Identification of the hydantoin alkaloids parazoanthines as novel CXCR4 antagonists by computational and in vitro functional characterization.

CXCR4 chemokine receptor represents an attractive pharmacological target due to its key role in cancer metastasis and inflammatory diseases. Starting from our previously-developed pharmacophoric model, we applied a combined computational and experimental approach that led to the identification of the hydantoin alkaloids parazoanthines, isolated from the Mediterranean Sea anemone Parazoanthus axinellae, as novel CXCR4 antagonists. Parazoanthine analogues were then synthesized to evaluate the contribution of functional groups to the overall activity. Within the panel of synthesized natural and non-natural parazoanthines, parazoanthine-B was identified as the most potent CXCR4 antagonist with an IC50 value of 9.3 nM, even though all the investigated compounds were able to antagonize in vitro the down-stream effects of CXC12, albeit with variable potency and efficacy. The results of our study strongly support this class of small molecules as potent CXCR4 antagonists in tumoral pathologies characterized by an overexpression of this receptor. Furthermore, their structure-activity relationships allowed the optimization of our pharmacophoric model, useful for large-scale in silico screening.

In vitro and in vivo characterization of stem-like cells from canine osteosarcoma and assessment of drug sensitivity.

Cancer stem cell (CSC) self-renewing and drug resistance cause treatment failure and tumor recurrence. Osteosarcoma is an aggressive bone tumor characterized by biological and molecular heterogeneity, possibly dependent on CSCs. CSC identification in osteosarcoma and their efficient targeting are still open questions. Spontaneous canine osteosarcoma shares clinical and biological features with the human tumors, representing a model for translational studies. We characterized three CSC-enriched canine osteosarcoma cultures. In serum-free conditions, these CSC cultures grow as anchorage-independent spheroids, show mesenchymal-like properties and in vivo tumorigenicity, recapitulating the heterogeneity of the original osteosarcoma. Osteosarcoma CSCs express stem-related factors (Sox2, Oct4, CD133) and chemokine receptors and ligands (CXCR4, CXCL12) involved in tumor proliferation and self-renewal. Standard drugs for osteosarcoma treatment (doxorubicin and cisplatin) affected CSC-enriched and parental primary cultures, showing different efficacy within tumors. Moreover, metformin, a type-2 diabetes drug, significantly inhibits osteosarcoma CSC viability, migration and self-renewal and, in co-treatment with doxorubicin and cisplatin, enhances drug cytotoxicity. Collectively, we demonstrate that canine osteosarcoma primary cultures contain CSCs exhibiting distinctive sensitivity to anticancer agents, as a reliable experimental model to assay drug efficacy. We also provide proof-of-principle of metformin efficacy, alone or in combination, as pharmacological strategy to target osteosarcoma CSCs.

Autophagy Activator Drugs: A New Opportunity in Neuroprotection from Misfolded Protein Toxicity.

The aim of this review is to critically analyze promises and limitations of pharmacological inducers of autophagy against protein misfolding-associated neurodegeneration. Effective therapies against neurodegenerative disorders can be developed by regulating the "self-defense" equipment of neurons, such as autophagy. Through the degradation and recycling of the intracellular content, autophagy promotes neuron survival in conditions of trophic factor deprivation, oxidative stress, mitochondrial and lysosomal damage, or accumulation of misfolded proteins. Autophagy involves the activation of self-digestive pathways, which is different for dynamics (macro, micro and chaperone-mediated autophagy), or degraded material (mitophagy, lysophagy, aggrephagy). All neurodegenerative disorders share common pathogenic mechanisms, including the impairment of autophagic flux, which causes the inability to remove the neurotoxic oligomers of misfolded proteins. Pharmacological activation of autophagy is typically achieved by blocking the kinase activity of mammalian target of rapamycin (mTOR) enzymatic complex 1 (mTORC1), removing its autophagy suppressor activity observed under physiological conditions; acting in this way, rapamycin provided the first proof of principle that pharmacological autophagy enhancement can induce neuroprotection through the facilitation of oligomers' clearance. The demand for effective disease-modifying strategies against neurodegenerative disorders is currently stimulating the development of a wide number of novel molecules, as well as the re-evaluation of old drugs for their pro-autophagic potential.

