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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.

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.

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.

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.

// 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.

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.

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.

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 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.

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.

Squamous cell carcinoma (SCC) of the head and neck is a common malignancy accounting for over 300,000 cases worldwide every year [1] and occurring at all ages including in children [2]. Of all malignancies locaded in the head and neck region (i.e., oral cavity, pharynx, larynx and paranasal sinuses), the OSCC in its strictest definition (i.e. in front of the tonsils) accounts for the vast majority of cases [1–4]. Surgery remains the first line of treatment for oral cancer, in combination with radiotherapy or chemotherapy [3]. Nevertheless, the poor outcome of many patients affected by OSCC underline the urgent need for innovative therapeutic approaches. In this respect, the development of selective tumor-biology based therapies, that can improve current therapy and allow more tolerable treatment regimens, has become a main field of interest in cancer research. Previous studies have shown that the TNF-family member TRAIL exhibits selective anti-tumor activity due to its unique ability to induce apoptosis in a variety of continuous cancer cell lines and primary tumor cells, displaying minimal or absent toxicity on most normal cells and tissues [5]. Like other members of the TNF family of proteins, TRAIL can be expressed as type II transmembrane protein or as a soluble protein [6] and, besides inducing apoptosis of cancer cells, it plays a variety of biological functions on immune cells and on other cells of hematopoietic origin [6]. There are several ongoing phase I and phase II clinical trials to evaluate the potential anti-cancer activity of soluble recombinant TRAIL in both solid tumors [7] and hematological malignancies [8]. On these bases, the aim of the present study was to investigate the expression of TRAIL in a large sample (n = 134) of paraffin-embedded OSCC specimens and to assess whether this has a prognostic significance for the survival of patients suffering from OSCC. In addition, by using three continuous cell lines (PE15D, PE46 and PE49) of OSCC origin, we analyzed the potential biological significance of the high TRAIL expression in OSCC cells and the response of OSCC cells to treatment in vitro with soluble recombinant TRAIL.

Background Previous studies have reported conflicting results on the frequency and potential pathogenetic role of Merkel-cell polyomavirus (MCPyV) in B-chronic lymphocytic leukemia (B-CLL). Objectives To evaluate the association of MCPyV to B-CLL and to investigate the occurrence of MCPyV infection in relationship to the natural history of B-CLL. Study design Samples of primary B-CLL peripheral blood mononuclear cells were obtained from two distinct University Hospitals of Italy from January 2010. For one B-CLL patient, it was possible to retrospectively examine the blood sample at diagnosis of B-CLL (March 2004) and several pathological tissues of cutaneous tumors occurring during the course of the disease. Results Only one out of 50 B-CLL blood samples examined was positive for MCPyV DNA. Retrospective analysis revealed that MCPyV DNA was absent in peripheral blood sample at diagnosis, becoming present only in advanced disease stages also in tonsil tissue as well as in a biopsy of differentiated squamous cell carcinoma. Conclusions The association with MCPyV seems to represent a rare and late event during the natural history of B-CLL.

The serine/threonine protein kinase C (PKC) family was first identified as intracellular receptor(s) for the tumor promoting agents phorbol esters. Thirty years after the discovery of PKC, the role of specific PKC isoforms has been described in relationship with an altered pattern of expression in different types of cancer and a good number of small molecule inhibitors (inhibitory peptides, antisense oligonucleotides or natural compounds) targeting PKC are now available. Despite all these achievements and a huge amount of basic research studies on the biochemical regulation of PKC, there has been a delay in clinical trials with drugs targeting PKC function. This delay is easily explained taking into account the extreme biological complexity of the PKC family of isoforms and the incomplete understanding of the specific role of each PKC isozyme in different types of cancers. Some of the difficulties in developing pharmacological compounds selectively tuning the different PKCs have started to be overcome. In this review, the growing evidences of the role of the PKC isoforms α, βII, δ, ε, ζ and ι is in promoting or counteracting tumor progression will be discussed in relationship with promising therapeutic perspectives.

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.

