The induction of Maspin expression by a glucosamine-derivative has an antiproliferative activity in prostate cancer cell lines

Mammary serine protease inhibitor or Maspin has been characterized as a class II tumor suppressor gene in several cancer types, among them prostate cancer (CaP). Androgen ablation is an effective therapy for CaP, but with short-term effectiveness, thus new therapeutic strategies are actively sought. The present study is aimed to explore the effects of a glucosamine derivative, 2-(N-Carbobenzyloxy)L-phenylalanylamido-2-deoxy-β-D-glucose (NCPA), on two CaP cell lines, PC3 and LNCaP. In particular we analyzed the impact of NCPA on Maspin production, cell viability and cell cycle progression and apoptosis/necrosis pathway activation has been determined in PC3 and LNCaP cell lines. NCPA is able to stimulate Maspin production in PC3 and not in LNCaP cell lines. NCPA blocks the PC3 cell cycle in G1 phase, by inhibiting Cyclin D1 production and induces the apoptosis, therefore interfering with aggressiveness of this androgen-insensitive cell line. Moreover, NCPA is able to induce the expression of Maspin in LNCaP cell line treated with androgen receptor inhibitor, Bicalutamide, and in turn to stimulate the apoptosis of these cells. These findings suggest that NCPA, stimulating the endogenous production of a tumor suppressor protein, could be useful in the design of new therapeutic strategies for treatment of CaP

Case report: The CCDC103 variant causes ultrastructural sperm axonemal defects and total sperm immotility in a professional athlete without primary ciliary diskinesia

Primary ciliary dyskinesia (PCD) is an inherited autosomal-recessive disorder characterized by abnormal ciliary motion, due to a defect in ciliary structure and/or function. This genetic condition leads to recurrent upper and lower respiratory infections, bronchiectasis, laterality defect, and subfertility. Male infertility is often associated with PCD, since the ultrastructure of the axoneme in the sperm tail is similar to that of the motile cilia of respiratory cells. We present the first reported case of a male patient from a non-consanguineous Italian family who exhibited a severe form of asthenozoospermia factor infertility but no situs inversus and absolutely no signs of the clinical respiratory phenotype, the proband being a professional basketball player. Whole-exome sequencing (WES) has identified a homozygote mutation (CCDC103 c.461 A > C, p.His154Pro) in the proband, while his brother was a heterozygous carrier for this mutation. Morphological and ultrastructural analyses of the axoneme in the sperm flagellum demonstrated the complete loss of both the inner and outer dynein arms (IDA and ODA, respectively). Moreover, immunofluorescence of DNAH1, which is used to check the assembly of IDA, and DNAH5, which labels ODA, demonstrated that these complexes are absent along the full length of the flagella in the spermatozoa from the proband, which was consistent with the IDA and ODA defects observed. Noteworthy, TEM analysis of the axoneme from respiratory cilia showed that dynein arms, although either IDAs and/or ODAs seldom missing on some doublets, are still partly present in each observed section. This case reports the total sperm immotility associated with the CCDC103 p.His154Pro mutation in a man with a normal respiratory phenotype and enriches the variant spectrum of ccdc103 variants and the associated clinical phenotypes in PCD, thus improving counseling of patients about their fertility and possible targeted treatments

