(+)-Lipoic Acid Reduces Lipotoxicity and Regulates Mitochondrial Homeostasis and Energy Balance in an In Vitro Model of Liver Steatosis.

Non-alcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipids within hepatocytes, which compromises liver functionality following mitochondrial dysfunction and increased production of reactive oxygen species (ROS). Lipoic acid is one of the prosthetic groups of the pyruvate dehydrogenase complex also known for its ability to confer protection from oxidative damage because of its antioxidant properties. In this study, we aimed to investigate the effects of lipoic acid on lipotoxicity and mitochondrial dynamics in an in vitro model of liver steatosis. HepG2 cells were treated with palmitic acid and oleic acid (1:2) to induce steatosis, without and with 1 and 5 µM lipoic acid. Following treatments, cell proliferation and lipid droplets accumulation were evaluated. Mitochondrial functions were assessed through the evaluation of membrane potential, MitoTracker Red staining, expression of genes of the mitochondrial quality control, and analysis of energy metabolism by HPLC and Seahorse. We showed that lipoic acid treatment restored membrane potential to values comparable to control cells, as well as protected cells from mitochondrial fragmentation following PA:OA treatment. Furthermore, our data showed that lipoic acid was able to determine an increase in the expression of mitochondrial fusion genes and a decrease in mitochondrial fission genes, as well as to restore the bioenergetics of cells after treatment with palmitic acid and oleic acid. In conclusion, our data suggest that lipoic acid reduces lipotoxicity and improves mitochondrial functions in an in vitro model of steatosis, thus providing a potentially valuable pharmacological tool for NAFLD treatment.

Thyroid Cancer Persistence in Patients with Unreliable Thyroglobulin Measurement: Circulating microRNA as Candidate Alternative Biomarkers.

BACKGROUND: We aimed to evaluate the role of circulating miRNAs as a biomarker of the persistence of papillary thyroid cancer (PTC) in patients with an "uninformative" thyroglobulin (Tg) measurement. METHODS: We prospectively enrolled 49 consecutive PTC patients with Tg-positive antibodies (TgAb) who had undergone a (near)-total thyroidectomy and 131I therapy (RIT). The serum thyroid stimulating hormone (TSH), Tg, and TgAb levels were measured before and at 6 and 12 months after RIT, respectively. The serum miRNA (221, 222, 375, 155, and 146b) levels were measured simultaneously. RESULTS: The response to the initial therapy was assessed according to the 2015 ATA criteria. A decrease in 50% or more of serum miRNA over time was observed in 41/49 PTC patients, who showed an excellent response (ER), but six and two patients were classified to have an indeterminate/incomplete biochemical or incomplete structural response to initial therapy. CONCLUSION: Serum miRNA kinetics emerge as a promising biomarker for the early detection of a persistent disease in PTC patients with uninformative Tg results.

New Insights into Cold Shock Proteins Effects in Human Cancer: Correlation with Susceptibility, Prognosis and Therapeutical Perspectives.

The microenvironment of the tumor cells is central to its phenotypic modification. One of the essential elements of this milieu is thermal regulation. An augment in local temperature has been reported to augment the tumor cell's responsiveness to chemoand radiation treatment. Cold shock proteins are RNA/DNA binding proteins identified by the existence of one or more cold shock domains. In humans, the best studied components of this group of proteins are called Y-box binding proteins, such as Y-box binding protein-1 (YB-1), but several other proteins have been recognized. Biological functions of these proteins extend from the control of transcription, translation and splicing to the regulation of exosomal RNA content. Several findings correlate an altered cold shock protein expression profile with tumor diseases. In this review we summarize the data for a causative participation of cold shock proteins in cancer onset and diffusion. Furthermore, the possible use of cold shock proteins for diagnostics, prognosis, and as targets for cancer treatment is exposed.

Methylome Analysis in Nonfunctioning and GH-Secreting Pituitary Adenomas.

