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PDF file - 133K, HIF-1alpha protein correlates with VEGFR-2, FGFR-2 and HGF.

PDF file - 17K, Real Time RT-PCR Primers.

PDF file - 166K, HIF-1alpha protein does not correlate with AKT, and VHL.

PDF file - 23K, Proteins identified in MMECs upon HIF-1�siRNA and panobinostat treatment (MALDITOFF mass spectrometry).

PDF file - 114K, Lack of HIF-1alpha expression by BM macrophages in normoxia condition.

PDF file - 2376K, Panobinostat effect on MMECs in the presence of BM cells and proteomic analysis of MMECs upon treatment with HIF-1α·siRNA or panobinostat.

Purpose: To investigate the role of hypoxia-inducible factor-1α (HIF-1α) in angiogenesis and drug resistance of bone marrow endothelial cells of patients with multiple myeloma.Experimental Design: HIF-1α mRNA and protein were evaluated in patients with multiple myeloma endothelial cells (MMEC) at diagnosis, at relapse after bortezomib- or lenalidomide-based therapies or on refractory phase to these drugs, at remission; in endothelial cells of patients with monoclonal gammapathies of undetermined significance (MGUS; MGECs), and of those with benign anemia (controls). The effects of HIF-1α inhibition by siRNA or panobinostat (an indirect HIF-1α inhibitor) on the expression of HIF-1α proangiogenic targets, on MMEC angiogenic activities in vitro and in vivo, and on overcoming MMEC resistance to bortezomib and lenalidomide were studied. The overall survival of the patients was also observed.Results: Compared with the other endothelial cell types, only MMECs from 45% of relapsed/refractory patients showed a normoxic HIF-1α protein stabilization and activation that were induced by reactive oxygen species (ROS). The HIF-1α protein correlated with the expression of its proangiogenic targets. The HIF-1α inhibition by either siRNA or panobinostat impaired the MMECs angiogenesis–related functions both in vitro and in vivo and restored MMEC sensitivity to bortezomib and lenalidomide. Patients with MMECs expressing the HIF-1α protein had shorter overall survival.Conclusions: The HIF-1α protein in MMECs may induce angiogenesis and resistance to bortezomib and lenalidomide and may be a plausible target for the antiangiogenic management of patients with well-defined relapsed/refractory multiple myeloma. It may also have prognostic significance. Clin Cancer Res; 20(4); 847–58. ©2013 AACR.

PDF file - 403K, HIF-1alpha protein negative MMECs are sensitive to the antiangiogenic effect of bortezomib and lenalidomide.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is often characterized by a life-threatening interstitial pneumonia requiring hospitalization. The aim of this retrospective cohort study is to identify hallmarks of in-hospital mortality in patients affected by Coronavirus Disease 19 (COVID-19). A total of 150 patients admitted for COVID-19 from March to June 2021 to “F. Perinei” Murgia Hospital in Altamura, Italy, were divided into survivors (n = 100) and non-survivors groups (n = 50). Blood counts, inflammation-related biomarkers and lymphocyte subsets were analyzed into two groups in the first 24 h after admission and compared by Student’s t-test. A multivariable logistic analysis was performed to identify independent risk factors associated with in-hospital mortality. Total lymphocyte count and CD3+ and CD4+ CD8+ T lymphocyte subsets were significantly lower in non-survivors. Serum levels of interleukin-6 (IL-6), lactate dehydrogenase (LDH), C-reactive protein (CRP) and procalcitonin (PCT) were significantly higher in non-survivors. Age > 65 years and presence of comorbidities were identified as independent risk factors associated with in-hospital mortality, while IL-6 and LDH showed a borderline significance. According to our results, markers of inflammation and lymphocytopenia predict in-hospital mortality in COVID-19.

There is a large body of evidence showing a strong correlation between carcinogenesis of several types of human tumors, including chronic lymphocytic leukemia (CLL), and oxidative stress (OS). The mechanisms by which OS may promote cancer pathogenesis have not been completely deciphered yet and, in CLL, as in other neoplasms, whether OS is a primary cause or simply a downstream effect of the disease is still an open question. It has been demonstrated that, in CLL, OS concomitantly results from increased reactive oxygen species (ROS) production, mainly ascribable to CLL cells mitochondrial activity, and impaired antioxidant defenses. Interestingly, OS evaluation in CLL patients, at diagnosis, seems to have a prognostic significance, thus getting new insights in the biological comprehension of the disease with potential therapeutic implications.

