Alessia Fidoamore

Glioblastoma Stem Cells Microenvironment: The Paracrine Roles of the Niche in Drug and Radioresistance

Among all solid tumors, the high-grade glioma appears to be the most vascularized one. In fact, “microvascular hyperplasia” is a hallmark of GBM. An altered vascular network determines irregular blood flow, so that tumor cells spread rapidly beyond the diffusion distance of oxygen in the tissue, with the consequent formation of hypoxic or anoxic areas, where the bulk of glioblastoma stem cells (GSCs) reside. The response to this event is the induction of angiogenesis, a process mediated by hypoxia inducible factors. However, this new capillary network is not efficient in maintaining a proper oxygen supply to the tumor mass, thereby causing an oxygen gradient within the neoplastic zone. This microenvironment helps GSCs to remain in a “quiescent” state preserving their potential to proliferate and differentiate, thus protecting them by the effects of chemo- and radiotherapy. Recent evidences suggest that responses of glioblastoma to standard therapies are determined by the microenvironment of the niche, where the GSCs reside, allowing a variety of mechanisms that contribute to the chemo- and radioresistance, by preserving GSCs. It is, therefore, crucial to investigate the components/factors of the niche in order to formulate new adjuvant therapies rendering more efficiently the gold standard therapies for this neoplasm.

Targeting CXCR1 on breast cancer stem cells: signaling pathways and clinical application modelling

In breast cancer it has been proposed that the presence of cancer stem cells may drive tumor initiation, progression and recurrences. IL-8, up-regulated in breast cancer, and associated with poor prognosis, increases CSC self-renewal in cell line models. It signals via two cell surface receptors, CXCR1 and CXCR2. Recently, the IL-8/CXCR1 axis was proposed as an attractive pathway for the design of specific therapies against breast cancer stem cells. Reparixin, a powerful CXCR1 inhibitor, was effective in reducing in vivo the tumour-initiating population in several NOD/SCID mice breast cancer models, showing that the selective targeting of CXCR1 and the combination of reparixin and docetaxel resulted in a concomitant reduction of the bulk tumour mass and CSC population. The available data indicate that IL-8, expressed by tumour cells and induced by chemotherapeutic treatment, is a key regulator of the survival and self-renewal of the population of CXCR1-expressing CSC. Consequently, this investigation on the mechanism of action of the reparixin/paclitaxel combination, was based on the observation that reparixin treatment contained the formation of metastases in several experimental models. However, specific data on the formation of breast cancer brain metastases, which carry remarkable morbidity and mortality to a substantial proportion of advanced breast cancer patients, have not been generated. The obtained data indicate a beneficial use of the drug combination reparixin and paclitaxel to counteract brain tumour metastasis due to CSC, probably due to the combined effects of the two drugs, the pro-apoptotic action of paclitaxel and the cytostatic and anti-migratory effects of reparixin.

Involvement of peroxisome proliferator-activated receptor β/δ (PPAR β/δ) in BDNF signaling during aging and in Alzheimer disease: Possible role of 4-hydroxynonenal (4-HNE)

Aging and many neurological disorders, such as AD, are linked to oxidative stress, which is considered the common effector of the cascade of degenerative events. In this phenomenon, reactive oxygen species play a fundamental role in the oxidative decomposition of polyunsaturated fatty acids, resulting in the formation of a complex mixture of aldehydic end products, such as malondialdehyde, 4-hydroxynonenal, and other alkenals. Interestingly, 4-HNE has been indicated as an intracellular agonist of peroxisome proliferator-activated receptor β/δ. In this study, we examined, at early and advanced AD stages (3, 9, and 18 months), the pattern of 4-HNE and its catabolic enzyme glutathione S-transferase P1 in relation to the expression of PPARβ/δ, BDNF signaling, as mRNA and protein, as well as on their pathological forms (i.e., precursors or truncated forms). The data obtained indicate a novel detrimental age-dependent role of PPAR β/δ in AD by increasing pro-BDNF and decreasing BDNF/TrkB survival pathways, thus pointing toward the possibility that a specific PPARβ/δ antagonist may be used to counteract the disease progression.

Nucleolin antagonist triggers autophagic cell death in human glioblastoma primary cells and decreased in vivo tumor growth in orthotopic brain tumor model

Nucleolin (NCL) is highly expressed in several types of cancer and represents an interesting therapeutic target. It is expressed at the plasma membrane of tumor cells, a property which is being used as a marker for several human cancer including glioblastoma. In this study we investigated targeting NCL as a new therapeutic strategy for the treatment of this pathology. To explore this possibility, we studied the effect of an antagonist of NCL, the multivalent pseudopeptide N6L using primary culture of human glioblastoma cells. In this system, N6L inhibits cell growth with different sensitivity depending to NCL localization. Cell cycle analysis indicated that N6L-induced growth reduction was due to a block of the G1/S transition with down-regulation of the expression of cyclin D1 and B2. By monitoring autophagy markers such as p62 and LC3II, we demonstrate that autophagy is enhanced after N6L treatment. In addition, N6L-treatment of mice bearing tumor decreased in vivo tumor growth in orthotopic brain tumor model and increase mice survival. The results obtained indicated an anti-proliferative and pro-autophagic effect of N6L and point towards its possible use as adjuvant agent to the standard therapeutic protocols presently utilized for glioblastoma.

