Inhibition of Phosphoinositide 3-Kinase/Protein Kinase B Signaling Hampers the Vasopressin-dependent Stimulation of Myogenic Differentiation.

Arginine-vasopressin (AVP) promotes muscle differentiation, hypertrophy, and regeneration through the combined activation of the calcineurin and Calcium/Calmodulin-dependent Protein Kinase (CaMK) pathways. The AVP system is impaired in several neuromuscular diseases, suggesting that AVP may act as a physiological factor in skeletal muscle. Since the Phosphoinositide 3-kinases/Protein Kinase B/mammalian Target Of Rapamycin (PI3K/Akt/mTOR) signaling plays a significant role in regulating muscle mass, we evaluated its role in the AVP myogenic effect. In L6 cells AKT1 expression was knocked down, and the AVP-dependent expression of mTOR and Forkhead box O3 (FoxO) was analyzed by Western blotting. The effect of the PI3K inhibitor LY294002 was evaluated by cellular and molecular techniques. Akt knockdown hampered the AVP-dependent mTOR expression while increased the levels of FoxO transcription factor. LY294002 treatment inhibited the AVP-dependent expression of Myocyte Enhancer Factor-2 (MEF2) and myogenin and prevented the nuclear translocation of MEF2. LY294002 also repressed the AVP-dependent nuclear export of histone deacetylase 4 (HDAC4) interfering with the formation of multifactorial complexes on the myogenin promoter. We demonstrate that the PI3K/Akt pathway is essential for the full myogenic effect of AVP and that, by targeting this pathway, one may highlight novel strategies to counteract muscle wasting in aging or neuromuscular disorders.

Pathological and Molecular Features of Glioblastoma and Its Peritumoral Tissue.

Glioblastoma (GBM) is one of the most aggressive and lethal human brain tumors. At present, GBMs are divided in primary and secondary on the basis of the mutational status of the isocitrate dehydrogenase (IDH) genes. In addition, IDH1 and IDH2 mutations are considered crucial to better define the prognosis. Although primary and secondary GBMs are histologically indistinguishable, they retain distinct genetic alterations that account for different evolution of the tumor. The high invasiveness, the propensity to disperse throughout the brain parenchyma, and the elevated vascularity make these tumors extremely recidivist, resulting in a short patient median survival even after surgical resection and chemoradiotherapy. Furthermore, GBM is considered an immunologically cold tumor. Several studies highlight a highly immunosuppressive tumor microenvironment that promotes recurrence and poor prognosis. Deeper insight into the tumor immune microenvironment, together with the recent discovery of a conventional lymphatic system in the central nervous system (CNS), led to new immunotherapeutic strategies. In the last two decades, experimental evidence from different groups proved the existence of cancer stem cells (CSCs), also known as tumor-initiating cells, that may play an active role in tumor development and progression. Recent findings also indicated the presence of highly infiltrative CSCs in the peritumoral region of GBM. This region appears to play a key role in tumor growing and recurrence. However, until recently, few studies investigated the biomolecular characteristics of the peritumoral tissue. The aim of this review is to recapitulate the pathological features of GBM and of the peritumoral region associated with progression and recurrence.

Levetiracetam enhances the temozolomide effect on glioblastoma stem cell proliferation and apoptosis.