Objective To evaluate the prognostic significance of oxidative stress (OS) and antioxidant defense status measurement in patients with chronic lymphocytic leukemia (CLL). Method d-ROMs test and BAP test were evaluated at diagnosis of 165 patients with CLL and correlated with clinical-biological features and prognosis. Results An increased oxidative damage (d-ROMs test) and a reduced antioxidant potential (BAP test), were found in CLL patients than normal controls (p

Neuroblastoma is the most commonly extra-cranial solid tumor of childhood frequently diagnosed. The nervous system-specific metabolite N-acetylaspartate (NAA) is synthesized from aspartate and acetyl-CoA in neurons, it is among the most abundant metabolites present in the central nervous system (CNS) and appears to be involved in many CNS disorders. The functional significance of the high NAA concentration in the brain remains uncertain, but it confers to NAA a unique clinical significance exploited in magnetic resonance spectroscopy. In the current study, we show that treatment of SH-SY5Y neuroblastoma-derived cell line with sub-cytotoxic physiological concentrations of NAA inhibits cell growth. This effect is partly due to enhanced apoptosis, shown by decrease of the anti-apoptotic factors survivin and Bcl-xL, and partly to arrest of the cell-cycle progression, linked to enhanced expression of the cyclin-inhibitors p53, p21Cip1/Waf1 and p27Kip1. Moreover, NAA-treated SH-SY5Y cells exhibited morphological changes accompanied with increase of the neurogenic markers TH and MAP2 and down-regulation of the pluripotency markers OCT4 and CXCR4/CD184. Finally, NAA-pre-treated SH-SY5Y cells resulted more sensitive to the cytotoxic effect of the chemotherapeutic drugs Cisplatin and 5-fluorouracil. To our knowledge, this is the first study demonstrating the neuronal differentiating effects of NAA in neuroblastoma cells. NAA may be a potential preconditioning or adjuvant compound in chemotherapeutic treatment.

JAK2 V617F mutational status is an essential diagnostic index in myeloproliferative neoplasms (MPNs). Although widely used for detection of JAK2 V617F mutation in peripheral blood (PB), sensitive real-time quantitative PCR (qPCR) presents some methodological limitations. Recently, emerging alternative technologies, like digital droplet PCR (ddPCR), have been reported to overcome some of qPCR&rsquo, s technical drawbacks. The purpose of this study was to compare the diagnostic utility of ddPCR to qPCR for JAK2 V617F detection and quantification in samples from MPNs patients. Sensitivity and specificity of qPCR and ddPCR in the detection of the mutation were assessed by using a calibrator panel of mutated DNA on 195 JAK2 positive MPN samples. Based on our results, ddPCR proved to be a suitable, precise, and sensitive method for detection and quantification of the JAK2 V617F mutation.

The iron chelator deferasirox (DFX) prevents complications related to transfusional iron overload in several haematological disorders characterized by marrow failure. It is also able to induce haematological responses in a percentage of treated patients, particularly in those affected by myelodysplastic syndromes. The underlying mechanisms responsible for this feature, however, are still poorly understood. In this study, we investigated the effect of DFX-treatment in human haematopoietic/progenitor stem cells, focussing on its impact on the redox balance, which proved to control the interplay between stemness maintenance, self-renewal and differentiation priming. Here we show, for the first time, that DFX treatment induces a significant diphenyleneiodonium-sensitive reactive oxygen species (ROS) production that leads to the activation of POU5F1 (OCT4), SOX2 and SOX17 gene expression, relevant in reprogramming processes, and the reduction of the haematopoietic regulatory proteins CTNNB1 (β-Catenin) and BMI1. These DFX-mediated events were accompanied by decreased CD34 expression, increased mitochondrial mass and up-regulation of the erythropoietic marker CD71 (TFRC) and were compound-specific, dissimilar to deferoxamine. Our findings would suggest a novel mechanism by which DFX, probably independently on its iron-chelating property but through ROS signalling activation, may influence key factors involved in self-renewal/differentiation of haematopoietic stem cells.

Toxoneuron nigriceps (Hymenoptera, Braconidae) is an endophagous parasitoid of the larval stages of the tobacco budworm, Heliothis virescens (Lepidoptera, Noctuidae). The bracovirus associated with this wasp (TnBV) is currently being studied. Several genes expressed in parasitised host larvae have been isolated and their possible roles partly elucidated. TnBVank1 encodes an ankyrin motif protein similar to insect and mammalian IκB, an inhibitor of the transcription nuclear factor κB (NF-κB). Here we show that, when TnBVank1 was stably expressed in polyclonal Drosophila S2 cells, apoptosis is induced. Furthermore, we observed the same effects in haemocytes of H. virescens larvae, after TnBVank1 in vivo transient transfection, and in haemocytes of parasitised larvae. Coimmunoprecipitation experiments showed that TnBVANK1 binds to ALG-2 interacting protein X (Alix/AIP1), an interactor of apoptosis-linked gene protein 2 (ALG-2). Using double-immunofluorescence labeling, we observed the potential colocalization of TnBVANK1 and Alix proteins in the cytoplasm of polyclonal S2 cells. When Alix was silenced by RNA interference, TnBVANK1 was no longer able to cause apoptosis in both S2 cells and H. virescens haemocytes. Collectively, these results indicate that TnBVANK1 induces apoptosis by interacting with Alix, suggesting a role of TnBVANK1 in the suppression of host immune response observed after parasitisation by T. nigriceps.