Biological and Biochemical Basis of the Differential Efficacy of First and Second Generation Somatostatin Receptor Ligands in Neuroendocrine Neoplasms.

Endogenous somatostatin shows anti-secretory effects in both physiological and pathological settings, as well as inhibitory activity on cell growth. Since somatostatin is not suitable for clinical practice, researchers developed synthetic somatostatin receptor ligands (SRLs) to overcome this limitation. Currently, SRLs represent pivotal tools in the treatment algorithm of neuroendocrine tumors (NETs). Octreotide and lanreotide are the first-generation SRLs developed and show a preferential binding affinity to somatostatin receptor (SST) subtype 2, while pasireotide, which is a second-generation SRL, has high affinity for multiple SSTs (SST5 > SST2 > SST3 > SST1). A number of studies demonstrated that first-generation and second-generation SRLs show distinct functional properties, besides the mere receptor affinity. Therefore, the aim of the present review is to critically review the current evidence on the biological effects of SRLs in pituitary adenomas and neuroendocrine tumors, by mainly focusing on the differences between first-generation and second-generation ligands.

Novel celecoxib analogues inhibit glial production of prostaglandin E2, nitric oxide, and oxygen radicals reverting the neuroinflammatory responses induced by misfolded prion protein fragment 90-231 or lipopolysaccharide.

We tested the efficacy of novel cyclooxygenase 2 (COX-2) inhibitors in counteracting glia-driven neuroinflammation induced by the amyloidogenic prion protein fragment PrP90-231 or lipopolysaccharide (LPS). In search for molecules with higher efficacy than celecoxib, we focused our study on its 2,3-diaryl-1,3-thiazolidin-4-one analogues. As experimental models, we used the immortalized microglial cell line N9, rat purified microglial primary cultures, and mixed cultures of astrocytes and microglia. Microglia activation in response to PrP90-231 or LPS was characterized by growth arrest, morphology changes and the production of reactive oxygen species (ROS). Moreover, PrP90-231 treatment caused the overexpression of the inducible nitric oxide synthase (iNOS) and COX-2, with the consequent nitric oxide (NO), and prostaglandin E2 (PGE2) accumulation. These effects were challenged by different celecoxib analogues, among which Q22 (3-[4-(sulfamoyl)phenyl]-2-(4-tolyl)thiazolidin-4-one) inhibited microglia activation more efficiently than celecoxib, lowering both iNOS and COX-2 activity and reducing ROS release. During neurodegenerative diseases, neuroinflammation induced by amyloidogenic peptides causes the activation of both astrocytes and microglia with these cell populations mutually regulating each other. Thus the effects of PrP90-231 and LPS were also studied on mixed glial cultures containing astrocytes and microglia. PrP90-231 treatment elicited different responses in the co-cultures induced astrocyte proliferation and microglia growth arrest, resulting in a differential ability to release proinflammatory molecules with the production of NO and ROS mainly attributable on microglia, while COX-2 expression was induced also in astrocytes. Q22 effects on both NO and PGE2 secretion were more significant in the mixed glial cultures than in purified microglia, demonstrating Q22 ability to revert the functional interaction between astrocytes and microglia. These results demonstrate that Q22 is a powerful drug able to revert glial neuroinflammatory responses and might represent a lead to explore the chemical space around celecoxib frameworks to design even more effective agents, paving the way to novel approaches to contrast the neuroinflammation-dependent toxicity.

Canine osteosarcoma cell lines contain stem-like cancer cells: biological and pharmacological characterization.