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

XAF-1 exists as a single gene with eight exons, which localizes to chromosome 17p13.2, a region just telomeric to the p53 gene. Although XAF-1 has been shown to have a potent proapoptotic activity and to act as a tumor suppressor in several solid tumors by inhibiting X-linked inhibitor of apoptosis at the mitochondrial level, its potential role in hematological malignancies has not been addressed. As 17p- deletion has been associated with poor prognosis in patients affected by B chronic lymphocytic leukemia (B-CLL), we have analyzed the steady-state messenger RNA (mRNA) levels of XAF-1 in a cohort of B-CLL samples, characterized for CLL-associated chromosomal aberrations using fluorescence in situ hybridization, Zap70 levels and p53 status. When assayed ex vivo for cytotoxic response to 10 µM of the non-genotoxic activator of the p53 pathway Nutlin-3, B-CLL samples showed a variable susceptibility to Nutlin-3 cytotoxicity upon 48 h of treatment. Our data suggest that XAF-1 might represent an important determinant in modulating the cytotoxic activity of Nutlin-3 and probably also of other pro-apoptotic drugs in B-CLL cells. In fact, p53del/mut B-CLL samples, which are less responsive to chemotherapeutic or Nutlin-3 treatment than p53wt B-CLL samples, showed low levels of XAF-1 mRNA. Moreover, modulating the steady-state mRNA levels of XAF-1 through IFN-β treatment or silencing the XAF-1 gene significantly affected the Nutlin-3-mediated cytotoxicity in selected samples of primary p53wt and p53del/mut B-CLL as well as in leukemic cell lines.

The small molecule inhibitor of the MDM2/p53 interaction Nutlin-3 significantly up-regulated the steady-state mRNA and protein levels of Notch1 in TP53wild-type (OCI, SKW6.4) but not in TP53deleted (HL-60) or TP53mutated (BJAB) leukemic cell lines. A direct demonstration that NOTCH1 was a transcriptional target of p53 in leukemic cells was obtained in experiments carried out with siRNA for p53. Moreover, inhibition of Notch1 expression using Notch1-specific siRNA significantly increased cytotoxicity in TP53wild-type leukemic cells. Of note, Nutlin-3 up-regulated Notch1 expression also in primary TP53wild-type B-chronic lymphocytic leukemia (B-CLL) cells and the combined use of Nutlin-3 plus pharmacological γ-secretase inhibitors of the Notch signaling showed a synergistic cytotoxicity in both TP53wild-type leukemic cell lines and primary B-CLL cells. A potential drawback of γ-secretase inhibitors was their ability to enhance osteoclastic maturation of normal circulating preosteoclasts induced by RANKL + M-CSF. Notwithstanding, Nutlin-3 completely suppressed osteoclastogenesis irrespective of the presence of γ-secretase inhibitors. Taken together, these data indicate that the p53-dependent up-regulation of Notch1 in response to Nutlin-3 represents an antiapoptotic feedback mechanism able to restrain the potential therapeutic efficacy of Nutlin-3 in hematologic malignancies. Therefore, therapeutic combinations of Nutlin-3 + γ-secretase inhibitors might potentiate the cytotoxicity of Nutlin-3 in p53wild-type leukemic cells.

Little is known on the ability of different epithelia to release soluble TNF-related apoptosis-inducing ligand (TRAIL) and the relevance of TRAIL secretion by epithelial cells is still incompletely understood. On these bases, we have measured the concentration of soluble TRAIL by ELISA in the conjunctival sac fluid. It was the highest ever detected in a biological fluid (mean value of 26,800 pg/ml), being approximately 20-fold greater than that found in human saliva and >200-fold greater than that detected in human serum. On the other hand, osteoprotegerin, the soluble decoy receptor of TRAIL, was almost undetectable in the conjunctival sac fluid. Of note, the levels of soluble TRAIL measured in conjunctival sac fluid were in the range able to induce in vitro apoptosis of lymphoma cells. By in situ immunohistochemistry, TRAIL protein expression was predominantly detected in the corneal epithelium and, to a less extent, in the conjunctival epithelium. By flow cytometry analysis, membrane-associated TRAIL was documented in isolated corneal epithelial cells obtained from patients undergoing photorefractive keratectomy (PRK). The key contribution provided by corneal epithelium to the production of soluble TRAIL was underscored in time-course experiments, in which a marked decrease of the levels of soluble TRAIL in the conjunctival sac fluid was demonstrated 1 day after PRK followed by a progressive recovery at days 5–30 after PRK. Taken together, our findings strongly support a major role of soluble TRAIL in protecting cornea and conjunctiva from tumor formation and/or invasion. J. Cell. Physiol. 218: 199–204, 2009. © 2008 Wiley-Liss, Inc.

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.