Pituitary adenomas (PAs), usually benign lesions, can sometimes present with "aggressive" features (rapid growth, local invasiveness, scarce response to conventional treatments). Despite the fact that a few genetic alterations have been associated to this clinical behavior, the role of epigenetic modifications, mainly methylation and miRNAs activity, is now opening new frontiers in this field. We evaluated the methylation profile of 21 PA (11 GH-omas, 10 nonfunctioning tumors-NFPAs) samples from TNS surgery and 5 normal pituitaries, collected at our neurosurgery between 2015 and 2017. DNA was extracted and sequenced, selecting 184,841 target regions. Moreover, methylation profiles were correlated with demographic, radiological, and clinicopathological features. NFPAs showed higher methylation levels vs. GH-omas, with 178 differentially methylated regions (DMRs) mainly consisting of noncoding and intronic sequences, and mostly localized in the open sea regions. We also found three hypermethylated genes (C7orf50, GNG7, and BAHCC1) involved in tumorigenesis processes and potentially influencing pituitary tumor pathophysiology. Among the clinicopathological features, only the maximum diameter resulted significantly higher in NFPAs. Our data provide further evidence of the complex epigenetic background of pituitary tumors. In line with the current literature, we confirmed a significant prevalence of hypermethylation in NFPAs vs. GH-omas, whose pathophysiological consequence is yet to be defined.

CXCL12/CXCR4 axis supports mitochondrial trafficking in tumor myeloma microenvironment.

Mesenchymal stromal cells (MSCs) within the protective microenvironment of multiple myeloma (MM) promote tumor growth, confer chemoresistance and support metabolic needs of plasma cells (PCs) even transferring mitochondria. In this scenario, heterocellular communication and dysregulation of critical signaling axes are among the major contributors to progression and treatment failure. Here, we report that myeloma MSCs have decreased reliance on mitochondrial metabolism as compared to healthy MSCs and increased tendency to deliver mitochondria to MM cells, suggesting that this intercellular exchange between PCs and stromal cells can be consider part of MSC pro-tumorigenic phenotype. Interestingly, we also showed that PCs promoted expression of connexin 43 (CX43) in MSCs leading to CXCL12 activation and stimulation of its receptor CXCR4 on MM cells favoring protumor mitochondrial transfer. Consistently, we observed that selective inhibition of CXCR4 by plerixafor resulted in a significant reduction of mitochondria trafficking. Moreover, intracellular expression of CXCR4 in myeloma PCs from BM biopsy specimens demonstrated higher CXCR4 colocalization with CD138+ cells of non-responder patients to bortezomib compared with responder patients, suggesting that CXCR4 mediated chemoresistance in MM. Taken together, our data demonstrated that CXCL12/CXCR4 axis mediates intercellular coupling thus suggesting that the myeloma niche may be exploited as a target to improve and develop therapeutic approaches.

Rare among Rare: Phenotypes of Uncommon CMT Genotypes.

(1) Background: Charcot-Marie-Tooth disease (CMT) is the most frequent form of inherited chronic motor and sensory polyneuropathy. Over 100 CMT causative genes have been identified. Previous reports found PMP22, GJB1, MPZ, and MFN2 as the most frequently involved genes. Other genes, such as BSCL2, MORC2, HINT1, LITAF, GARS, and autosomal dominant GDAP1 are responsible for only a minority of CMT cases. (2) Methods: we present here our records of CMT patients harboring a mutation in one of these rare genes (BSCL2, MORC2, HINT1, LITAF, GARS, autosomal dominant GDAP1). We studied 17 patients from 8 unrelated families. All subjects underwent neurologic evaluation and genetic testing by next-generation sequencing on an Ion Torrent PGM (Thermo Fischer) with a 44-gene custom panel. (3) Results: the following variants were found: BSCL2 c.263A > G p.Asn88Ser (eight subjects), MORC2 c.1503A > T p.Gln501His (one subject), HINT1 c.110G > C p.Arg37Pro (one subject), LITAF c.404C > G p.Pro135Arg (two subjects), GARS c.1660G > A p.Asp554Asn (three subjects), GDAP1 c.374G > A p.Arg125Gln (two subjects). (4) Expanding the spectrum of CMT phenotypes is of high relevance, especially for less common variants that have a higher risk of remaining undiagnosed. The necessity of reaching a genetic definition for most patients is great, potentially making them eligible for future experimentations.

Nusinersen Modulates Proteomics Profiles of Cerebrospinal Fluid in Spinal Muscular Atrophy Type 1 Patients.

