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Borgo Vercelli, Italy

Mellai M.,Neuro Bio Oncology Center | Monzeglio O.,Neuro Bio Oncology Center | Piazzi A.,University of Piemonte Orientale | Caldera V.,Neuro Bio Oncology Center | And 6 more authors.
Journal of Neuro-Oncology | Year: 2012

MGMT (O6-methylguanine-DNA methyltransferase) promoter hypermethylation is a helpful prognostic marker for chemotherapy of gliomas, although with some controversy for low-grade tumors. The objective of this study was to retrospectively investigate MGMT promoter hypermethylation status for a series of 350 human brain tumors, including 275 gliomas of different malignancy grade, 21 glioblastoma multiforme (GBM) cell lines, and 75 non-glial tumors. The analysis was performed by methylation-specific PCR and capillary electrophoresis. MGMT expression at the protein level was also evaluated by both immunohistochemistry (IHC) and western blotting analysis. Associations of MGMT hypermethylation with IDH1/IDH2 mutations, EGFR amplification, TP53 mutations, and 1p/19q co-deletion, and the prognostic significance of these, were investigated for the gliomas. MGMT promoter hypermethylation was identified in 37.8% of gliomas, but was not present in non-glial tumors, with the exception of one primitive neuroectodermal tumor (PNET). The frequency was similar for all the astrocytic gliomas, with no correlation with histological grade. Significantly higher values were obtained for oligodendrogliomas. MGMT promoter hypermethylation was significantly associated with IDH1/IDH2 mutations (P = 0.0207) in grade II-III tumors, whereas it had a borderline association with 1p deletion (P = 0.0538) in oligodendrogliomas. No other association was found. Significant correlation of MGMT hypermethylation with MGMT protein expression was identified by IHC in GBMs and oligodendrogliomas (P = 0.0001), but not by western blotting. A positive correlation between MGMT protein expression, as detected by either IHC or western blotting, was also observed. The latter was consistent with MGMT promoter hypermethylation status in GBM cell lines. In low-grade gliomas, MGMT hypermethylation, but not MGMT protein expression, was associated with a trend, only, toward better survival, in contrast with GBMs, for which it had favorable prognostic significance. © Springer Science+Business Media, LLC. 2012. Source


Mellai M.,Neuro Bio Oncology Center | Cattaneo M.,National Research Council Italy | Storaci A.M.,National Research Council Italy | Annovazzi L.,Neuro Bio Oncology Center | And 5 more authors.
Oncotarget | Year: 2015

The suppressor of Lin-12-like (C. elegans) (SEL1L) is involved in the endoplasmic reticulum (ER)-associated degradation pathway, malignant transformation and stem cells. In 412 formalin-fixed and paraffin-embedded brain tumors and 39 Glioblastoma multiforme (GBM) cell lines, we determined the frequency of five SEL1L single nucleotide genetic variants with regulatory and coding functions by a SNaPShotTM assay. We tested their possible association with brain tumor risk, prognosis and therapy. We studied the in vitro cytotoxicity of valproic acid (VPA), temozolomide (TMZ), doxorubicin (DOX) and paclitaxel (PTX), alone or in combination, on 11 GBM cell lines, with respect to the SNP rs 12435998 genotype. The SNP rs12435998 was prevalent in anaplastic and malignant gliomas and in meningiomas of all histologic grades, but unrelated to brain tumor risks. In GBM patients, the SNP rs12435998 was associated with prolonged overall survival (OS) and better response to TMZ-based radio-chemotherapy. GBM stem cells with this SNP showed lower levels of SEL1L expression and enhanced sensitivity to VPA. Source


Riganti C.,University of Turin | Salaroglio I.C.,University of Turin | Pinzon-Daza M.L.,University of Turin | Pinzon-Daza M.L.,El Rosario University | And 9 more authors.
Cellular and Molecular Life Sciences | Year: 2014