Although usually referred to as a structural actin-binding protein, LIM and SH3 domain-containing protein may actually be dynamically involved in the control of a wide spectrum of cellular processes, by virtue of its interaction with several molecular partners. Alongside being ubiquitously expressed in physiological conditions, LIM and SH3 domain-containing protein is overexpressed in a growing number of human cancers, in which it may actively contribute to their aggressiveness by promoting cell proliferation and migration. In view of the recent findings, implicating the protein in cancer progression, we discuss here the most relevant discoveries highlighting the role of this versatile protein in various human tumors. The correlation between LIM and SH3 domain-containing protein expression levels in cancer and the poor outcome and metastatic behavior of tumors denotes the clinical significance of this protein and hints its potential value as a new cancer prognostic or even diagnostic biomarker. This may be decisive not only to optimize existing pharmacological regimes but also to delineate novel, more efficacious therapeutic and/or preventive approaches.

Penile cancer is a rare disease, with an incidence that is higher in less developed countries and is in the range of 1 - 10 per 100000 men worldwide. Early diagnosis is essential for cure, as 5 year cancer-specific survival is 90 - 100 % in patients with intraepithelial neoplasms and in those with low-grade superficial tumors without lymphovascular invasion, but it drops to 30% in men with multiple mobile or bilateral inguinal lymph nodes. The EGFR family plays a major role in penile cancer biology, with distinct receptors being involved in HPV-positive and -negative tumors. A number of anti-EGFR agents were used in penile cancer patients outside the context of a clinical trial, mainly as a salvage treatment after failure of first-line chemotherapy. A total of 28 patients received anti-EGFR monoclonal antibodies, with 50% of them showing a response to treatment, and a median PFS of ∼ 3 months. The rarity of the disease poses great challenge in terms of education and awareness of the general population, planning of preventive measures on a large scale, as well as conduction of prospective trials and approval of high-cost biological therapy.

Gastric cancer (GC) remains one of the major causes of cancer-related mortality worldwide. As for other types of cancers, several limitations to the success of current therapeutic GC treatments may be due to cancer drug resistance that leads to tumor recurrence and metastasis. Increasing evidence suggests that cancer stem cells (CSCs) are among the major causative factors of cancer treatment failure. The research of molecular CSC mechanisms and the regulation of their properties have been intensively studied. To date, molecular gastric cancer stem cell (GCSC) characterization remains largely incomplete. Among the GCSC-targeting approaches to overcome tumor progression, recent studies have focused their attention on microRNA (miRNA). The miRNAs are short non-coding RNAs which play an important role in the regulation of numerous cellular processes through the modulation of their target gene expression. In this review, we summarize and discuss recent findings on the role of miRNAs in GCSC regulation. In addition, we perform a meta-analysis aimed to identify novel miRNAs involved in GCSC homeostasis.

We synthesized 3-aroyl-1-arylpyrrole (ARAP) derivatives as potential anticancer agents having different substituents at the pendant 1-phenyl ring. Both the 1-phenyl ring and 3-(3,4,5-trimethoxyphenyl)carbonyl moieties were mandatory to achieve potent inhibition of tubulin polymerization, binding of colchicine to tubulin, and cancer cell growth. ARAP 22 showed strong inhibition of the P-glycoprotein-overexpressing NCI-ADR-RES and Messa/Dx5MDR cell lines. Compounds 22 and 27 suppressed in vitro the Hedgehog signaling pathway, strongly reducing luciferase activity in SAG treated NIH3T3 Shh-Light II cells, and inhibited the growth of medulloblastoma D283 cells at nanomolar concentrations. ARAPs 22 and 27 represent a new potent class of tubulin polymerization and cancer cell growth inhibitors with the potential to inhibit the Hedgehog signaling pathway.

Hepatitis-C-virus-related infective diseases are worldwide spread pathologies affecting primarily liver. The infection is often asymptomatic, but when chronically persisting can lead to liver scarring and ultimately to cirrhosis, which is generally apparent after decades. In some cases, cirrhosis will progress to develop liver failure, liver cancer, or life-threatening esophageal and gastric varices. HCV-infected cells undergo profound metabolic dysregulation whose mechanisms are yet not well understood. An emerging feature in the pathogenesis of the HCV-related disease is the setting of a pro-oxidative condition caused by dysfunctions of mitochondria which proved to be targets of viral proteins. This causes deregulation of mitochondria-dependent catabolic pathway including fatty acid oxidation. Nuclear receptors and their ligands are fundamental regulators of the liver metabolic homeostasis, which are disrupted following HCV infection. In this contest, specific attention has been focused on the peroxisome proliferator activated receptors given their role in controlling liver lipid metabolism and the availability of specific pharmacological drugs of potential therapeutic utilization. However, the reported role of PPARs in HCV infection provides conflicting results likely due to different species-specific contests. In this paper we summarize the current knowledge on this issue and offer a reconciling model based on mitochondria-related features.