Gastroprotective Effects of L-Lysine Salification of Ketoprofen in Ethanol-Injured Gastric Mucosa

Ketoprofen L‐lysine salt (KLS), a NSAID, is widely used for its analgesic efficacy and tolerability. L‐lysine salification was reported to increase the solubility and the gastric absorption and tolerance of ketoprofen. Since the management of NSAIDs gastrotoxicity still represents a major limitation in prolonged therapies, mainly when gastric lesions are present, this study investigated the gastro‐protective activity of L‐lysine by using a well‐established model of gastric mucosa injury, the ethanol‐gastric injury model. Several evidences show that the damaging action of ethanol could be attributed to the increase of ROS, which plays a key role in the increase of lipid peroxidation products, including malonyldialdehyde and 4‐hydroxy‐2‐nonenal. With the aim to unravel the mechanism of L‐lysine gastroprotection, cellular MDA levels and 4‐HNE protein adducts as markers of lipid peroxidation and a panel of key endogenous gastro‐protective proteins were assayed. The data obtained indicate a gastroprotective effect of L‐lysine on gastric mucosa integrity. J. Cell. Physiol. 230: 813–820, 2015.

Roles of PPAR transcription factors in the energetic metabolic switch occurring during adult neurogenesis

ABSTRACT PPARs are a class of ligand-activated transcription factors belonging to the superfamily of receptors for steroid and thyroid hormones, retinoids and vitamin D that control the expression of a large number of genes involved in lipid and carbohydrate metabolism and in the regulation of cell proliferation, differentiation and death. The role of PPARs in the CNS has been primarily associated with lipid and glucose metabolism; however, these receptors are also implicated in neural cell differentiation and death, as well as neuronal maturation. Although it has been demonstrated that PPARs play important roles in determining NSCs fate, less is known about their function in regulating NSCs metabolism during differentiation. In order to identify the metabolic events, controlled by PPARs, occurring during neuronal precursor differentiation, the glucose and lipid metabolism was followed in a recognized model of neuronal differentiation in vitro, the SH-SY5Y neuroblastoma cell line. Moreover, PPARs distribution were also followed in situ in adult mouse brains. The concept of adult neurogenesis becomes relevant especially in view of those disorders in which a loss of neurons is described, such as Alzheimer disease, Parkinson disease, brain injuries and other neurological disorders. Elucidating the crucial steps in energetic metabolism and the involvement of PPARγ in NSC neuronal fate (lineage) may be useful for the future design of preventive and/or therapeutic interventions.

PPARβ/δ and γ in a Rat Model of Parkinson's Disease: Possible Involvement in PD Symptoms

Parkinsons disease is one of the most common neurologic disorder, affecting about 1–4% of persons older than 60 years. Among the proposed mechanisms of PD generation, free radical damage is believed to play a pivotal role in the development and/or progression of the disease. Recently, PPARs, a class of transcription factors involved in several pathways both in physiological and pathological conditions, have been linked by us and others to neurodegeneration. Particularly, PPARγ and its ligands have been indicated as potential therapeutic targets for the treatment of several pathological conditions associated with neuroinflammation within the CNS. The anti‐inflammatory function of PPARγ has attracted attention since agonists exert a broad spectrum of protective effects in several animal models of neurological diseases, including psychiatric diseases. On the other hand a detrimental role for PPARβ/δ has been proposed in Alzheimer, being closely related to the decrease of BDNF and Trkfl. On these bases, in this work we used a 6‐OHDA hemi‐lesioned rat model, inducing loss of dopaminergic neurons, to study the effects of the lesion at three time points from the lesion (1, 2, and 3 weeks), in relevant areas of PD motor symptoms, such as substantia nigra and globus pallidus and in the area of reward and mood control, the nucleus accumbens. In particular, it was studied: (i) the expression of BDNF and its downstream signals; (ii) the modulation of PPARs levels. The results obtained indicate the possible use of a dual PPARβ/δ antagonist/PPARγ agonist to counteract primary and secondary signs of PD neurodegeneration. J. Cell. Biochem. 116: 844–855, 2015.

Hypoxia modulation of peroxisome proliferator-activated receptors (PPARs) in human glioblastoma stem cells. Implications for therapy.