Cancer stem cells (CSCs) represent a small subpopulation of cells responsible for tumor formation and progression, drug resistance, tumor recurrence and metastasization. CSCs have been identified in many human tumors including osteosarcoma (OSA). CSC distinctive properties are the expression of stem cell markers, sustained growth, self-renewal and tumorigenicity. Here we report the isolation of stem-like cells from two canine OSA cultures, characterized by self-renewal, evaluated by sphere formation ability, differential marker expression, and in vitro proliferation when cultured in a medium containing EGF and bFGF. Current therapies for OSA increased survival time, but prognosis remains poor, due to the development of drug resistance and metastases. Chemotherapy shrinks the tumor mass but CSCs remain unaffected, leading to tumor recurrence. Metformin, a drug for type 2 diabetes, has been shown to possess antitumor properties affecting CSC survival in different human and animal cancers. Here we show that metformin has a significant antiproliferative effect on canine OSA stem-like cells, validating this in vitro model for further pre-clinical drug evaluations. In conclusion, our results demonstrate the feasibility of obtaining CSC-enriched cultures from primary canine OSA cells as a promising model for biological and pharmacological studies of canine and human OSAs.

In vitro and in vivo antiproliferative activity of metformin on stem-like cells isolated from spontaneous canine mammary carcinomas: translational implications for human tumors.

BACKGROUND: Cancer stem cells (CSCs) are considered the cell subpopulation responsible for breast cancer (BC) initiation, growth, and relapse. CSCs are identified as self-renewing and tumor-initiating cells, conferring resistance to chemo- and radio-therapy to several neoplasias. Nowadays, th (about 10mM)e pharmacological targeting of CSCs is considered an ineludible therapeutic goal. The antidiabetic drug metformin was reported to suppress in vitro and in vivo CSC survival in different tumors and, in particular, in BC preclinical models. However, few studies are available on primary CSC cultures derived from human postsurgical BC samples, likely because of the limited amount of tissue available after surgery. In this context, comparative oncology is acquiring a relevant role in cancer research, allowing the analysis of larger samples from spontaneous pet tumors that represent optimal models for human cancer. METHODS: Isolation of primary canine mammary carcinoma (CMC) cells and enrichment in stem-like cell was carried out from fresh tumor specimens by culturing cells in stem-permissive conditions. Phenotypic and functional characterization of CMC-derived stem cells was performed in vitro, by assessment of self-renewal, long-lasting proliferation, marker expression, and drug sensitivity, and in vivo, by tumorigenicity experiments. Corresponding cultures of differentiated CMC cells were used as internal reference. Metformin efficacy on CMC stem cell viability was analyzed both in vitro and in vivo. RESULTS: We identified a subpopulation of CMC cells showing human breast CSC features, including expression of specific markers (i.e. CD44, CXCR4), growth as mammospheres, and tumor-initiation in mice. These cells show resistance to doxorubicin but were highly sensitive to metformin in vitro. Finally, in vivo metformin administration significantly impaired CMC growth in NOD-SCID mice, associated with a significant depletion of CSCs. CONCLUSIONS: Similarly to the human counterpart, CMCs contain stem-like subpopulations representing, in a comparative oncology context, a valuable translational model for human BC, and, in particular, to predict the efficacy of antitumor drugs. Moreover, metformin represents a potential CSC-selective drug for BC, as effective (neo-)adjuvant therapy to eradicate CSC in mammary carcinomas of humans and animals.

Isolation of stem-like cells from spontaneous feline mammary carcinomas: phenotypic characterization and tumorigenic potential.

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-α and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs.

Development and validation of a GC-MS method for determination of metformin in normal brain and in glioblastoma tissues.

