Distinct cellular responses induced by saporin and a transferrin-saporin conjugate in two different human glioblastoma cell lines.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults, with a median survival of ~12-18 months post-diagnosis. GBM usually recurs within 12 months post-resection, with poor prognosis. Thus, novel therapeutic strategies to target and kill GBM cells are urgently needed. The marked difference of tumour cells with respect to normal brain cells renders glioblastoma a good candidate for selective targeted therapies. Recent experimental strategies focus on over expressed cell surface receptors. Targeted toxins represent a new class of selective molecules composed by a potent protein toxin and a carrier ligand. Targeted toxins approaches against glioblastoma were under investigation in phase I and II clinical trials with several immunotoxins (IT)/ligand toxins such as IL4-Pseudomonas aeruginosa exotoxin A (IL4-PE, NBI-3001), tumour growth factor fused to PE38, a shorter PE variant, (TGF)alpha-TP-38, IL13-PE38, and a transferrin-C diphtheriae toxin mutant (Tf-CRM107). In this work, we studied the effects of the plant ribosome-inactivating saporin and of its chimera transferrin-saporin against two different GBM cell lines. The data obtained here indicate that cell proliferation is affected by the toxin treatments but that different mechanisms are used, directly linked to the presence of an active or inactive p53. A model is proposed for these alternative intracellular pathways.

Glycosilated nucleolin as marker for human gliomas.

Nucleolin is a multifunctional DNA and RNA binding protein involved in regulation of gene transcription, chromatin remodeling, RNA metabolism, and ribosomal RNA synthesis. Nucleolin seems to be over-expressed in highly proliferative cells and is involved in many aspect of gene expression: DNA recombination and replication, RNA transcription by RNA polymerase I and II, rRNA processing, mRNA stabilization, cytokinesis, and apoptosis. Although nucleolin is localized predominantly in the nucleolus, it has also been shown to be localized in a phosphorylated/glycolsilated form on the cell surface of different cells. Numerous articles dealing with surface nucleolin targeting for tumor therapy have been recently published. However, at present, no extensive informations are so far available for the presence of nucleolin in human gliomas. In the present work we investigated on the presence and localization of nucleolin in glioma on glioma specimens at different grade of malignancy and on primary glioma cell cultures derived by surgical resection, trying to correlate the presence of glycosilated membrane nucleolin with the malignancy grade. To this purpose an antibody produced by us against gp273 protein, demonstrated to recognized the glycosilated surface nucleolin, has been used. The results obtained demonstrate that surface nucleolin increase with the malignancy grade thus suggesting that it may constitute a histopathological marker for glioma grading and a possible tool for targeted therapy.

Hypoxia induces peroxisome proliferator-activated receptor α (PPARα) and lipid metabolism peroxisomal enzymes in human glioblastoma cells.

Glioblastoma multiforme (GBM) represents the most severe type of glioma, the most common brain tumor. Their malignancy shows a relationship with an increased proliferation and a poorly organized tumor vascularization, an event that leads to inadequate blood supply, hypoxic areas and at last to the formation of necrotic areas, a feature of glioblastoma. Hypoxic/necrotic tumors are more resistant to chemotherapy and radiation therapies, thus it is crucial to formulate new therapeutic approaches that can render these tumors more sensitive to the action of conventional therapies. It has been demonstrated that under hypoxia, gliomas accumulate lipid droplets and that this event is positively correlated with the degree of malignancy, glioblastoma being the most endowed with lipid droplets. We have previously demonstrated in ex vivo glioma specimens a grade-dependent lipid metabolism perturbation. Here we studied the lipid pathways and the presence of stemness markers in glioma primary cultures, obtained from surgical specimens of patients affected by glioma at different grade of malignancy, GBM primary cultures cultured under both hypoxic and normoxic conditions, as well as normal human astrocytes. The results obtained demonstrate that hypoxia plays a crucial role in regulating the expression of lipid metabolism peroxisomal enzymes, the lipid droplets accumulation as well as the transcription factor PPARα.

Signal transduction pathways involved in PPARß/δ-induced neuronal differentiation.

