key role in mediating the response to extracellular Tat. Moreover, the ability of Tat to activate the transcriptional activity of c-fos promoter was consistently decreased by pretreatment with the ERK/MAPK kinase inhibitor PD98058. Activation of c-fos is functional as demonstrated by induction of the AP-1 transcription factor, which is involved in the regulation of critical genes for the activation of T lymphocytes, such as interleukin 2. The Tat-mediated induction of c-fos and AP-1 in uninfected lymphoid T cells may contribute to explain the immune hyperactivation that characterizes the progression to autoimmuno deficiency syndrome and constitutes the optimal environment for HIV-1 replication, occurring predominantly in activated/proliferating CD4+ T cells.

The expression and function of surface TRAIL and TRAIL receptors were investigated in primary megakaryocytic cells, generated in serum-free liquid phase from peripheral human CD34+ cells. The surface expression of both TRAIL and “death receptor” TRAIL-R2 became detectable starting from the early phase of megakaryocytic differentiation (day 6 of culture) and persisted at later (days10–14) culture times. On the other hand, “death receptor” TRAIL-R1, “decoy receptors” TRAIL-R3, and TRAIL-R4 were barely detectable or undetectable at any time point examined. Addition of recombinant TRAIL at day 6 of culture increased the rate of spontaneous apoptosis of CD34+/CD41dim megakaryoblasts and it significantly decreased the total output of mature megakaryocytic cells evaluated after additional 4–8 days of culture. Conversely, addition in culture of TRAIL-R2-Fc chimera, which blocked the interaction between endogenous TRAIL and TRAIL-R2 on the surface of cultured megakaryocytic cells, increased the total megakaryocytic cell count. In addition, recombinant TRAIL promoted a small but reproducible increase of maturation in the surviving megakaryocytic cell population, evaluated by both phenotypic analysis and morphology. A similar pro-maturation effect was observed when TRAIL was added to bone marrow-derived CD61+ megakaryocytic cells. Thus, our data suggest a role of TRAIL as a regulator of megakaryocytopoiesis. © 2005 Wiley-Liss, Inc.

The regulation of the circulating levels of TNF-related apoptosis-inducing ligand (TRAIL), a cytokine of the TNF family, playing a key role in the immune surveillance against cancer, is incompletely understood.The objective of the study was to investigate the potential link between TRAIL and 17β-estradiol.Circulating TRAIL levels were measured by an ELISA in plasma samples (n = 246) of healthy, age-matched (range 30-70 y) men and women and in the sera (n = 180) of females belonging to different physiopathological conditions (childhood, pregnancy, under gonadotropin treatment, menopause) characterized by different levels of circulating 17β-estradiol.TRAIL plasma levels in women with aged younger than 50 years were significantly lower compared with age-matched men, whereas in woman 50 years old or older, TRAIL levels were significantly higher compared with the age-matched men and with the younger women. Moreover, an analysis of women with different conditions revealed a significant inverse correlation between the serum levels of TRAIL and 17β-estradiol, with the lowest levels of TRAIL being observed during pregnancy and the highest in childhood and in postmenopausal women. Moreover, gonadotropin treatment in women undergoing assisted reproduction was accompanied by an acute decrease of serum TRAIL levels. Finally, in vitro treatment with 17β-estradiol decreased the TRAIL expression levels in peripheral blood mononuclear cells.Our data suggest that 17β-estradiol plays a role in regulating TRAIL circulating levels. The demonstration that postmenopausal women exhibit the highest TRAIL levels is of particular interest in light of a previous large study population showing that TRAIL is positively correlated to the overall survival.

// 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.

Besides its well-established oncosuppressor activity, a key function of p53 in regulating metabolic pathways has been recently identified. Nevertheless, the role of p53 with respect to diabetes mellitus (DM) appears highly controversial. To address this issue, we have used the cis-imidazoline compound Nutlin-3, an inhibitor of MDM2/p53 interaction, which represents a potent and selective non-genotoxic activator of the p53 pathway both in in vivo and in vitro experimental settings. Experimental DM was induced by intraperitoneal injections of low concentrations of streptozotocin (STZ) in C57BL/6N mice (n = 20). A group of control vehicle-injected mice (n = 10) and of STZ-treated mice (n = 10) was co-injected with Nutlin-3. Mice co-injected with STZ + Nutlin-3 exhibited attenuated features of DM with respect to animals treated with STZ alone. Indeed, STZ + Nutlin-3-treated mice were characterized by significantly (p < 0.05) lower levels of hyperglycemia, reduced weight loss, and increased spleen weight. In addition, STZ alone promoted a marked decrease in the levels of several circulating cytokines, including interleukin-12 (IL-12)p40. On the other hand, co-injection of STZ + Nutlin-3 significantly (p < 0.01) counteracted IL-12p40 down-modulation. In vitro experiments performed on the RAW264.7 macrophagic cell line model, used as cellular source of IL-12p40, demonstrated that Nutlin-3 treatment increased IL-12p40 release, strongly suggesting a direct effect of Nutlin-3 on the immune system. Overall, these data demonstrate that systemic administration of Nutlin-3 ameliorates the severity of STZ-induced DM and increases the levels of circulating IL-12p40.