Metformin hydrochloride (MH) has recently been repurposed as an anticancer agent, showing antiproliferative activity in vitro and in vivo. In particular, experimental evidence has suggested its potential clinical efficacy in glioblastoma (GBM), a very aggressive tumor frequently characterized by gloomy prognosis. Unfortunately, the published literature concerning experimental applications of MH in glioblastoma animal models report no data on metformin levels reached in the brain, which, considering the high hydrophilicity of the drug, are likely very low. Therefore, new sensitive analytical methods to be applied on biological tissues are necessary to improve our knowledge of MH in vivo biodistribution and biological effects on tumors. In this research work, a GC-MS method for MH quantification in brain tissues is proposed. MH has been derivatized using N-methyl-bis(trifluoroacetamide), as already described in the literature, but the derivatization conditions have been optimized; moreover, deuterated MH has been selected as the best internal standard, after a comparative evaluation including other internal standards employed in published methods. After ascertaining method linearity, its accuracy, precision, specificity, repeatability, LOD and LOQ (0.373 µM and 1.242 µM, respectively, corresponding to 0.887 and 2.958 pmol/mg of wet tissue) have been evaluated on mouse brain tissue samples, obtained through a straightforward preparation procedure involving methanolic extraction from lyophilized brain homogenates and solid phase purification. The method has been validated on brain samples obtained from mice, either healthy or xenografted with GBM cells, receiving metformin dissolved in the drinking water. This analytical method can be usefully applied in preclinical studies aiming at clarifying MH mechanism of action in brain tumors.

Stem-like signatures in human meningioma cells are under the control of CXCL11/CXCL12 chemokine activity.

BACKGROUND: Meningiomas are mainly benign brain tumors, although about 20% of histologically benign cases are clinically aggressive and recur after resection. We hypothesize that meningioma brain invasiveness and recurrence may be related to the presence of cancer stem cells and their high responsiveness to the CXCL12-CXCR4/CXCR7 chemokine axis. The aim of this study was to isolate meningioma stem cells from human samples, characterize them for biological features related to malignant behavior, and to identify the role of CXCR4/CXCR7 in these processes. METHODS: Meningioma stem cells were isolated from patient-derived primary cultures in stem cell-permissive conditions, and characterized for phenotype, self-renewal, proliferation and migration rates, vasculogenic mimicry (VM), and in vivo tumorigenesis, in comparison with differentiated meningioma cells and stem-like cells isolated from normal meninges. These cell populations were challenged with CXCL12 and CXCL11 and receptor antagonists to define the chemokine role in stem cell-related functions. RESULTS: Stem-like cells isolated from meningioma cultures display higher proliferation and migration rates, and VM, as compared to meningioma non-stem cells or cells isolated from normal meninges and were the only tumorigenic population in vivo. In meningioma cells, these stem-like functions were under the control of the CXCR4/CXCR7 chemokine axis. CONCLUSIONS: We report a role for CXCL11 and CXCL12 in the control of malignant features in stem-like cells isolated from human meningioma, providing a possible basis for the aggressive clinical behavior observed in subsets of these tumors. CXCR4/CXCR7 antagonists might represent a useful approach for meningioma at high risk of recurrence and malignant progression.

In vitro study of uptake and synthesis of creatine and its precursors by cerebellar granule cells and astrocytes suggests some hypotheses on the physiopathology of the inherited disorders of creatine metabolism.