Introduction Hepatocellular carcinoma (HCC) represents the sixth leading cancer and the third most common cause of death from cancer. Many different aetiological factors are involved in the development of HCC, which may be modulated by both estrogens and androgens hormones during its initiation, progression and metastasis. The misuse of anabolic androgenic steroids (AAS) is associated with serious adverse effects to the liver, including cellular adenomas and adenocarcinomas, and is considered a factor risk of developing hepatic sex hormone related tumours. The purpose of this study was to investigate the role of Nandrolone, one of the most commonly used AAS, in regulating proliferation and differentiation of HCC. Material and methods Human HCC cell line HepG2 was treated with Nandrolone, a synthetic androgen ligand, for 48 hs and its viability and proliferation was assessed by MTS and cell cycle analysis, respectively. The expression of protein involved in cell cycle regulation and differentiation markers were analysed by western blot and real time PCR. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were performed using Seahorse XF96 extracellular flux analyzer. Respiratory chain complex activities were assayed spectrophotometrically. Stemness surface markers expression was detected by FACSCalibur flow cytometer. Results and discussions Nandrolone treatment caused cell growth inhibition associated to a downregulation of cyclin D1 and an upregulation of the cyclin-dependent kinase inhibitors p21Waf1/Cip1 leading to cell cycle arrest in the G2 phase. Moreover, a significant overall impairment of mitochondrial functions, resulting in a reduced OCR and impairment of OXPHOS complexes activities were also observed, thus suggesting a role in the control of the metabolic reprogramming. Finally, a significant increase of the stemness markers was detected following Nandrolone treatment, also confirmed in additional human stem cell types and in an in vivo mouse model. Conclusion Nandrolone shows a strong anti-proliferative effect in differentiated tumour cells, promoting cancer cells stemness through cellular metabolic reprogramming. These results could have important public health implications in order to improve the primary prevention such as revising altered lifestyles, like AAS abuse.

Introduction Recently, several studies have highlighted the key role of cancer stem cells (CSC) in tumour initiation, metastasis, and relapses. The CSC pool generally exhibits higher resistance to conventional chemo-radiotherapy and a different cell metabolism. Aim of our study is to investigate the main metabolic differences in the human osteosarcoma stem-like cells (3ABOS) and differentiated osteosarcoma cells (MG63) to unveil new metabolic therapeutic targets. Material and methods Metabolic analyses were performed with Seahorse Bioanalyzer. Live cell imaging for ROS content, mitochondrial membrane potential and morphology, were performed by confocal microscopy by using DCF-DA, Mito-Tracker Red and NAO as selective probes, respectively. Protein expression was revealed by qPCR and western blot. Results and discussions Our results showed a significant reduction of the mitochondrial oxygen consumption rate in 3ABOS compared with MG63 cells. Next, we assessed the specific contributions of glucose, fatty acid and glutamine to the respiratory phenotype, unveiling larger reliance on oxidation of these three main fuels with a significant reduction in mitochondrial flexibility in 3ABOS. The lower OXPHOS is compensated by a shift in glucose metabolism demonstrated by increased extracellular acidification rate.These results were further supported by a significant reduction of 3ABOS proliferation in glucose shortage. According to this scenario, confocal microscopy highlighted reduced mitochondrial membrane potential, and increased ROS content in 3ABOS compared to MG63. Additionally, 3ABOS displayed a lower mitochondrial DNA amount associated with more elongated mitochondrial network confirmed by both live cell imaging and Mitofusin expression analysis. Moreover, members of the NADPH oxidases family resulted to be differently expressed in the two cell lines, thus suggesting a potential role of ROS mediated signalling in cancer cell phenotype. Conclusion Overall our results demonstrated that the oxidative metabolic phenotype hallmarks cancer biology. Further investigations are ongoing to define specific drugs acting on metabolic target and their effectiveness as a therapeutic approach.