Spinal muscular atrophy (SMA) type 1 is a severe infantile autosomal-recessive neuromuscular disorder caused by a survival motor neuron 1 gene (SMN1) mutation and characterized by progressive muscle weakness. Without supportive care, SMA type 1 is rapidly fatal. The antisense oligonucleotide nusinersen has recently improved the natural course of this disease. Here, we investigated, with a functional proteomic approach, cerebrospinal fluid (CSF) protein profiles from SMA type 1 patients who underwent nusinersen administration to clarify the biochemical response to the treatment and to monitor disease progression based on therapy. Six months after starting treatment (12 mg/5 mL × four doses of loading regimen administered at days 0, 14, 28, and 63), we observed a generalized reversion trend of the CSF protein pattern from our patient cohort to that of control donors. Notably, a marked up-regulation of apolipoprotein A1 and apolipoprotein E and a consistent variation in transthyretin proteoform occurrence were detected. Since these multifunctional proteins are critically active in biomolecular processes aberrant in SMA, i.e., synaptogenesis and neurite growth, neuronal survival and plasticity, inflammation, and oxidative stress control, their nusinersen induced modulation may support SMN improved-expression effects. Hence, these lipoproteins and transthyretin could represent valuable biomarkers to assess patient responsiveness and disease progression.

SIRT2 and SIRT3 expression correlates with redox imbalance and advanced clinical stage in patients with multiple myeloma.

OBJECTIVES: Sirtuins comprise seven family elements (SIRT1-7) involved in various cell signalling pathways comprising cancer inhibition and tumorigenesis. The present study aims to evaluate SIRT2 and SIRT3 gene expression and potential redox reactions in patients with multiple myeloma (MM) at onset and its correlation with disease status, extent and presence of organ damage secondary to myeloma. DESIGN & METHODS: Total RNA was extracted from 17 MM patients and 10 controls to assess gene expression using real-time PCR. The NAD+/NADH ratio as well as the levels of glutathione peroxidase (GPx) and hydrogen peroxide (HP) in peripheral blood mononuclear cells (PBMCs) were determined using established biochemical assays. RESULTS: SIRT2 and SIRT3 expression is reduced in MM patients compared to healthy controls. Correlational analysis demonstrated that SIRT2 reduction is associated with advanced clinical stage and with more advanced bone lesions than in the remaining patients. SIRT3 expression is correlated with lytic bone lesions. Biochemical analysis indicated an imbalance of oxidative stress biomarkers with low concentrations of the antioxidant enzyme GPx, low amounts of NAD + and higher concentrations of pro-oxidant enzyme HP in PBMCs of MM patients compared to controls. Moreover, MM patients with bone lesions had lower concentrations of NAD + and GPx in PBMCs than patients without signs of bone disease. In addition, MM patients had higher quantities of intracellular HP than controls. CONCLUSIONS: Our results demonstrate that SIRT2 and SIRT3 are downregulated in MM and that lower concentrations correlate with an advanced stage of disease and redox imbalance. We conclude that SIRT2 and SIRT3 together with oxidative stress biomarkers, may be useful for improved risk stratification of MM patients.

A Phase 1/2 Study of Flavocoxid, an Oral NF-κB Inhibitor, in Duchenne Muscular Dystrophy.

Flavocoxid is a blended extract containing baicalin and catechin with potent antioxidant and anti-inflammatory properties due to the inhibition of the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) enzymes, nuclear factor-κB (NF-κB), tumor necrosis factor (TNF)-alpha, and the mitogen-activated protein kinases (MAPKs) pathways. This phase 1/2 study was designed to assess the safety and tolerability of flavocoxid in patients with Duchenne muscular dystrophy (DMD). Thirty-four patients were recruited: 17 were treated with flavocoxid at an oral dose of 250 or 500 mg, according to body weight, for one year; 17 did not receive flavocoxid and served as controls. The treatment was well tolerated and nobody dropped out. Flavocoxid induced a significant reduction in serum interleukin (IL)-1 beta and TNF-alpha only in the group of DMD boys on add-on therapy (flavocoxid added to steroids for at least six months). The decrease in IL-1 beta was higher in younger boys. The serum H2O2 concentrations significantly decreased in patients treated with flavocoxid alone with a secondary reduction of serum glutathione peroxidase (GPx) levels, especially in younger boys. The exploratory outcome measures failed to show significant effects but there was a trend showing that the younger boys who received treatment were faster at performing the Gowers' maneuver, while the older boys who received treatment were faster at doing the 10-m walk test (10MWT). Therefore, a double-blind, placebo-controlled study for at least two/three years is warranted to verify flavocoxid as a steroid substitute or as add-on therapy to steroids.