Low delivery of many anticancer drugs across the blood-brain barrier (BBB) is a limitation to the success of chemotherapy in glioblastoma. This is because of the high levels of ATP-binding cassette transporters like P-glycoprotein (Pgp/ABCB1), which effluxes drugs back to the bloodstream. Temozolomide is one of the few agents able to cross the BBB; its effects on BBB cells permeability and Pgp activity are not known. We found that temozolomide, at therapeutic concentration, increased the transport of Pgp substrates across human brain microvascular endothelial cells and decreased the expression of Pgp. By methylating the promoter of Wnt3 gene, temozolomide lowers the endogenous synthesis of Wnt3 in BBB cells, disrupts the Wnt3/glycogen synthase kinase 3/β-catenin signaling, and reduces the binding of β-catenin on the promoter of mdr1 gene, which encodes for Pgp. In co-culture models of BBB cells and human glioblastoma cells, pre-treatment with temozolomide increases the delivery, cytotoxicity, and antiproliferative effects of doxorubicin, vinblastine, and topotecan, three substrates of Pgp that are usually poorly delivered across BBB. Our work suggests that temozolomide increases the BBB permeability of drugs that are normally effluxed by Pgp back to the bloodstream. These findings may pave the way to new combinatorial chemotherapy schemes in glioblastoma. © 2013 Springer Basel. Source


Cattaneo M.,Istituto di Ricovero e Cura a Carattere Scientifico Multimedica | Cattaneo M.,National Research Council Italy | Baronchelli S.,Istituto di Ricovero e Cura a Carattere Scientifico Multimedica | Baronchelli S.,National Research Council Italy | And 10 more authors.
Journal of Biological Chemistry | Year: 2014

Valproic acid (VPA), an histone deacetylase inhibitor, is emerging as a promising therapeutic agent for the treatments of gliomas by virtue of its ability to reactivate the expression of epigenetically silenced genes. VPA induces the unfolded protein response (UPR), an adaptive pathway displaying a dichotomic yin yang characteristic; it initially contributes in safeguarding the malignant cell survival, whereas long-lasting activation favors a proapoptotic response. By triggering UPR, VPA might tip the balance between cellular adaptation and programmed cell death via the deregulation of protein homeostasis and induction of proteotoxicity. Here we aimed to investigate the impact of proteostasis on glioma stem cells (GSC) using VPA treatment combined with subversion of SEL1L, a crucial protein involved in homeostatic pathways, cancer aggressiveness, and stem cell state maintenance. We investigated the global expression of GSC lines untreated and treated with VPA, SEL1L interference, and GSC line response to VPA treatment by analyzing cell viability via MTT assay, neurosphere formation, and endoplasmic reticulum stress/UPR-responsive proteins. Moreover, SEL1L immunohistochemistry was performed on primary glial tumors. The results show that (i) VPA affects GSC lines viability and anchorage-dependent growth by inducing differentiative programs and cell cycle progression, (ii) SEL1L down-modulation synergy enhances VPA cytotoxic effects by influencing GSCs proliferation and self-renewal properties, and (iii) SEL1L expression is indicative of glioma proliferation rate, malignancy, and endoplasmic reticulum stress statuses. Targeting the proteostasis network in association to VPA treatment may provideanalternative approachtodeplete GSC and improve glioma treatments. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Source


Annovazzi L.,Neuro Bio Oncology Center | Caldera V.,Neuro Bio Oncology Center | Mellai M.,Neuro Bio Oncology Center | Riganti C.,University of Turin | And 4 more authors.
International Journal of Oncology | Year: 2015

Therapeutic resistance in glioblastoma multiforme (GBM) has been linked to a subpopulation of cells with stem cell-like properties, the glioma stem cells (GSCs), responsible for cancer progression and recurrence. This study investigated the in vitro cytotoxicity of three chemotherapeutics, temozolomide (TMZ), doxorubicin (Dox) and paclitaxel (PTX) on glioma cell lines, by analyzing the molecular mechanisms leading to DNA repair and cell resistance, or to cell death. The drugs were tested on 16 GBM cell lines, grown as neurospheres (NS) or adherent cells (AC), by studying DNA damage occurrence by Comet assay, the expression by immunofluorescence and western blotting of checkpoint/repair molecules and apoptosis. The three drugs were able to provoke a genotoxic injury and to inhibit dose- and time-dependently cell proliferation, more evidently in AC than in NS. The first cell response to DNA damage was the activation of the damage sensors (p-ATM, p-53BP1, γ-H2AX), followed by repair effectors; the expression of checkpoint/repair molecules appeared higher in NS than in AC. The non-homologous repair pathway (NHEJ) seemed more involved than the homologous one (HR). Apoptosis occurred after long treatment times, but only a small percentage of cells in NS underwent death, even at high drug concentration, whereas most cells survived in a quiescent state and resumed proliferation after drug removal. In tumor specimens, checkpoint/repair proteins were constitutively expressed in GBMs, but not in low-grade gliomas. © 2015, Spandidos Publications. All rights reserved. Source

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