Introduction Targeting metabolism represents a new approach to treat cancer, expecially when conventional chemoterapy fails. In this study, we tested a metabolic approach to treat PC, investigating, in vitro and in vivo, its response to DCA treatment. Material and methods Two PC cell lines, BXPC3 and PANC1, were treated with DCA 4 and 10 mM for 24 hour. Cell viability and proliferation were assessed by MTS assay and xCELLigence, apoptosis and ROS by flow-cytometry; pPDH Ser293 /tot PDH, LC3B, DRP1, MFN1, MNF2, OPA1 and TOMM20 protein expression was evaluated by western blotting, lin28 gene expression by qPCR. The oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) were measured by Seahorse Technology. Ultra-low attachment plates were used to form spheroids. In vivo, DCA was administered to BXPC3-luc tumor-bearing nude mice. After measuring bioluminescence signalling, the tumour masses were harvested, photographed and weighed. Results and discussions DCA treatment reduced cell proliferation, decreasing cell survival with an increase in ROS production and apoptosis in both cell lines. Despite PDH activation by dephosphorylation, DCA did not restore bioenergetic profile but decreased OCR, a measure of oxidative phosphorylation efficiency. ECAR was not affected, suggesting that the glicolytic capacity was not modified by DCA treatment. These observations led us to explore mitophagy, whose activation was confirmed by LC3B protein overexpression and TOMM20 downregulation, and mitochondrial dynamics also altered following DCA treatment as shown by the downregulation of MFN1, MFN2, OPA1, key proteins of mitochondrial fusion. Interestingly, DCA was able to negatively affect the cancer stem cell (CSCs) fraction in both cell lines, reducing the expression of stemness genes such as Lin28 and inhibiting spheroid formation. When added to 3D cultures already formed, it was able to downregulate stemness genes expression, leading to a significant size reduction and affecting spheroid viability. Finally, DCA efficacy was confirmed in a xenograft pancreatic cancer mouse model in which DCA treatment displayed a significant retarded progression of PC, reducing diameter of the tumour mass. Conclusion Our data suggest that DCA is able to strongly affect PC cells metabolism counteracting mitochondrial activity. This effect is not related to PDH activity stimulation. In addition, the ability of DCA to hit CSCs offers a further rationale to candidate this drug for PC treatment, trying to reach a complete tumour eradication.

Introduction Pancreatic adenocarcinoma is a tumour with poor prognosis. Usually diagnosed at a late stage, the high mortality is linked to resistance to conventional chemotherapy. Combination therapy and targeted therapies proved to be not very effective. Thus, a better understanding of the molecular mechanisms underlying drug resistance in pancreatic cancer could lead to the development of more effective therapeutic strategies. Material and methods BX-PC3 pancreatic tumour cells were treated with increasing doses of NAA (2,4,8 and 16 mM) for 72 hour and cell viability was assessed by xCELLigence system technology. The gene expression profile induced by NAA treatment in BX-PC3 cells was examined using Real-Time qPCR. Anti-proliferative and differentiating effects of NAA in BX-PC3 treated cells were evaluated by flow cytometric analysis. Acetyl CoA levels after 72 hour NAA treatment was mesured by HPLC/HRMS. The expression of proteins involved in acetylation mechanism were measured by Western Blotting. The metabolic analysis were performed by the Seahorse Bioanalyzer. The effects of NAA in 3D cultures were studied morphologically by inverted microscope. Results and discussions NAA treatment in BX-PC3 pancreatic tumour cells elicited anti-proliferative and differentiating effects evident with the arrest of proliferation and decreased expression of specific stemness markers such as cMyc, Klf4, Lin28 and Oct4. Exposure of cells to NAA induced down-regulation of CD133 and CD184 surface markers, arrest of cell cyle at G0/G1 phase, associated to increased levels of p53, p21 and p27 genes. Moreover, NAA-treated BX-PC3 cells showed decreased levels of the central metabolite Coenzyme A, which correlates with alterations in protein acetylation. In addition, an overall impairment of mitochondrial function was observed following NAA treatment, resulting in a revised feeding of metabolic substrates. Finally, NAA showed a strong effect on tumour spheroid growth, with reduction in colony size. Conclusion To our knowledge, this is the first study that demonstrates the differentiating effects of NAA treatment in pancreatic tumour cells and its ability to reduce the size of 3D pancreatic carcinoma spheroids.

The Nef protein of the human immunodeficiency virus type 1 (HIV-1) plays a crucial role in AIDS pathogenesis by modifying host cell signaling pathways. We investigated the effects of Nef on the NADPH oxidase complex, a key enzyme involved in the generation of reactive oxygen species during the respiratory burst in human monocyte/macrophages. We have recently shown that the inducible expression of HIV-1 Nef in human macrophages cell line modulates in bi-phasic mode the superoxide anion release by NADPH oxidase, inducing a fast increase of the superoxide production, followed by a delayed strong inhibition mediated by Nef-induced soluble factor(s). Our study is focused on the molecular mechanisms involved in Nef-mediated activation of NADPH oxidase and superoxide anion release. Using U937 cells stably transfected with different Nef alleles, we found that both Nef membrane localization and intact SH3-binding domain are needed to induce superoxide release. The lack of effect during treatment with a specific MAPK pathway inhibitor, PD98059, demonstrated that Nef-induced superoxide release is independent of Erk1/2 phosphorylation. Furthermore, Nef induced the phosphorylation and then the translocation of the cytosolic subunit of NADPH oxidase complex p47(phox) to the plasma membrane. Adding the inhibitor PP2 prevented this process, evidencing the involvement of the Src family kinases on Nef-mediated NADPH oxidase activation. In addition, LY294002, a specific inhibitor of phosphoinositide 3-kinase (PI3K) inhibited both the Nef-induced p47(phox) phosphorylation and the superoxide anion release. These data indicate that Nef regulates the NADPH oxidase activity through the activation of the Src kinases and PI3K.