Serum levels of miRNA-21-5p in vitiligo patients and effects of miRNA-21-5p on SOX5, beta-catenin, CDK2 and MITF protein expression in normal human melanocytes.

BACKGROUND: Epigenetics of vitiligo was evaluated in few studies. In particular, the role of miR-21, a microRNA involved in various processes, including melanogenesis, was never investigated. OBJECTIVE: Evaluation of serum levels of miR-21-5p in vitiligo patients and miR-21-5p effects on melanogenesis. METHODS: We measured serum levels of miR-21-5p in 40 patients affected by nonsegmental vitiligo and 40 sex- and age-matched healthy controls. Next, normal human melanocytes were transfected with miR-21-5p to study the effects of this microRNA, which targeted some proteins involved in melanogenesis pathway like SOX5, beta-catenin, cyclin-dependent kinase 2 (CDK2), and MITF. RESULTS: The expression of miR-21-5p in vitiligo patients was 3.6-4454.4 fold (mean 990.4 ± 1397.9) higher than in controls. The relative expression of miR-21-5p was directly and significantly correlated with disease severity, defined by VASI (Vitiligo Area and Severity Index) score (Rho = 0.89, p = 10-7), but not other individual or clinical characteristics. In the second part of the study, a significant reduction of SOX5, beta-catenin and CDK2 protein expression and increase of MITF protein expression was observed in cultured melanocytes after 24 h trasfection with miR-21-5p. CONCLUSION: According to literature, miR-21-5p upregulation and consequent SOX5 downregulation should upregulate melanogenesis, while vitiligo is characterized by skin depigmentation. Our results suggest that current knowledge of the pathogenesis of vitiligo is probably incomplete. Clinical manifestations could result from an altered balance between metabolic pathways with contrasting effects. In this view, miR-21-5p upregulation might be a tentative compensation mechanism. Further studies appear necessary to confirm and better understand our results and their importance.

Advances in Treatment of ATTRv Amyloidosis: State of the Art and Future Prospects.

Hereditary amyloid transthyretin (ATTRv) amyloidosis with polyneuropathy is a progressive disease that is transmitted as an autosomal dominant trait and characterized by multiple organ failure, including axonal sensory-motor neuropathy, cardiac involvement, and autonomic dysfunction. Liver transplantation (LT) and combined heart-liver transplantation, introduced in the 1990s, have been the only therapies for almost two decades. In 2011, tafamidis meglumine became the first specific drug approved by regulatory agencies, since then the attention toward this disease has progressively increased and several drugs with different mechanisms of action are now available. This review describes the drugs already on the market, those that have shown interesting results although not yet approved, and those currently being tested.

microRNA-10 and -221 modulate differential expression of Hippo signaling pathway in human astroglial tumors.

Gliomas represent over 70% of all brain tumors, they are highly invasive and structurally vascular neoplasms. Despite the latest technological advance in neuro-surgery the survival of patients with high-grade glioma remains poor. The lack of robust treatment options has propelled the search for new markers that may able allow the identification of patients who can benefit from molecularly targeted therapies. The Hippo signaling pathway is considered as a key regulator of tissue homeostasis, cell proliferation and apoptosis, and alterations of this pathway seem to contribute to tumorigenesis. Yes-associated protein (YAP1) is a downstream target of the Hippo pathway which acts as a transcription co-activator. In cancer, YAP1 has been reported to function either as an oncogene or tumor suppressor, depending on the cell context. The aim of this study was to examine the expression of YAP1, Survivin and LATS1 kinase activity in human astroglial tumors with different grades of malignancy. Moreover, we also investigated the expression of miR-221 and miR-10b and their relationship with core molecules of the Hippo pathway. Our results showed the overexpression of YAP1 and Survivin as well as a decreased activity of large tumor suppressor 1 (LATS1) in high-grade glioblastoma versus anaplastic astrocytoma and low-grade glioma. Furthermore, we also demonstrated that miR-221 and miR-10b are specifically involved in Hippo signaling via LATS1 regulation and that their knockdown significantly decreased glioma cell proliferation. This preliminary data confirmed the crucial role of the Hippo pathway in cancer and suggested that miR-221 and miR-10b could be potential therapeutic targets for glioma treatment.