BACKGROUND: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor in which cancer cells with stem cell-like features, called cancer stem cells (CSCs), were identified. Two CSC populations have been previously identified in GBM, one derived from the GBM area called enhanced lesion (GCSCs) and the other one from the brain area adjacent to the tumor margin (PCSCs) that greatly differ in their growth properties and tumor-initiating ability. To date the most effective chemotherapy to treat GBM is represented by alkylating agents such as temozolomide (TMZ), whose activity can be regulated by histone deacetylases (HDACs) inhibitors through the modulation of O6-methylguanine-DNA methyltransferase (MGMT) expression. Levetiracetam (LEV), a relatively new antiepileptic drug, modulates HDAC levels ultimately silencing MGMT, thus increasing TMZ effectiveness. However, an improvement in the therapeutic efficacy of TMZ is needed. METHODS: Cell proliferation was investigated by BrdU cell proliferation assay and by Western Blot analysis of PCNA expression. Apoptosis was evaluated by Western Blot and Immunofluorescence analysis of the cleaved Caspase-3 expression. MGMT and HDAC4 expression was analyzed by Western Blotting and Immunofluorescence. Statistical analysis was performed using the Student's t test and Mann-Whitney test. RESULTS: Here we evaluated the effect of TMZ on the proliferation rate of the IDH-wildtype GCSCs and PCSCs derived from six patients, in comparison with the effects of other drugs such as etoposide, irinotecan and carboplatin. Our results demonstrated that TMZ was less effective compared to the other agents; hence, we verified the possibility to increase the effect of TMZ by combining it with LEV. Here we show that LEV enhances the effect of TMZ on GCSCs proliferation (being less effective on PCSCs) by decreasing MGMT expression, promoting HDAC4 nuclear translocation and activating apoptotic pathway. CONCLUSIONS: Although further studies are needed to determine the exact mechanism by which LEV makes GBM stem cells more  sensitive to TMZ, these results suggest that the clinical therapeutic efficacy of TMZ in GBM might be enhanced by the combined treatment with LEV.

Cancer stem cells from peritumoral tissue of glioblastoma multiforme: the possible missing link between tumor development and progression.

In glioblastoma multiforme (GBM), cancer stem cells (CSCs) are thought to be responsible for gliomagenesis, resistance to treatment and recurrence. Unfortunately, the prognosis for GBM remains poor and recurrence frequently occurs in the peritumoral tissue within 2 cm from the tumor edge. In this area, a population of CSCs has been demonstrated which may recapitulate the tumor after surgical resection. In the present study, we aimed to characterize CSCs derived from both peritumoral tissue (PCSCs) and GBM (GCSCs) in order to deepen their significance in GBM development and progression. The stemness of PCSC/GCSC pairs obtained from four human GBM surgical specimens was investigated by comparing the expression of specific stem cell markers such as Nestin, Musashi-1 and SOX2. In addition, the growth rate, the ultrastructural features and the expression of other molecules such as c-Met, pMet and MAP kinases, involved in cell migration/invasion, maintenance of tumor stemness and/or resistance to treatments were evaluated. Since it has been recently demonstrated the involvement of the long non-coding RNAs (lncRNAs) in the progression of gliomas, the expression of H19 lncRNA, as well as of one of its two mature products miR-675-5p was evaluated in neurospheres. Our results show significant differences between GCSCs and PCSCs in terms of proliferation, ultrastructural peculiarities and, at a lower extent, stemness profile. These differences might be important in view of their potential role as a therapeutic target.

Pivotal role of human stearoyl-CoA desaturases (SCD1 and 5) in breast cancer progression: oleic acid-based effect of SCD1 on cell migration and a novel pro-cell survival role for SCD5.

The influence of cell membrane fluidity on cancer progression has been established in different solid tumors. We previously reported that "cancer-associated fibroblasts" (CAFs) induced epithelial-mesenchymal transition and increased cell membrane fluidity and migration in poorly (MCF-7) and highly invasive (MDA-MB-231) breast cancer cells. We also found that the membrane fluidity regulating enzyme stearoyl-CoA desaturase 1 (SCD1) was upregulated in tumor cells co-cultured with CAFs and established its essential role for both intrinsic and CAF-driven tumor cell motility. Here, we further explored the mechanisms involved in the SCD1-based modulation of breast cancer cell migration and investigated the role of the other human SCD isoform, SCD5. We showed that the addition of oleic acid, the main SCD1 product, nullified the inhibitory effects produced on MCF-7 and MDA-MB-231 cell migration by SCD1 depletion (pharmacological or siRNA-based). Conversely, SCD5 seemed not involved in the regulation of cancer cell motility. Interestingly, a clear induction of necrosis was observed as a result of the depletion of SCD5 in MCF-7 cells, where the expression of SCD5 was found to be upregulated by CAFs. The necrotic effect was rescued by a 48-h treatment of cells with oleic acid. These results provide further insights in understanding the role of SCD1 in both intrinsic and CAF-stimulated mammary tumor cell migration, unveiling the metabolic basis of this desaturase-triggered effect. Moreover, our data suggest the ability of CAFs to promote the maintenance of tumor cell survival by the induction of SCD5 levels.