Gliobastoma (GB), the most common adult brain tumor, infiltrates normal brain area rendering impossible the complete surgical resection, resulting in a poor median survival (14–15 months), despite the aggressive multimodality treatments post‐surgery, such as radiation and chemo‐therapy. GB is characterized by hypoxic and necrotic regions due to a poorly organized tumor vascularization, leading to inadequate blood supply and consequently to hypoxic and necrotic areas. We have previously shown that, under hypoxia GB primary cells increased the expression of stemness markers as well as the expression of the nuclear receptor peroxisome proliferator‐activated receptor α (PPARα) and also the crucial role played by PPARs in mouse neural stem cells maintenance and differentiation. Due to the importance of lipid signaling in cell proliferation and differentiation, in this work, we analyzed the expression of PPARs in GB neurospheres both in normoxic and hypoxic conditions. The results obtained suggest a differential regulation of the three PPARs by hypoxia, thus indicating a possible therapeutic strategy to counteract GB recurrencies. J. Cell. Biochem. 113: 3342–3352, 2012.

Energy metabolism in glioblastoma stem cells: PPARα a metabolic adaptor to intratumoral microenvironment

Glioblastoma (GB), the most-common cancer in the adult brain, despite surgery and radio/ chemotherapy, is to date almost incurable. Many hypoxic tumors, including GB, show metabolic reprogramming to sustain uncontrolled proliferation, hypoxic conditions and angiogenesis. Peroxisome Proliferator-activated Receptors (PPAR), particularly the α isotype, have been involved in the control of energetic metabolism. Herein, we characterized patient-derived GB neurospheres focusing on their energetic metabolism and PPARα expression. Moreover, we used a specific PPARα antagonist and studied its effects on the energetic metabolism and cell proliferation/survival of GB stem cells. The results obtained demonstrate that tumor neurospheres are metabolically reprogrammed up-regulating glucose transporter, glucose uptake and glycogen and lipid storage, mainly under hypoxic culture conditions. Treatment with the PPARα antagonist GW6471 resulted in decreased cell proliferation and neurospheres formation. Therefore, PPARα antagonism arises as a potent new strategy as adjuvant to gold standard therapies for GB for counteracting recurrences and opening the way for pre-clinical trials for this class of compounds. When tumor neurospheres were grown in hypoxic conditions in the presence of different glucose concentrations, the most diluted one (0.25g/L) mimicking the real concentration present in the neurosphere core, PPARα increase/PPARγ decrease, increased proliferation and cholesterol content, decreased glycogen particles and LDs were observed. All these responses were reverted by the 72 h treatment with the PPARα antagonist.Glioblastoma (GB), the most-common cancer in the adult brain, despite surgery and radio/ chemotherapy, is to date almost incurable. Many hypoxic tumors, including GB, show metabolic reprogramming to sustain uncontrolled proliferation, hypoxic conditions and angiogenesis. Peroxisome Proliferator-activated Receptors (PPAR), particularly the α isotype, have been involved in the control of energetic metabolism. Herein, we characterized patient-derived GB neurospheres focusing on their energetic metabolism and PPARα expression. Moreover, we used a specific PPARα antagonist and studied its effects on the energetic metabolism and cell proliferation/survival of GB stem cells. The results obtained demonstrate that tumor neurospheres are metabolically reprogrammed up-regulating glucose transporter, glucose uptake and glycogen and lipid storage, mainly under hypoxic culture conditions. Treatment with the PPARα antagonist GW6471 resulted in decreased cell proliferation and neurospheres formation. Therefore, PPARα antagonism arises as a potent new strategy as adjuvant to gold standard therapies for GB for counteracting recurrences and opening the way for pre-clinical trials for this class of compounds. When tumor neurospheres were grown in hypoxic conditions in the presence of different glucose concentrations, the most diluted one (0.25g/L) mimicking the real concentration present in the neurosphere core, PPARα increase/PPARγ decrease, increased proliferation and cholesterol content, decreased glycogen particles and LDs were observed. All these responses were reverted by the 72 h treatment with the PPARα antagonist.

The PPARβ/δ Agonist GW0742 Induces Early Neuronal Maturation of Cortical Post-Mitotic Neurons: Role of PPARβ/δ in Neuronal Maturation

Increasing evidences support that signaling lipids participate in synaptic plasticity and cell survival, and that the lipid signaling is closely associated with neuronal differentiation, learning, and memory and with pathologic events, such as epilepsy and Alzheimers disease. The Peroxisome Proliferator‐Activated Receptors (PPAR) are strongly involved in the fatty acid cell signaling, as many of the natural lypophylic compounds are PPAR ligands. We have previously shown that PPARβ/δ is the main isotype present in cortical neuron primary cultures and that during neuronal maturation, PPARβ/δ is gradually increased and activated. To get more insight into the molecular mechanism by which PPARβ/δ may be involved in neuronal maturation processes, in this work a specific PPARβ/δ agonist, GW0742 was used administered alone or in association with a specific PPARβ/δ antagonist, the GSK0660, and the parameters involved in neuronal differentiation and maturation were assayed. The data obtained demonstrated the strong involvement of PPARβ/δ in neuronal maturation, triggering the agonist an anticipation of neuronal differentiation, and the antagonist abolishing the observed effects. These effects appear to be mediated by the activation of BDNF pathway. J. Cell. Physiol. 231: 597–606, 2016.