// Vitalba Ruggieri 1 , Francesca Agriesti 1 , Rosella Scrima 2 , Ilaria Laurenzana 1 , Donatella Perrone 2 , Tiziana Tataranni 1 , Carmela Mazzoccoli 1 , Lorenzo Lo Muzio 2 , Nazzareno Capitanio 2 , Claudia Piccoli 1, 2 1 Laboratory of Pre-Clinical and Translational Research, IRCCS, CROB, Rionero in Vulture, Potenza, Italy 2 Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy Correspondence to: Nazzareno Capitanio, e-mail: nazzareno.capitanio@unifg.it Claudia Piccoli, e-mail: claudia.piccoli@unifg.it Keywords: oral squamous cell carcinoma, dichloroacetate, oxidative metabolism, mitochondria, reactive oxygen species Received: August 31, 2014 Accepted: November 11, 2014 Published: November 25, 2014 ABSTRACT Reprogramming of metabolism is a well-established property of cancer cells that is receiving growing attention as potential therapeutic target. Oral squamous cell carcinomas (OSCC) are aggressive and drugs-resistant human tumours displaying wide metabolic heterogeneity depending on their malignant genotype and stage of development. Dichloroacetate (DCA) is a specific inhibitor of the PDH-regulator PDK proved to foster mitochondrial oxidation of pyruvate. In this study we tested comparatively the effects of DCA on three different OSCC-derived cell lines, HSC-2, HSC-3, PE15. Characterization of the three cell lines unveiled for HSC-2 and HSC-3 a glycolysis-reliant metabolism whereas PE15 accomplished an efficient mitochondrial oxidative phosphorylation. DCA treatment of the three OSCC cell lines, at pharmacological concentrations, resulted in stimulation of the respiratory activity and caused a remarkably distinctive pro-apoptotic/cytostatic effect on HSC-2 and HSC-3. This was accompanied with a large remodeling of the mitochondrial network, never documented before, leading to organelle fragmentation and with enhanced production of reactive oxygen species. The data here presented indicate that the therapeutic efficacy of DCA may depend on the specific metabolic profile adopted by the cancer cells with those exhibiting a deficient mitochondrial oxidative phosphorylation resulting more sensitive to the drug treatment.

The DNA glycosylase MUTYH (mutY homolog (Escherichia coli)) counteracts the mutagenic effects of 8-oxo-7,8-dihydroguanine (8-oxodG) by removing adenine (A) misincorporated opposite the oxidized purine. Biallelic germline mutations in MUTYH cause the autosomal recessive MUTYH-associated adenomatous polyposis (MAP). Here we designed new tools to investigate the biochemical defects and biological consequences associated with different MUTYH mutations in human cells. To identify phenotype(s) associated with MUTYH mutations, lymphoblastoid cell lines (LCLs) were derived from seven MAP patients harboring missense as well as truncating mutations in MUTYH. These included homozygous p.Arg245His, p.Gly264TrpfsX7 or compound heterozygous variants (p.Gly396Asp/Arg245Cys, p.Gly396Asp/Tyr179Cys, p.Gly396Asp/Glu410GlyfsX43, p.Gly264TrpfsX7/Ala385ProfsX23 and p.Gly264TrpfsX7/Glu480del). DNA glycosylase assays of MAP LCL extracts confirmed that all these variants were defective in removing A from an 8-oxoG:A DNA substrate, but retained wild-type OGG1 activity. As a consequence of this defect, MAP LCLs accumulated DNA 8-oxodG in their genome and exhibited a fourfold increase in spontaneous mutagenesis at the PIG-A gene compared with LCLs from healthy donors. They were also hypermutable by KBrO3--a source of DNA 8-oxodG--indicating that the relatively modest spontaneous mutator phenotype associated with MUTYH loss can be significantly enhanced by conditions of oxidative stress. These observations identify accumulation of DNA 8-oxodG and a mutator phenotype as likely contributors to the pathogenesis of MUTYH variants.

Hepatitis C virus (HCV) is the major reason for liver transplantation and the main cause of liver-related morbidity and mortality in a great number of countries. As for the other viruses, this pathogen interferes in more than one process and in more than one way with host cell biology. A mounting body of evidence points, in particular, toward the drastic alterations of mitochondrial physiology and functions that virus is able to induce, albeit the mechanisms have partly remained elusive. Role of the mitochondria in immunity and in quality control systems, as autophagy, as well as the strategies that HCV has evolved to evade and even to manipulate mitochondrial surveillance for its benefit, highlights the importance of deepening the mechanisms that modulate this virus-mitochondrion interaction, not only to intensify our knowledge of the HCV infection pathogenesis but also to design efficient antiviral strategies.