Characterization of inflammatory cell infiltrate of scleroderma skin: B cells and skin score progression.

BACKGROUND: The purpose of this study was to investigate the frequency and the distribution of inflammatory cell infiltrate in two sets of cutaneous biopsies derived from clinically affected and unaffected skin in patients with systemic sclerosis (SSc) and to test correlation between the cell infiltrate and the progression of skin involvement. METHODS: Skin was immunohistochemically assessed to identify CD68, CD3, CD20 and CD138-positive (+) cells in clinically affected and unaffected skin in 28 patients with SSc. Patients were followed for 6 months and the characteristics of the infiltrate were analyzed according to disease duration, clinical features and skin involvement progression. RESULTS: In all SSc cutaneous specimens, cellular infiltrates were found in a perivascular location predominantly in the mid and deeper portions of the dermis. All the analyzed biopsies showed a CD3+ and CD68+ cell infiltrate and the mean number of CD3+ and of CD68+ cells was higher in clinically involved skin (CD3+, 71.7 ± 34.6 and CD68+, 26.3 ± 8.4, respectively) than in clinically uninvolved skin (CD3+, 45.7 ± 36.0 and CD68+, 13.6 ± 6.1, respectively) (p < 0.001 for both comparisons). CD20+ cells were found in 17 (60.7%) patients and in these patients the mean number of CD20+ cells was higher in clinically involved (4.7 ± 5.9) than in uninvolved skin (1.9 ± 2.9), (p = 0.04). There was a greater number of CD20+ cells in patients with early SSc compared with patients with long-standing disease. CD138+ cells were found in 100% of biopsies of clinically involved skin and in 89.3% of biopsies of uninvolved skin. The mean number of CD138+ cells was higher in clinically involved skin (3.6 ± 2.3) than in clinically uninvolved skin (1.9 ± 1.7), (p < 0.001). Seven patients experienced more than 20% worsening in the skin score after 6 months of follow up; all of them had a CD20+ skin infiltrate on biopsy of clinically involved skin. CONCLUSIONS: Our results confirm that mononuclear cells are present in the skin of all patients with SSc, underlining the role of inflammatory cell infiltrates in skin involvement in SSc. B cells in the skin seem to characterize patients with early diffuse skin disease and to correlate with skin progression.

Analysis of angiogenesis related factors in glioblastoma, peritumoral tissue and their derived cancer stem cells.

The formation of new blood vessels represents a crucial event under both physiological and pathological circumstances. In this study, we evaluated by immunohistochemistry, and/or Western blotting and/or quantitative real time-PCR the expression of HIF1α, HIF2α, VEGF, VEGFR1 and VEGFR2 in surgical glioblastoma multiforme (GBM) and peritumoral tissue samples obtained from 50 patients as well as in cancer stem cells (CSCs) isolated from GBM (GCSCs) and peritumoral tissue (PCSCs) of 5 patients. We also investigated the contribution of both GCSCs and PCSCs on the behavior of endothelial cells (ECs) in vitro. Immunohistochemistry demonstrated the expression of angiogenesis markers in both GBM and peritumoral tissue. In addition, in vitro tube formation assay indicated that both GCSCs and PCSCs stimulate EC proliferation as well as tube-like vessel formation. An increased migration aptitude was mainly observed when ECs were cultured in the presence of GCSCs rather than in the presence of PCSCs. These findings suggest that relevant neoangiogenetic events may occur in GBM. In particular, VEGF/VEGFR co-expression in PCSCs leads to hypothesize the involvement of an autocrine signaling. Moreover, our results suggest that both GCSCs and PCSCs own the skill of activating the "angiogenic switch" and the capability of modulating EC behavior, indicating that both cell types are either responsive to angiogenic stimuli or able to trigger angiogenic response. Together with our previous findings, this study adds a further piece to the challenging puzzle of the characterization of peritumoral tissue and of the definition of its real role in GBM pathophysiology.