Circulating microRNAs Profile in Patients With Transthyretin Variant Amyloidosis.

Transthyretin variant amyloidosis (ATTRv) is a rare autosomal dominant disease characterized by the accumulation of amyloid in many organs, mostly causing a sensory-motor neuropathy, cardiomyopathy, and dysautonomia. The aim of the study was to report microRNAs (miRNAs) expression profile identified in the blood of ATTRv patients. Ten ATTRv patients, 10 asymptomatic carriers of transthyretin variant (TTRv), 10 patients with Charcot-Marie-Tooth (CMT) disease, and 10 healthy controls were studied. Human Schwann cells cultures were used to study the regulatory effects of miR-150-5p on the expression of cAMP response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF). ATTRv patients had 33 miRNAs up-regulated and 48 down-regulated versus healthy controls; 9 miRNAs were up-regulated and 30 down-regulated versus CMT patients; 19 miRNAs were up-regulated and 38 down-regulated versus asymptomatic TTRv carriers. Twelve out of the 19 upregulated miRNAs had a fold increase higher than 100. The validation experiment indicated miR-150-5p as a valuable biomarker to differentiate ATTRv patients from asymptomatic TTRv carriers (AUC: 0.9728; p < 0.0001). Schwann cells culture model demonstrated that miR-150-5p is a powerful negative regulator of CREB, BDNF, and NGF genes. Identification of deregulated miRNAs can help in understanding the complex pathomechamism underlying the development of ATTRv and related multisystemic pathology. Further investigations are needed on the role of circulating miR-150-5p to predict the shift of TTRv carriers from an asymptomatic status to symptoms appearance.

Effect of exercise on telomere length and telomere proteins expression in mdx mice.

In Duchenne muscular dystrophy (DMD), telomere shortening has been postulated to contribute to the failure of regenerative activity promoting the premature senescence of satellite cells. The aim of the present study was to investigate the telomere length and the expression of telomeric repeat-binding factor-1 (TRF1), poly (ADP-ribose) polymerase-1 (PARP1) and mouse telomerase reverse transcriptase (MTERT) in gastrocnemius, tibialis anterior and diaphragm muscles of the murine model of DMD, the mdx mouse and whether a chronic protocol of forced exercise impacts on them. Our results confirmed a telomere shortening in mdx muscles, more evident in the diaphragm, in which exercise induced a greater shortening than in wild-type mice. Moreover, we showed for the first time in mdx an increased TRF1 and PARP1 expression and an augmented activity of MTERT, further enhanced by exercise. These results reinforce the hypothesis that a deregulation of mechanisms involved in telomere length occurs and may pave the way for the test of compounds targeting proteins modulating telomere maintenance as a novel strategy to treat dystrophinopathies.

Circulating miRNAs expression as potential biomarkers of mild traumatic brain injury.

TBI is the main cause of death and disability in individuals aged 1-45 in Western countries. One of the main challenges of TBI at present is the lack of specific diagnostic biomarkers, especially for mild TBI (mTBI), which remains currently difficult to value in clinical practice. In this context MiRNAs may be important mediators of the profound molecular and cellular changes that occur after TBI in both the short and the long term. Recently, plasma miRNAs profiling in human TBI, have revealed dynamic temporal regulation of miRNA expression within the cortex. Aim of this study was to select a specific miRNAs panel for mTBI, by focusing the research on the prognostic meaning of miRNAs in the hours following the trauma, in order to be able to use this MIRNAs as potential biomarkers useful for monitoring the follow up of mild TBI. Serum levels of 17 miRNAs were measured by RT-quantitative polymerase chain reaction (qPCR) in 20 patients with mTBI at three different time-points (0 h, 24 h, 48 h) and in 10 controls. For 15 miRNAs we found a significant differences in the comparison among the three time points: for each of these miRNAs the values were greater at baseline and progressively reduced at 24 h and 48 h. These data allow us to consider the miRNAs included in panel as sensitive and specific biomarkers for mTBI, useful in monitoring the post-trauma period.