BACKGROUND: The discovery of the inherited disorders of creatine (Cr) synthesis and transport in the last few years disclosed the importance of blood Cr supply for the normal functioning of the brain. These putatively rare diseases share a common pathogenetic mechanism (the depletion of brain Cr) and similar phenotypes characterized by mental retardation, language disturbances, seizures and movement disorders. In the effort to improve our knowledge on the mechanisms regulating Cr pool inside the nervous tissue, Cr transport and synthesis and related gene transcripts were explored in primary cultures of rat cerebellar granule cells and astrocytes. METHODS: Cr uptake and synthesis were explored in vitro by incubating monotypic primary cultures of rat type I astrocytes and cerebellar granule cells with: a) D3-Creatine (D3Cr) and D3Cr plus ß-guanidinopropionate (GPA, an inhibitor of Cr transporter), and b) labelled precursors of Guanidinoacetate (GAA) and Cr (Arginine, Arg; Glycine, Gly). Intracellular D3Cr and labelled GAA and Cr were assessed by ESI-MS/MS. Creatine transporter (CT1), L-arginine:glycine amidinotransferase (AGAT), and S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT) gene expression was assessed in the same cells by real time PCR. RESULTS: D3Cr signal was extremely high in cells incubated with this isotope (labelled/unlabelled Cr ratio reached about 10 and 122, respectively in cerebellar granule cells and astrocytes) and was reduced by GPA. Labelled Arg and Gly were taken up by the cells and incorporated in GAA, whose concentration paralleled that of these precursors both in the extracellular medium and inside the cells (astrocytes). In contrast, the increase of labelled Cr was relatively much more limited since labelled Cr after precursors' supplementation did not exceed 2,7% (cerebellar granule cells) and 21% (astrocytes) of unlabelled Cr. Finally, AGAT, GAMT and SLC6A8 were expressed in both kind of cells. CONCLUSIONS: Our results confirm that both neurons and astrocytes have the capability to synthesize and uptake Cr, and suggest that at least in vitro intracellular Cr can increase to a much greater extent through uptake than through de novo synthesis. Our results are compatible with the clinical observations that when the Cr transporter is defective, intracellular Cr is absent despite the brain should be able to synthesize it. Further research is needed to fully understand to what extent our results reflect the in vivo situation.

CXCR4 expression in feline mammary carcinoma cells: evidence of a proliferative role for the SDF-1/CXCR4 axis.

BACKGROUND: Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation. RESULTS: A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4 immunoreactivity. CXCR4 score was statistically significantly associated with the histological features of the samples, showing an increase accordingly with the degree of neoplastic transformation (from normal tissue to metastatic lesions). Finally, in the primary cultures obtained from 6 primary feline mammary carcinomas CXCR4 expression was detected in all cells and its activation by SDF-1 in vitro treatment caused a significant increase in the proliferation rate in 5 out of 6 tumours. CONCLUSIONS: These results indicate that malignant feline mammary tumours commonly express CXCR4, with a higher level in malignant tumours, and, in most of the cases analysed, metastatic cells display stronger immunoreactivity for CXCR4 than the corresponding primary tumours. Moreover, CXCR4 activation in primary cultures of feline mammary carcinomas causes increase in the proliferative rate. Thus, SDF-1/CXCR4 system seems to play a tumorigenic in feline mammary gland malignancy and in vitro cultures from these tumour samples may represent an experimental model to investigate the biological and pharmacological role of this chemokinergic axis.

Role of prion protein aggregation in neurotoxicity.

In several neurodegenerative diseases, such as Parkinson, Alzheimer's, Huntington, and prion diseases, the deposition of aggregated misfolded proteins is believed to be responsible for the neurotoxicity that characterizes these diseases. Prion protein (PrP), the protein responsible of prion diseases, has been deeply studied for the peculiar feature of its misfolded oligomers that are able to propagate within affected brains, inducing the conversion of the natively folded PrP into the pathological conformation. In this review, we summarize the available experimental evidence concerning the relationship between aggregation status of misfolded PrP and neuronal death in the course of prion diseases. In particular, we describe the main findings resulting from the use of different synthetic (mainly PrP106-126) and recombinant PrP-derived peptides, as far as mechanisms of aggregation and amyloid formation, and how these different spatial conformations can affect neuronal death. In particular, most data support the involvement of non-fibrillar oligomers rather than actual amyloid fibers as the determinant of neuronal death.