Progenitor/Stem Cell Markers in Brain Adjacent to Glioblastoma: GD3 Ganglioside and NG2 Proteoglycan Expression.

Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM, peritumor tissue (brain adjacent to tumor, BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased, regardless of the tumor cell presence, whereas GD3 correlated with neoplastic infiltration. In BAT, NG2 was coexpressed with a-smooth muscle actin (a-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM.

Gene expression profile of glioblastoma peritumoral tissue: an ex vivo study.

The gene expression pattern of glioblastoma (GBM) is well documented but the expression profile of brain adjacent to tumor is not yet analysed. This may help to understand the oncogenic pathway of GBM development. We have established the genome-wide expression profiles of samples isolated from GBM tumor mass, white matter adjacent to tumor (apparently free of tumor cells), and white matter controls by using the Affymetrix HG-U133 arrays. Array-CGH (aCGH) was also performed to detect genomic alterations. Among genes dysregulated in peritumoral white matter, 15 were over-expressed, while 42 were down-regulated when compared to white matter controls. A similar expression profile was detected in GBM cells. Growth, proliferation and cell motility/adhesion-associated genes were up-regulated while genes involved in neurogenesis were down-regulated. Furthermore, several tumor suppressor genes along with the KLRC1 (a member of natural killer receptor) were also down-regulated in the peritumoral brain tissue. Several mosaic genomic lesions were detected by aCGH, mostly in tumor samples and several GBM-associated mosaic genomic lesions were also present in the peritumoral brain tissue, with a similar mosaicism pattern. Our data could be explained by a dilution of genes expressed from tumor cells infiltrating the peritumour tissue. Alternatively, these findings could be substained by a relevant amount of "apparently normal" cells presenting a gene profile compatible with a precancerous state or even "quiescent" cancer cells. Otherwise, the recurrent tumor may arise from both infiltrating tumor cells and from an interaction and recruitment of apparently normal cells in the peritumor tissue by infiltrating tumor cells.

Epithelial-stromal interactions in human breast cancer: effects on adhesion, plasma membrane fluidity and migration speed and directness.

Interactions occurring between malignant cells and the stromal microenvironment heavily influence tumor progression. We investigated whether this cross-talk affects some molecular and functional aspects specifically correlated with the invasive phenotype of breast tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration) by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer-associated fibroblasts, CAFs) in 2D or 3D (nodules) cultures. Confocal immunofluorescence analysis of the epithelial adhesion molecule E-cadherin on frozen nodule sections demonstrated that NFs and CAFs, respectively, induced or inhibited its expression in MCF-7 cells. An increase in the mesenchymal adhesion protein N-cadherin was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of cancer cells. CAFs, in turn, promoted N-cadherin up-regulation in MDA-MB-231 cells and its de novo expression in MCF-7 cells. Beyond promotion of "cadherin switching", another sign of the CAF-triggered epithelial-mesenchymal transition (EMT) was the induction of vimentin expression in MCF-7 cells. Plasma membrane labeling of monolayer cultures with the fluorescent probe Laurdan showed an enhancement of the membrane fluidity in cancer cells co-cultured with NFs or CAFs. An increase in lipid packing density of fibroblast membranes was promoted by MCF-7 cells. Time-lapsed cell tracking analysis of mammary cancer cells co-cultured with NFs or CAFs revealed an enhancement of tumor cell migration velocity, even with a marked increase in the directness induced by CAFs.Our results demonstrate a reciprocal influence of mammary cancer and fibroblasts on various adhesiveness/invasiveness features. Notably, CAFs' ability to promote EMT, reduction of cell adhesion, increase in membrane fluidity, and migration velocity and directness in mammary cancer cells can be viewed as an overall progression- and invasion-promoting effect.