Charcot-Marie-Tooth disease: experience from a large Italian tertiary neuromuscular center.

INTRODUCTION: Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disease. Thanks to the advances of the latest generation sequencing, more than 80 causative genes have been reported to date. METHODS: In this retrospective, observational study, we have analyzed clinical, electrophysiological, and genetic data of CMT patients in care at Neuromuscular Center of Messina University Hospital, Messina, Italy, for at least 22 years (from 1994 to 2016). Our center is the only reference center for genetic neuropathies in Sicily and in the southern part of Calabria. RESULTS: We reviewed the clinical records of 566 patients with the aim to evaluate how many patients received a genetic diagnosis and the distribution of the genetic subtypes. About 352/566 (62.19%) received a genetic diagnosis. The most frequent genetic diagnoses were CMT1A/PMP22 duplication (51.13%), followed by HNPP/PMP22 deletion (15.05%), CMT1B/MPZ mutation (10.22%), CMTX/GJB1 mutation (9.37%), and CMT2F/HSPB1 (4%). Other rare mutations included MFN2 mutation (n. 8 pts), BSCL2 mutation (n.8 pts), PMP22 point mutation (n.7 pts), GDAP1 mutation (n.4 pts), GARSmutation (n. 2 pts), TRPV4 mutation (n. 2 pts), LITAF mutation (n.1 pt), and NEFL mutation (n. 1 pt). CONCLUSIONS: Our study provides further data on frequency of CMT genes, subtypes in a wide Mediterranean area and contributes to help clinicians in addressing the genetic testing workup.

MicroRNAs expression in pituitary tumors: differences related to functional status, pathological features, and clinical behavior.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at post-transcriptional level, having a role in many biological processes, such as control of cell proliferation, cell cycle, and cell death. Altered miRNA expression has been reported in many neoplasms, including pituitary adenomas (PAs). PURPOSE: In this study, we aimed to evaluate the expression of 20 miRNAs involved in pathways relevant to pituitary pathophysiology, in PAs and normal pituitary tissue and to correlate their expression profile with clinical and pathological features. METHODS: Pituitary tumor samples were obtained during transphenoidal surgery from patients with non-functioning (NFPA, n = 12) and functioning (n = 11, 5 GH-, 3 ACTH-, 3 PRL-omas) PAs. The expression of selected miRNAs in PAs and in normal pituitary was analyzed by RT-qPCR. miRNAs expression was correlated with demographic, clinical, and neuroradiological data and with histopathological features including pituitary hormones immunostaining, Ki-67 proliferation index, and p53 immunohistochemistry evaluation. RESULTS: All evaluated miRNAs except miR-711 were expressed in both normal and tumor pituitary tissue. Seventeen miRNAs were significantly down-regulated in pituitary tumors compared to normal pituitary. miRNAs were differentially expressed in functioning PAs or in NFPAs, as in the latter group miR-149-3p (p = 0.036), miR-130a-3p (p = 0.014), and miR-370-3p (p = 0.026) were significantly under expressed as compared to functioning tumors. Point-biserial correlation analysis demonstrated a negative correlation between miR-26b-5p and Ki-67 (p = 0.031) and between miR-30a-5p and 'atypical' morphological features (p = 0.038) or cavernous sinus invasion (p = 0.049), while 508-5p was inversely correlated with clinical aggressiveness (p = 0.043). CONCLUSIONS: In this study, we found a significant down-regulation of 17 miRNAs in PAs vs normal pituitary, with differential expression profile related to functional status and tumor aggressiveness.

Role of Erythropoietin in Cerebral Glioma: An Innovative Target in Neuro-Oncology.