THE UTILITY OF INTERPHASE FLUORESCENCE IN SITU HYBRIDIZATION IN THE DIAGNOSIS OF LYMPHOMAS

Lymphomas are characterized by heterogeneous biology, pathologic features, and clinical outcome. They are classified based on the normal counterpart, or cell of origin, from which they arise. Because lymphocytes have physiologic immune functions that vary both by lineage and by stage of differentiation; the classification of lymphomas coming from these normal lymphoid populations is complex. Genomic instability is a feature of lymphomas due to aberrant alterations at genetic, epigenetic, transcriptional, protein, and dysregulated oncogenic signaling pathways. Detection of specific chromosomal abnormalities is essential in diagnosing of several lymphoproliferative disorders. Interphase FISH investigates cytogenetic alterations, the gold standard technique localizes fluorescent signals to specific interphase non-dividing cells. Translocations and rearrangements are the common chromosomal alterations of lymphomas involving proto-oncogenes and tumor suppressors. The common abnormalities include: t (11;14) (q13; q32) in mantle cell lymphoma and in some cases of plasma cell myeloma; t(14;18)(q32;q21) in follicular lymphoma; t(8;14)(q21;q32) in Burkitt lymphoma; t(11;18)(q21;q21) in MALT lymphoma; BCL6 rearrangement in Large B-cell Diffuse Lymphoma (LBCL); IRF4/DUSP22 rearrangement in LBCL; t(2;5)(p23;q35) NPM-ALK in T-cell lymphomas. Less commonly are deletions, trisomy 12 or partial trisomy 12q13 in Chronic Lymphocytic Leukaemia (CLL). The WHO has recently introduced a particular type of lymphoma morphologically similar to Burkitt Lymphoma but without t (8;14) (q24; q32): instead, the following entity is cytogenetically characterized by a peculiar pattern of an 11q aberration consisting of a gain in 11q23.2-23.3 followed by a telomeric loss in 11q24.1-qter. This study aims to standardize Interphase FISH for diagnosing lymphoma associated with immunophenotypic features, focusing our attention on particular cases showing interested genic abnormalities, such as 11q alterations and t (11;14) in Precursor T-Lymphoblastic Transformation of Mantle Cell Lymphoma. During this study, we have also performed ImmunoFISH, a method combining immunolabelling with fluorescent in situ hybridization (FISH) to simultaneously detect the nucleo-cytoplasmic distribution of proteins and specific nucleotide sequences within the chromosomes

Effects of a glucosamine-derivative on prostate cancer cell line PC3

Prostate Cancer (CaP) is the most common form of male tumor and is the second leading cause of cancer death. Androgen ablation has proved to be an effective therapy for metastatic prostate cancer, but the regression of metastatic lesions lasts only 18 to 24 months. Mammary serine protease inhibitor or Maspin is a 42 kDa, a non-inhibitory member of the serine protease inhibitor superfamily, and has been characterized as a class II tumor suppressor gene in several cancer types, among them prostate cancer (CaP), due to its ability to inhibit metastasis. In normal prostate epithelial cells, Maspin is highly expressed whereas in prostate cancer cell lines its expression is almost completely suppressed. Previously, it has been demonstrated that NCPA, a glucosamine-derivative synthetized in our laboratory, was able to inhibit IKKα nuclear translocation and to stimulate the production and nuclear localization of Maspin in an osteosarcoma cell line: 143B. IKKα, one of two catalytic subunits of NF-B transcriptional factors, enhances tumor promotion by repressing, among other mechanisms, maspin promoter. Tumor-suppressing and anti-metastatic activities of Maspin have been attributed to its ability to inhibit both invasiveness and cell cycle progression and to stimulate apoptosis of tumor cells. Aim of this presentation is to analyze the ability of NCPA to affect metastatic and proliferation activity of PC3, which is an androgen-insensitive prostate cancer cell line

Glucosamine and its peptidyl-derivative NAPA: novel therapeutic strategy for chondrocytes matrix remodeling

Cartilage degradation, due to an imbalance between anabolic and catabolic rate of chondrocyte metabolism, is the main feature of Osteoarthritis (OA). To date, OA is mainly treated with Non Steroidal Anti-Inflammatory Drugs (NSAIDs), in order to reduce arthritis-related symptoms. In the last decades an increasing number of patients have started to use supplements, such as Glucosamine (GlcN) and chondroitin sulfate, as potential chondroprotective agents. Several in vivo clinical trials as well as in vitro experiments have been performed reporting inconsistent outcomes. Previously, in our lab we analyzed the anabolic effects of GlcN and its N-acetyl-phenylalanine derivative (NAPA) in a rabbit OA model, finding that intra-articular administration of GlcN and NAPA was very effective in reducing cartilage changes in injured rabbit knee. GlcN and NAPA intra-articular administration allows higher concentrations to be reached in the joints compared to oral administration, thus providing an explanation for the ability of both molecules to interfere with OA progression. We also studied the effects of GlcN and NAPA on inflammatory pathways, finding that both molecules can interfere with MAP kinase and NF-kB pathways, by interfering with IKK activity. Finally, we studied the effectiveness of GlcN and NAPA one the biosynthetic activity and hence the matrix production of human primary chondrocytes cultured in micromasses, which represent a good tridimensional culture model. We explored the ability of GlcN and NAPA to stimulate the synthesis of collagen type II (Coll II), Aggrecan (ACAN) and Small Leucine-Rich Proteoglycans (SLRPs). After 6 weeks, micromasses stimulated with GlcN + NAPA still showed a large amount of ECM compared to untreated cells. Moreover, Collagen type II was more abundant and better organized compared to that produced by untreated cells. Finally, cells resulted viable in both treated and untreated micromasses, even if in the middle of untreated micromasses, few dead cells were observed, whereas in the treated micromasses only viable cells and cells completely surrounded by ECM were detected