Soluble E-cadherin and IL-6 serum levels in patients affected by prostate cancer before and after prostatectomy.

Prostate specific antigen (PSA) is still the best available tumour marker in prostate cancer (PCa), but presents some limits. Therefore, there is a need for novel markers in the detection and management of PCa. The 80-kDa soluble form of E-cadherin (sE-cad) and the cytokine IL-6 are being discussed as supplemental serum markers for PCa. In this study, sE-cad and IL-6 serum levels were determined in patients with pathological localized or locally advanced PCa without any previous treatment. These patients underwent radical retropubic prostatectomy (RRP) in accordance with the EAU Guidelines on Prostate Cancer. The molecules were determined via immunoenzymatic assays in samples collected before and after surgery. Statistical analysis was performed by Student's t-test and Pearson's correlation test. sE-cad levels were 6.0 ± 2.7 and 4.6 ± 2.3 µg/ml, before and after RRP, respectively. A highly statistically significant decrease in sE-cad concentrations after RRP was observed (p<0.0001), in 50/61 patients (82%). sE-cad levels before and after surgery were correlated (Pearson's correlation coefficient, r=0.6993, p<0.0001). sE-cad values detected after surgery were higher in patients with PSA levels >10 ng/ml (p<0.05). sE-cad levels before RRP were significantly higher in patients with G3 tumours compared to those with G2 tumours (p<0.02). Finally, sE-cad concentrations both before and after surgery were higher in tumours with Gleason score =7 compared to those with Gleason score <7 (p<0.002 and p<0.05, respectively). Preliminary data from 20 patients indicated a statistically significant increase in IL-6 levels after RRP (11.2 vs. 7.2 pg/ml, p<0.001). This is the first study on the reduction in sE-cad levels after RRP in PCa patients. Moreover, it shows that preoperative sE-cad concentrations are higher in patients with less differentiated PCa. Promising findings of this pilot study may lead to investigation of sE-cad in a larger study with follow-up.

New findings on anaplasmosis caused by infection with Anaplasma phagocytophilum.

Ixodes ricinus (I. ricinus) is one of the vectors of Anaplasma phagocytophilum (A. phagocytophilum) in Europe, in which rates of infection range from 1.9% to 34%. In 1998, human granulocytic ehrlichiosis-like (HGE-like) Ehrlichia DNA was detected in Italy, by PCR technique in one I. ricinus nymph out of 55 ticks that were examined. In 1996, 6.3% of 310 human sera in high-risk subjects from Italy were found positive for antibodies to Ehrlichia phagocytophila (E. phagocytophila). In the same year, the authors reported the first case of equine granulocytic ehrlichiosis. In 1997, only 2 out of 563 equine blood samples examined were found positive for antibodies to E. phagocytophila in the Latium region. In 1998, serological positivity was not observed in 14 symptomatic race horses. In 2002, a symptomatic horse living in Rome was found positive for Ehrlichia equi (E. equi) antibodies, as confirmed by PCR. E. equi was also demonstrated in horses by detection of specific antibodies from two asymptomatic ponies. We tested 128 sera from sheep in different flocks, and antibodies to E. phagocytophila were detected in 17 sera (13.3%) of these sheep. From 2000 to 2004, 147 dog sera were tested for antibodies against A. phagocytophilum, and 7 of these sera were positive (4.8%). These data confirm the presence of the infection in human, domestic animals, and pets in Italy. Studies are under way to correlate the distributions of the disease and tick vector, I. ricinus.