BACKGROUND: Erythropoietin (EPO) is a cytokine primarily involved in the regulation of erythropoiesis. In response to hypoxia-ischemia, hypoxia-inducible factor 1 induces EPO production, which, in turn, inhibits apoptosis of erythroid progenitor cells. By the same mechanism and acting through other signaling pathways, EPO exerts neuroprotective effects. Increased resistance to hypoxia and decreased apoptosis are thought to be important mechanisms for tumor progression, including malignant glioma. Because recent studies have demonstrated that EPO and its receptor (EPOR) are expressed in several tumors and can promote tumor growth, in the present study, we investigated EPO and EPOR expression in human glioma and the effect of EPO administration in a rat model of glioma implantation. METHODS: Using Western blotting and immunohistochemical analysis, we examined the expression of EPO, EPOR, platelet endothelial cell adhesion molecule, and Ki-67 in human glioma specimens and experimentally induced glioma in rats. In the experimental setting, a daily dose of recombinant human EPO (rHuEPO) or saline solution were administered for 21 days in Fischer rats subjected to 9L cell line implantation. RESULTS: In both human and animal specimens, we found an increase in EPOR expression as long as the lesion presented with an increasing malignant pattern. A significant direct correlation was found between the expression of EPOR and Ki-67 and EPOR and platelet endothelial cell adhesion molecule in low- and high-grade gliomas. The rats treated with rHuEPO presented with significantly larger tumor spread compared with the saline-treated rats. CONCLUSIONS: The results of our study have shown that the EPO/EPOR complex might play a significant role in the aggressive behavior of high-grade gliomas. The larger tumor spread in rHuEPO-treated rats suggests a feasible role for EPO in the aggressiveness and progression of malignant glioma.

Hippo signaling pathway is altered in Duchenne muscular dystrophy.

Hippo signaling pathway is considered a key regulator of tissue homeostasis, cell proliferation, apoptosis and it is involved in cancer development. In skeletal muscle, YAP, a downstream target of the Hippo pathway, is an important player in myoblast proliferation, atrophy/hypertrophy regulation, and in mechano-trasduction, transferring mechanical signals into transcriptional responses. We studied components of Hippo pathway in muscle specimens from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy type 2A and type 2B and healthy subjects. Only DMD muscles had decreased YAP1 protein expression, increased LATS1/2 kinase activity, low Survivin mRNA expression and high miR-21 expression. In light of our novel results, a schematic model is postulated: low levels of YOD1 caused by increased inhibition by miR-21 lead to an increase of LATS1/2 activity which in turn augments phosphorylation of YAP. Reduced amount of active YAP, which is also a target of increased miR-21, causes decreased nuclear expression of YAP-mediated target genes. Since it is known that YAP has beneficial roles in promoting tissue repair and regeneration after injury so that its activation may be therapeutically useful, our results suggest that some components of Hippo pathway could become novel therapeutic targets for DMD treatment.

Silencing of telomere-binding protein adrenocortical dysplasia (ACD) homolog enhances radiosensitivity in glioblastoma cells.

Adrenocortical dysplasia (ACD) is a shelterin protein involved in the maintenance of telomere length and in cancer radioresistance. This study investigated the expression profile of ACD in human gliomas and its role in radioresistance of glioma cells. The expression of ACD was analyzed in 62 different grades of glioma tissues and correlated with prognosis. A radioresistant cell line was generated from U87MG cells. For mechanistic studies, ACD was inhibited by small interfering RNA-targeting ACD and the effect on cell radioresistance, telomerase activity, cyclinD1, caspase-3, hTERT, and BIRC1 was evaluated. Clonogenic assay was performed after irradiation, to investigate the effect of ACD silencing on radiation sensitivity. ACD expression appeared strongly upregulated in higher grade gliomas, and its expression was significantly correlated to grading and poor prognosis. In glioma cell lines, ACD expression pattern was similar to those observed in glioma tissues. In irradiated cells, ACD expression was increased in an ionizing radiation dose-dependent manner. A higher expression of ACD was observed in the radioresistant clones than parental cells. Silencing of ACD led to the enhanced radiation sensitivity, decreased telomerase activity and cyclin D1 expression, reduced expression of BIRC1, and finally to the upregulation of caspase-3. This study represents the first report, which demonstrated the expression pattern of ACD in gliomas and its prognostic value. Our results suggested that ACD is involved in glioblastoma radioresistance, likely through the modulation of telomerase activity, proliferation, and apoptosis. ACD might represent a potential molecular biomarker and a novel therapeutic target in glioblastoma.