Deferasirox (DFX) is an iron chelator used to prevent and treat complications related to transfusional iron overload in myelodisplastic syndrome patients (MDS). Intriguingly, DFX treatment induces haematological responses in a consistent percentage of patients whereas other chelators, like deferoxamine (DFO) do not show such an appreciable effect. A body of literature documents a general reduction of oxidative stress in patients treated with DFX but little is known about the direct effect of DFX treatment on hematopoietic stem cells (HSC). Consolidated evidence highlights the importance of redox signalling in the homeostasis of fundamental processes, particularly in controlling the balance between self-renewal and differentiation of stem cells. In this setting, reactive species of oxygen (ROS) would act as secondary messengers, modulating the expression of master transcription factors and regulatory proteins leading or (pre)conditioning stem cells towards differentiation. In the present study we investigated the effect of DFX and DFO on ROS production in hematopoietic stem/progenitor cells (HSPCs) in order to identify a molecular mechanism explaining the differential effect of iron chelators in rescuing altered hematopoiesis. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy subjects and incubated for 24 hours with DFO and DFX in a range of doses between 6.2mM and 200mM. Human HSPCs, isolated upon informed consent from peripheral blood of G-CSF-treated healthy donors by immuno-selection against the specific markers CD133 and CD34, were treated with 100 mM DFX or DFO for 24 hours. To completely abrogate ROS production, cells were co-incubated with diphenil iodide (DPI) 100mM, a known inhibitor of the main proteins able to generate ROS: the flavo-oxidases of the respiratory chain and the NADPH oxidases. Cell viability was determined by trypan blue staining. ROS levels were analyzed by laser scanning confocal microscopy (LSCM) and flow cytometry after the incubation at 37°C for 15 minutes with the intracellular H2O2 specific probe dichlorodihhyrofluorescein-diacetate (H2DCFDA) 10mM. b-catenin and BMI1 protein levels were assessed by western blotting. Data were presented as mean±s.e.m. and were compared by unpaired Student T-Test; a p The flow cytometry analysis on PBMCs revealed that DFX, surprisingly, was able to induce ROS production in a dose-dependent manner with a significant increase at 100mM and 200mM compared to CTRL (p=0.02) whereas DFO treatment didn’t show any effect compared to CTRL. Since the cell viability resulted to be not affected by both drugs, the dose of 100mM was chosen for the following experiments performed with HSPCs where the ability of DFX to cause a significant increase of H2O2 was confirmed. A deeper analysis aimed to clarify the localization of ROS released following DFX treatment was performed by LSCM: a significant up-regulation of mitochondrial ROS induced by DFX and not by DFO was clearly observed compared to untreated samples. Importantly, the addition of DPI strongly reduced the entity of the signal detected by DCF (p=0.01 versus DFX). In order to understand a possible link between ROS production and the ability of DFX to restore the hematopoiesis, the expression of b-catenin and BMI1, both of them sensitive to the intracellular redox state and involved in the hematopoietic function, was analysed by western blotting. It was shown that DFX significantly reduced the expression of both b-catenin, strictly linked to hematopoietic function and BMI1 (p=0.02 versus CTRL), a regulatory protein sustaining immaturity and required for the maintaining of adult self-renewing hematopoietic stem cells. Conversely, no significant variation was observed by DFO treatment. Interestingly, the b-catenin and BMI-1expression downregulations, specifically induced by DFX, were completely reversed by ROS abrogation obtained by DPI treatment. Our results show, for the first time, that DFX treatment, independently on its iron-chelating property, is able to induce ROS production that in turn influences key factors involved in self-renewal/differentiation of hematopoietic stem cells. In this scenario, the modulation of ROS, because of their ability to restore the hematopoietic function, could be taken in account as potential further pharmacological target in MDS treatment. Disclosures: No relevant conflicts of interest to declare.