Proteostasis unbalance in prion diseases: Mechanisms of neurodegeneration and therapeutic targets.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are progressive neurodegenerative disorders of the central nervous system that affect humans and animals as sporadic, inherited, and infectious forms. Similarly to Alzheimer's disease and other neurodegenerative disorders, any attempt to reduce TSEs' lethality or increase the life expectancy of affected individuals has been unsuccessful. Typically, the onset of symptoms anticipates the fatal outcome of less than 1 year, although it is believed to be the consequence of a decades-long process of neuronal death. The duration of the symptoms-free period represents by itself a major obstacle to carry out effective neuroprotective therapies. Prions, the infectious entities of TSEs, are composed of a protease-resistant protein named prion protein scrapie (PrPSc) from the prototypical TSE form that afflicts ovines. PrPSc misfolding from its physiological counterpart, cellular prion protein (PrPC), is the unifying pathogenic trait of all TSEs. PrPSc is resistant to intracellular turnover and undergoes amyloid-like fibrillation passing through the formation of soluble dimers and oligomers, which are likely the effective neurotoxic entities. The failure of PrPSc removal is a key pathogenic event that defines TSEs as proteopathies, likewise other neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's disease, characterized by alteration of proteostasis. Under physiological conditions, protein quality control, led by the ubiquitin-proteasome system, and macroautophagy clears cytoplasm from improperly folded, redundant, or aggregation-prone proteins. There is evidence that both of these crucial homeostatic pathways are impaired during the development of TSEs, although it is still unclear whether proteostasis alteration facilitates prion protein misfolding or, rather, PrPSc protease resistance hampers cytoplasmic protein quality control. This review is aimed to critically analyze the most recent advancements in the cause-effect correlation between PrPC misfolding and proteostasis alterations and to discuss the possibility that pharmacological restoring of ubiquitin-proteasomal competence and stimulation of autophagy could reduce the intracellular burden of PrPSc and ameliorate the severity of prion-associated neurodegeneration.

Chloride intracellular channel 1 activity is not required for glioblastoma development but its inhibition dictates glioma stem cell responsivity to novel biguanide derivatives.

BACKGROUND: Chloride intracellular channel-1 (CLIC1) activity controls glioblastoma proliferation. Metformin exerts antitumor effects in glioblastoma stem cells (GSCs) inhibiting CLIC1 activity, but its low potency hampers its translation in clinical settings. METHODS: We synthesized a small library of novel biguanide-based compounds that were tested as antiproliferative agents for GSCs derived from human glioblastomas, in vitro using 2D and 3D cultures and in vivo in the zebrafish model. Compounds were compared to metformin for both potency and efficacy in the inhibition of GSC proliferation in vitro (MTT, Trypan blue exclusion assays, and EdU labeling) and in vivo (zebrafish model), migration (Boyden chamber assay), invasiveness (Matrigel invasion assay), self-renewal (spherogenesis assay), and CLIC1 activity (electrophysiology recordings), as well as for the absence of off-target toxicity (effects on normal stem cells and toxicity for zebrafish and chick embryos). RESULTS: We identified Q48 and Q54 as two novel CLIC1 blockers, characterized by higher antiproliferative potency than metformin in vitro, in both GSC 2D cultures and 3D spheroids. Q48 and Q54 also impaired GSC self-renewal, migration and invasion, and displayed low systemic in vivo toxicity. Q54 reduced in vivo proliferation of GSCs xenotransplanted in zebrafish hindbrain. Target specificity was confirmed by recombinant CLIC1 binding experiments using microscale thermophoresis approach. Finally, we characterized GSCs from GBMs spontaneously expressing low CLIC1 protein, demonstrating their ability to grow in vivo and to retain stem-like phenotype and functional features in vitro. In these GSCs, Q48 and Q54 displayed reduced potency and efficacy as antiproliferative agents as compared to high CLIC1-expressing tumors. However, in 3D cultures, metformin and Q48 (but not Q54) inhibited proliferation, which was dependent on the inhibition dihydrofolate reductase activity. CONCLUSIONS: These data highlight that, while CLIC1 is dispensable for the development of a subset of glioblastomas, it acts as a booster of proliferation in the majority of these tumors and its functional expression is required for biguanide antitumor class-effects. In particular, the biguanide-based derivatives Q48 and Q54, represent the leads to develop novel compounds endowed with better pharmacological profiles than metformin, to act as CLIC1-blockers for the treatment of CLIC1-expressing glioblastomas, in a precision medicine approach.