Objective Although there is a large amount of data on the role of preadipocytes in promoting the release of proinflammatory cytokines and chemokines in response to macrophage-derived cytokines, the direct role of insulin and saturated fatty acids in modulating the release of inflammatory cytokines by cells differentiating along the adipocytic lineage is less understood. Methods 3T3-L1 murine preadipocyte cells were cultured for 3 d in a proliferating medium in the presence or absence of insulin 0.2 nmol/L plus palmitic acid 1 μmol/L. In parallel, 3T3-L1 cells were cultured in a differentiation medium containing dexamethasone, insulin, and isobutyl methyl xanthine in the absence or presence of palmitic acid for 3 d. The levels of several cytokines were evaluated in the culture supernatants by a bead-based multiplex immunoassay. Results Under the proliferation conditions, insulin plus palmitic acid promoted a significant increase in the release of interleukin-6, keratinocyte-derived chemokine (KC)/chemokine ligand-1 (CXCL-1), monocyte chemotactic protein-1 (MCP-1), and regulated and normal T cells expressed and presumably secreted (RANTES) by 3T3-L1 preadipocytes. Of note, the release of KC/CXCL-1, MCP-1, and RANTES increased significantly with adipocytic differentiation, and the addition of palmitic acid to differentiating 3T3-L1 cells resulted in a further significant promotion of KC/CXCL1, MCP-1, and RANTES, coupled to the increase of additional cytokines. Conclusions Taken together, these data show that a restricted common group of cytokines/chemokines (KC/CXCL1, MCP-1, and RANTES) is upregulated in proliferating and differentiating 3T3-L1 adipocytes in response to insulin and palmitic acid and that differentiating adipocytes respond with an increased range of cytokines with respect to proliferating 3T3-L1 preadipocytes.

BACKGROUND: Both the multi-kinase inhibitor sorafenib and the small molecule inhibitor of the MDM2/p53 interaction, nutlin-3, used alone, have shown promising anti-leukemic activity in acute myeloid leukemia cells. Thus, in this study we investigated the effect of the combination of sorafenib plus nutlin-3 in acute myeloid leukemia. DESIGN AND METHODS: Primary acute myeloid leukemia blasts (n=13) and FLT3(wild-type)/p53(wild-type) (OCI-AML3), FLT3(mutated)/p53(wild-type) (MOLM), FLT3(mutated)/p53(mutated) (MV4-11), FLT3(wild-type)/p53(deleted) (HL60) or FLT3(wild-type)/p53(mutated) (NB4) acute myeloid cell lines were exposed to sorafenib, used alone or in association with nutlin-3 at a 1:1 ratio, in a range of clinically achievable concentrations (1-10 μM). Induction of apoptosis and autophagy was evaluated by transmission electron microscopy and by specific flow cytometry analyses. The levels of Mcl-1, p53 and Bak proteins were analyzed by western blotting. Knock-down of Bax and Bak gene expression was performed in transfection experiments with specific short interfering RNA. RESULTS: The sorafenib+nutlin-3 drug combination exhibits synergistic cytotoxicity in primary acute myeloid leukemia blasts and in acute myeloid leukemia cell lines with maximal cytotoxicity in FLT3(mutated) MV4-11 and MOLM, followed by the FLT3(wild-type) OCI-AML3, HL60 and NB4 cell lines. The cytotoxic activity of sorafenib+nutlin-3 was characterized by an increase of both apoptosis and autophagy. Moreover, Bax and Bak showed prominent roles in mediating the decrease of cell viability in response to the drug combination in p53(wild-type) OCI-AML3 and p53(deleted) HL-60 cells, respectively, as demonstrated in transfection experiments performed with specific short interfering RNA. CONCLUSIONS: Our data demonstrate that acute myeloid leukemia cells show a variable but overall good susceptibility to the innovative therapeutic combination of sorafenib+nutlin-3, which differentially involves the pro-apoptotic Bcl-2 family members Bax and Bak in p53(wild-type) and p53(deleted) cells.