To potentiate the response of acute myeloid leukemia (AML) to TRAIL cytotoxicity, we have adopted a strategy of combining nutlin-3, a potent non-genotoxic activator of the p53 pathway, with recombinant TRAIL. The rationale for using such a combination was that deletions and/or mutations of the p53 gene occur in only 5-10% of AML and that TRAIL and nutlin-3 activate the extrinsic and intrinsic pathways of apoptosis, respectively. TRAIL induced a rapid increase of apoptosis when added to OCI M4-type and MOLM M5- type AML cells, carrying a wild-type p53, as well as to NB4 M3-type AML, carrying a mutated p53. On the other hand, the small molecule activator of the p53 pathway nutlin-3 induced p53 accumulation, cell cycle arrest and a slow progressive increase of apoptosis in OCI and MOLM but not in NB4. Of note, nutlin-3 up-regulated the surface expression of TRAIL-R2 and synergized with TRAIL in inducing apoptosis in OCI and MOLM as well as in primary M4-type and M5-type AML blasts, but not in NB4 cells. Moreover, while nutlin- 3 up-regulated the expression of cyclin dependent kinase inhibitor p21, a p53-target gene mediating cell cycle block and showing antiapoptotic activity, the simultaneous addition of TRAIL plus nutlin-3 induced the caspase-dependent cleavage of p21. The relevance of p21 down-regulation for sensitizing AML cells to apoptosis was underscored in knocking-down experiments with small interfering RNAs. Our data suggest that the combined treatment of nutlin-3 plus TRAIL might offer a novel therapeutic strategy for AML.

B chronic lymphocytic leukemia (B-CLL) cells express several members of the tumor necrosis factor (TNF) family, such as CD40L, CD30L, and TRAIL. By using the cDNA microarray technology, B-CLL samples were found to overexpress receptor activator of nuclear factor kB (NF-kB) ligand (RANKL), as compared to normal CD19(+) B cells. These findings were validated at the protein level by Western blot and flow cytometry analyses. Moreover, unlike primary normal B cells, leukemic B-CLL cells showed surface expression of RANK, the cognate transmembrane receptor of RANKL. When added in vitro to B-CLL cultures, either alone or in association with chlorambucil or fludarabine, recombinant RANKL did not significantly modulate cell viability, and it minimally affected the IL-8 expression/release. On the other hand, treatment with RANK-Fc chimera potently upregulated the release of IL-8 in the B-CLL culture supernatants, suggesting involvement of reverse signaling through transmembrane RANKL in IL-8 induction. In turn, exposure of B-CLL cells to recombinant IL-8 significantly decreased spontaneous apoptosis as well as chlorambucil- and fludarabine-mediated cytoxicity in B-CLL cells. Since IL-8 has been implicated in progression of B-CLL disease, our findings suggest that, by upregulating IL-8, the RANKL/RANK system may contribute to the pathogenesis of B-CLL.

Myelodysplastic syndromes (MDS) are characterized by impaired erythropoiesis, possibly caused by proapoptotic cytokines. We focused our study on the cytokine TRAIL (TNF-related apoptosis-inducing ligand), which has been shown to exhibit an anti-differentiation activity on erythroid maturation. Immunocytochemical analysis of bone marrow mononuclear cells (BMMC) showed an increased expression of TRAIL in MDS patients with respect to acute myeloid leukemia (AML) patients and normal BM donors. TRAIL expression was increased predominantly in myeloid precursors of granulocytic lineage and in a subset of monocytes and pro-erythroblasts. Significant levels of soluble TRAIL were released in 21 of 68 BMMC culture supernatants from MDS patients. On the other hand, TRAIL was detected less frequently in the culture supernatants of AML (4 of 33) and normal BMMC (0 of 22). Analysis of peripheral blood parameters revealed significantly lower levels of peripheral red blood cells and hemoglobin in the subset of patients whose BMMC released TRAIL in culture supernatants compared to the subgroup of patients who did not release TRAIL. Moreover, TRAIL-positive BMMC culture supernatants inhibited the differentiation of normal glycophorin A+ erythroblasts generated in serum-free liquid phase. Thus, increased expression and release of TRAIL at the bone marrow level is likely to impair erythropoiesis and to contribute to the degree of anemia, the major clinical feature of MDS.