Abstract 2299 Introduction Hematopoietic stem cells (HSCs) constitute a reservoir of undifferentiated cells that can be committed, upon appropriate stimuli, in the haematic lineages. Although residing in a bone-marrow hypoxic microenvironment (niche) and mainly relying on anaerobic glycolysis, HSCs are endowed with mitochondria. Recently, specific interest has been focused on HSCs mitochondria and on their role as reactive oxygen species (ROS) generators during the early phases of commitment. Indeed, consolidated evidences highlight the importance of redox signalling in the homeostasis of fundamental processes in cell adaptive biology and particularly in controlling the balance between self-renewal and differentiation of stem cells. HSCs constitutively generate low levels of ROS produced by both mitochondrial respiratory chain and NADPH oxidases (NOXs). ROS would act as secondary messengers, modulating the expression of master transcription factors leading or (pre)conditioning stem cells towards differentiation. Myelodisplastic syndrome (MDS) is characterized by disturbance of the HSC differentiation and most MDS patients are treated with iron chelators to compensate for the iron overload consequent to the blood cell transfusion-based standard therapy. Intriguingly, a robust percentage of patients, treated with the iron chelator deferasirox (DFX), recover correct HSCs differentiation whereas other chelators, like deferoxamine (DFO) did not. In the presented study we investigated the effect of DFX and DFO on the redox homeostasis of hematopoietic stem/progenitor cells (HSPCs) in order to get insights on the differential effect of iron chelators in rescuing altered hematopoiesis. Materials and Methods Human HSPCs were isolated from peripheral blood (PB) or bone marrow (BM) of G-CSF-treated or untreated healthy donors, respectively, by immuno-selection against the specific markers CD133 and CD34 and resulted >99% immunophenotypically homogeneous. Mitochondrial respiratory activity was measured in intact HSPCs by high resolution oxymetry. Morpho-functional features of HSPC-mitochondria, expression of the HSPC-surface commitment markers and ROS level were analyzed by laser scanning confocal microscopy (LSCM) and flow cytometry using specific probes or antibodies. HSPCs were treated with 100 mM DFX or DFO for 24 hrs. Results Measurement of oxygen consumption rate as well as molecular analyses in PB-HSPCs confirmed a poor mitochondrial oxidative phosphorylation phenotype. LSCM imaging of mitochondria in either PB- and BM-HSCs displayed a punctuate rather than interconnected network. However, co-staining of mitochondria and CD34/CD133 stemness-markers revealed a striking inverse correlation. Finally HSPCs produced DCF-detectable and DPI-inhibitable ROS attributable to constitutive NOX activity and related to stabilization of the hypoxia-inducibile factor (HIF1a) under normoxic condition. DFX treatment of HSPCs resulted in a significant up-regulation of ROS level whereas no significant change was observed following DFO treatment. Importantly, the DFX-mediated ROS production was insensitive to treatment with low NOX-inhibiting concentration of DPI but was abrogated by high concentration of DPI thus pointing to mitochondria as ROS source. Conclusions Our results show that HSCs in the early phase of commitment undergo a progressive increase of mitochondrial mass indicating the need of a bioenergetic up-regulation to cope with the oncoming energy-demanding proliferative/differentiative phenotype. Redox signaling, mediated by ROS production and likely triggered by changes in the environmental oxygen tension, appears to be essential in regulating HSC self-renewal and preservation of pluripotency. DFX treatment, by modulating ROS production, might lead to activation of redox-sensitive key factors able to restore the hematopoietic function in MDS patients. This effect seemingly is independent on the iron-chelating property of DFX but pertains to additional pharmacological properties that warren further investigation. Disclosures: No relevant conflicts of interest to declare.

// Francesca Grasso 1,2,* , Vitalba Ruggieri 3,* , Gabriele De Luca 1 , Paola Leopardi 1 , Maria Teresa Mancuso 4 , Ida Casorelli 5 , Pietro Pichierri 1 , Peter Karran 6 and Margherita Bignami 1 1 Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome, Italy 2 Department of Science, University Roma Tre, Rome, Italy 3 Laboratory of Pre-Clinical and Translational Research, IRCCS, Referral Cancer Center of Basilicata, Rionero in Vulture, Italy 4 Laboratory of Radiation Biology and Biomedicine, Agenzia Nazionale per le Nuove Tecnologie, l’Energia e lo Sviluppo Economico Sostenibile (ENEA) CR-Casaccia, Rome, Italy 5 Department of Immunohematology and Transfusion Unit, Azienda Ospedaliera Sant’Andrea, Rome, Italy 6 Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, UK * These authors contributed equally to this work Correspondence: Margherita Bignami, email: // Keywords : MUTYH, 6-thioguanine, azathioprine, UVA Received : October 22, 2014 Accepted : December 01, 2014 Published : December 02, 2014 Abstract The therapeutic thiopurines, including the immunosuppressant azathioprine (Aza) cause the accumulation of the UVA photosensitizer 6-thioguanine (6-TG) in the DNA of the patients’ cells. DNA 6-TG and UVA are synergistically cytotoxic and their interaction causes oxidative damage. The MUTYH DNA glycosylase participates in the base excision repair of oxidized DNA bases. Using Mutyh -nullmouse fibroblasts (MEFs) we examined whether MUTYH provides protection against the lethal effects of combined DNA 6-TG/UVA. Surprisingly, Mutyh -null MEFs were more resistant than wild-type MEFs, despite accumulating higher levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG).Their enhanced 6-TG/UVA resistance reflected the absence of the MUTYH protein and MEFs expressing enzymatically-dead human variants were as sensitive as wild-type cells. Consistent with their enhanced resistance, Mutyh -null cells sustained fewer DNA strand breaks and lower levels of chromosomal damage after 6-TG/UVA. Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, M utyh -null cells failed to arrest in S-phase at late time points. MUTYH-dependent toxicity was also apparent in vivo . Mutyh -/- mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG. Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh -/- mice whereas similarly treated wild-type animals remained tumor-free.