Neuroblastomas are pediatric tumors originating from neuroblasts in the developing peripheral nervous system. The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of survival and differentiation of specific neuronal populations in the central and peripheral nervous system. Patients whose neuroblastoma tumors express high levels of BDNF and TrkB have an unfavorable prognosis. We have previously reported on the neuronal differentiating activity of peroxisome proliferator-activated receptors (PPAR)ß/δ natural and synthetic ligands by modulating BDNF/TrkB pathway, suggesting their potential use as new therapeutic strategies for neuroblastoma. The validation of new therapeutic agents implies the understanding of their mechanisms of action. Herein, we report the effects of activated-PPARß/δ on signal transduction pathways known to be involved in neuronal differentiation, such as ERK1,2 and BDNF pathways. The results obtained, using also PPARß/δ silencing, indicating a neuronal differentiating effect PPARß/δ-dependent through BDNF-P75-ERK1,2 pathways, further support a role for PPARß/δ in neuronal differentiation and pointing towards PPARß/δ as a modulator of pathways crucial for neuronal differentiation. These findings open new perspectives in the formulation of potential therapeutic approaches to be used as adjuvant treatment with the standard therapies.

Lipid metabolism impairment in human gliomas: expression of peroxisomal proteins in human gliomas at different grades of malignancy.

Gliomas are histologically graded by cellularity, cytological atypia, necrosis, mitotic figures, and vascular proliferation, features associated with biologically aggressive behaviour. However, abundant evidence suggests the presence of unrecognized, clinically relevant subclasses of the diffuse gliomas, both in respect to their underlying molecular phenotype and their clinical response to therapy. It is well-known that patient prognosis and therapeutic decisions rely on accurate pathological grading. Recently, it was reported that human gliomas accumulate lipid droplets during progression, suggesting a lipid metabolism impairment. Considering the crucial role of peroxisomes in lipid metabolism, in the present work we studied the expression profiles of proteins either exclusively localized to peroxisomes, such as peroxin14 (PEX14), peroxisomal membrane protein 70Kda (PMP70), acyl-CoA oxidase, thiolase, or partially associated to peroxisomes such as Hydroxymethylglutaryl-CoA reductase (HMGCoA-red) and peroxisomal-related proteins, namely PPARalpha, in human glioma specimens at different grades of malignancy. Moreover, Nile red staining of lipid droplets, thin layer chromatography (TLC) and proton nuclear magnetic resonance spectroscopy (NMR) were carried out in order to correlate the biochemical results with the lipid content of tumor tissues. The results obtained indicate that correlating the malignancy grade with the expression of peroxisomal genes and proteins, may constitute a sensitive tool to highlight possible subtypes not recognized by the classical histological techniques.

Biomolecular characterization of human glioblastoma cells in primary cultures: differentiating and antiangiogenic effects of natural and synthetic PPARgamma agonists.

Gliomas are the most commonly diagnosed malignant brain primary tumors. Prognosis of patients with high-grade gliomas is poor and scarcely affected by radiotherapy and chemotherapy. Several studies have reported antiproliferative and/or differentiating activities of some lipophylic molecules on glioblastoma cells. Some of these activities in cell signaling are mediated by a class of transcriptional factors referred to as peroxisome proliferator-activated receptors (PPARs). PPARgamma has been identified in transformed neural cells of human origin and it has been demonstrated that PPARgamma agonists decrease cell proliferation, stimulate apoptosis and induce morphological changes and expression of markers typical of a more differentiated phenotype in glioblastoma and astrocytoma cell lines. These findings arise from studies mainly performed on long-term cultured transformed cell lines. Such experimental models do not exactly reproduce the in vivo environment since long-term culture often results in the accumulation of further molecular alterations in the cells. To be as close as possible to the in vivo condition, in the present work we investigated the effects of PPARgamma natural and synthetic ligands on the biomolecular features of primary cultures of human glioblastoma cells derived from surgical specimens. We provide evidence that PPARgamma agonists may interfere with glioblastoma growth and malignancy and might be taken in account as novel antitumoral drugs.