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.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF-alpha induced monocytic maturation of primary normal CD34-derived myeloid precursors and of the M2/M3-type acute myeloid leukemia HL-60 cell line, associated to increased nuclear factor (NF)-kappaB activity and nuclear translocation of p75, p65, and p50 NF-kappaB family members. Consistently, both cytokines also induced the degradation of the NF-kappaB inhibitors, IkappaBalpha and IkappaB epsilon, and up-regulated the surface expression of TRAIL-R3, a known NF-kappaB target. However, NF-kappaB activation and IkappaB degradation occurred with different time-courses, since TNF-alpha was more potent, rapid, and transient than TRAIL. Of the two TRAIL receptors constitutively expressed by HL-60 (TRAIL-R1 and TRAIL-R2), only the former was involved in IkappaB degradation, as demonstrated by using agonistic anti-TRAIL receptor antibodies. Moreover, NF-kappaB nuclear translocation induced by TRAIL but not by TNF-alpha was abrogated by z-IETD-fmk, a caspase-8-specific inhibitor. The key role of NF-kappaB in mediating the biological effects of TNF-alpha and TRAIL was demonstrated by the ability of unrelated pharmacological inhibitors of the NF-kappaB pathway (parthenolide and MG-132) to abrogate TNF-alpha- and TRAIL-induced monocytic maturation. These findings demonstrate that NF-kappaB is essential for monocytic maturation and is activated via distinct pathways, involving or not involving caspases, by the related cytokines TRAIL and TNF-alpha.

Treatment of the human HL-60 cell line with tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) resulted in rapid (6-24 hours) cytotoxicity associated with progressive maturation of the surviving cells along the monocytic lineage. The occurrence of monocytic maturation was demonstrated by a significant increase of both CD14 and CD11b surface expression, the acquisition of morphologic features typical of mature monocytes, and phagocytic capacity in TRAIL-treated cultures. By using selective pharmacologic inhibitors, it was possible to demonstrate that activation of the caspase cascade played a crucial role in mediating TRAIL cytotoxicity and monocytic maturation of HL-60 cells. Moreover, experiments performed using agonistic polyclonal antibodies, which mimic the interactions between TRAIL and each TRAIL receptor, indicated that TRAIL-R1 was responsible for mediating the TRAIL-induced maturation. Importantly, the maturational effects of TRAIL were observed also in primary normal CD34+ cells, seeded in serum-free liquid cultures for 4 to 8 days in the presence of SCF + GM−CSF. After treatment with TRAIL for 3 additional days, a significant increase in CD14 and CD11b expression, coupled with an increased number of mature monocytes and macrophages, was noticed in the absence of cytotoxicity. These data disclose a novel role for TRAIL as a positive regulator of myeloid differentiation. Moreover, the dichotomous effect of TRAIL on malignant cells (early induction of apoptosis and monocytic maturation of the surviving cells) might have important therapeutic implications for the treatment of acute myeloid leukemia.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) up-regulated the expression of constitutive cyclooxygenase (COX)-1 protein in HL-60 cells without affecting COX-2. The TRAIL-mediated COX-1 up-regulation was accompanied by a significant increase of the PGE2 synthesis and release, which was suppressed by the COX-1 inhibitor valeryl salicylate but not by the COX-2 inhibitor NS-398. Experiments carried out by adding exogenous PGE2 to HL-60 cells indicated that PGE2 was not involved in TRAIL cytotoxicity and rather showed a dose-dependent protection against TRAIL-induced apoptosis. Importantly, the ability of TRAIL to increase PGE2 production was also observed in normal, human CD34-derived myeloid cells and in freshly isolated peripheral blood CD14+ monocytes. Moreover, in contrast to HL-60 cells, primary, normal cells were not susceptible to TRAIL cytotoxicity. These data indicate that the ability of TRAIL to up-regulate eicosanoid production and release is not confined to malignant leukemic cells, but it may also play a role in normal hematopoiesis.

In natural killer (NK) cells, interleukin-2 (IL-2) differentially regulates the expression of several transcription factors, including JunB and c-fos. The cAMP response element binding protein, CREB, is a key transcriptional regulator of a large number of genes containing the octanucleotide CRE consensus sequence in their upstream regulatory regions. We studied here the functional role of CREB in the IL-2-mediated transcriptional regulation of c-fos in human NK cells. Our results show that IL-2 activates CREB in human NK cells and that CREB activation hasa prominent regulatory role on the IL-2-induced expression of functional c- fos and AP-1 in NK cells. We identify two domains of the c-fos promoter, containing three CRE sites, which are critical for the transcriptional activity induced by IL-2. The first domain is located within the first 220 nucleotides of the c-fos promoter, while the second encompasses the nucleotides - 440 and - 220. Our results show that CREB has a relevant role in the cytokine-mediated activation of NK cells, and are particularly remarkable in the light of the several genes that are positively regulated by c-fos and AP-1, such as IFN-gamma, IL-2 and GM-CSF genes.

Background Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells. Cytometry 44:57–64, 2001. © 2001 Wiley-Liss, Inc.