An adrenal cortical adenoma with neuroendocrine-type granules mimicking pheochromocytoma

Clinical and biochemical presentation of adrenal tumors may rarely conflict with their histologic features. In the present report, we describe a rare case of adrenal neoplasm clinically and biochemically labeled as pheochromocytoma which at histologic examination resulted adrenal cortical tumor. The neoplasm was examined with the electron microscope which revealed the presence of electron-dense neuroendocrine-type granules next to intracytoplasmic lipid droplets. The patient underwent laparoscopic left adrenalectomy which leads to normalization of 24 h urinary metanephrine and normetanephrine. This exceptional entity should be taken into consideration when the clinical and laboratory features conflict with the histological examination. The pathologist can clarify the mixed nature of the tumor by means of the identification of neuroendocrine granules at the electron microscope examination

Analysis of Primary Cilium Expression and Hedgehog Pathway Activation in Mesothelioma Throws Back Its Complex Biology

The primary cilium (PC) is a sensory organelle present on the cell surface, modulating the activity of many pathways. Dysfunctions in the PC lead to different pathologic conditions including cancer. Hedgehog signaling (Hh) is regulated by PC and the loss of its control has been observed in many cancers, including mesothelioma. Malignant pleural mesothelioma (MPM) is a fatal cancer of the pleural membranes with poor therapeutic options. Recently, overexpression of the Hh transcriptional activator GL1 has been demonstrated to be associated with poor overall survival (OS) in MPM. However, unlike other cancers, the response to G-protein-coupled receptor smoothened (SMO)/Hh inhibitors is poor, mainly attributable to the lack of markers for patient stratification. For all these reasons, and in particular for the role of PC in the regulation of Hh, we investigated for the first time the status of PC in MPM tissues, demonstrating intra- and inter-heterogeneity in its expression. We also correlated the presence of PC with the activation of the Hh pathway, providing uncovered evidence of a PC-independent regulation of the Hh signaling in MPM. Our study contributes to the understanding MPM heterogeneity, thus helping to identify patients who might benefit from Hh inhibitors

MYC protein expression scoring and its impact on the prognosis of aggressive B-cell lymphoma patients

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Tumor microenvironment of Burkitt lymphoma: different immune signatures with different clinical behavior

: Burkitt lymphoma (BL) is characterized by tumor microenvironment (TME) in which macrophages represent the main component, determining a distinct histological appearance known as "starry sky" pattern. However, in some instances, BL may exhibit a granulomatous reaction that has been previously linked to a favorable prognosis and spontaneous regression. The aim of our study was to deeply characterize the immune landscape of 7 cases of EBV + BL with granulomatous reaction compared to 8 cases of EBV + BL and 8 EBV- BL, both with typical "starry sky" pattern, by Gene expression profiling performed on the NanoString nCounter platform. Subsequently, the data were validated by multiplex and combined immunostaining. Based on unsupervised clustering of differentially expressed genes, BL samples formed 3 distinct clusters differentially enriched in BL with a diffuse granulomatous reaction (cluster 1), EBV+ BL with typical starry sky pattern (Cluster 2), EBV - BL with typical "starry sky" (cluster 3). We observed variations in the immune response signature among BL with granulomatous reaction and BL with typical "starry sky", both EBV + and EBV -. The TME signature in BL with diffuse granulomatous reaction showed a proinflammatory response, while BLs with "starry sky" were characterized by up-regulation of M2- polarization and pro-tumor response. Moreover, the analysis of additional signatures revealed an up-regulation of Dark zone-signature and epigenetic-signature in BL with typical "starry sky". Tumor associated macrophages (TAM) and epigenetic regulators may be promising targets for additional therapies in BL lymphoma opening novel immunotherapeutic strategies