Experimental Evidence and Clinical Implications of Pituitary Adenoma Stem Cells.

Pituitary adenomas, accounting for 15% of diagnosed intracranial neoplasms, are usually benign and pharmacologically and surgically treatable; however, the critical location, mass effects and hormone hypersecretion sustain their significant morbidity. Approximately 35% of pituitary tumors show a less benign course since they are highly proliferative and invasive, poorly resectable, and likely recurring. The latest WHO classification of pituitary tumors includes pituitary transcription factor assessment to determine adenohypophysis cell lineages and accurate designation of adenomas, nevertheless little is known about molecular and cellular pathways which contribute to pituitary tumorigenesis. In malignant tumors the identification of cancer stem cells radically changed the concepts of both tumorigenesis and pharmacological approaches. Cancer stem cells are defined as a subset of undifferentiated transformed cells from which the bulk of cancer cells populating a tumor mass is generated. These cells are able to self-renew, promoting tumor progression and recurrence of malignant tumors, also conferring cytotoxic drug resistance. On the other hand, the existence of stem cells within benign tumors is still debated. The presence of adult stem cells in human and murine pituitaries where they sustain the high plasticity of hormone-producing cells, allowed the hypothesis that putative tumor stem cells might exist in pituitary adenomas, reinforcing the concept that the cancer stem cell model could also be applied to pituitary tumorigenesis. In the last few years, the isolation and phenotypic characterization of putative pituitary adenoma stem-like cells was performed using a wide and heterogeneous variety of experimental models and techniques, although the role of these cells in adenoma initiation and progression is still not completely definite. The assessment of possible pituitary adenoma-initiating cell population would be of extreme relevance to better understand pituitary tumor biology and to identify novel potential diagnostic markers and pharmacological targets. In this review, we summarize the most updated studies focused on the definition of pituitary adenoma stem cell phenotype and functional features, highlighting the biological processes and intracellular pathways potentially involved in driving tumor growth, relapse, and therapy resistance.

Cross talk between mesenchymal and glioblastoma stem cells: Communication beyond controversies.

Mesenchymal stem cells (MSCs) can be isolated from bone marrow or other adult tissues (adipose tissue, dental pulp, amniotic fluid, and umbilical cord). In vitro, MSCs grow as adherent cells, display fibroblast-like morphology, and self-renew, undergoing specific mesodermal differentiation. High heterogeneity of MSCs from different origin, and differences in preparation techniques, make difficult to uniform their functional properties for therapeutic purposes. Immunomodulatory, migratory, and differentiation ability, fueled clinical MSC application in regenerative medicine, whereas beneficial effects are currently mainly ascribed to their secretome and extracellular vesicles. MSC translational potential in cancer therapy exploits putative anti-tumor activity and inherent tropism toward tumor sites to deliver cytotoxic drugs. However, controversial results emerged evaluating either the therapeutic potential or homing efficiency of MSCs, as both antitumor and protumor effects were reported. Glioblastoma (GBM) is the most malignant brain tumor and its development and aggressive nature is sustained by cancer stem cells (CSCs) and the identification of effective therapeutic is required. MSC dualistic action, tumor-promoting or tumor-targeting, is dependent on secreted factors and extracellular vesicles driving a complex cross talk between MSCs and GBM CSCs. Tumor-tropic ability of MSCs, besides providing an alternative therapeutic approach, could represent a tool to understand the biology of GBM CSCs and related paracrine mechanisms, underpinning MSC-GBM interactions. In this review, recent findings on the complex nature of MSCs will be highlighted, focusing on their elusive impact on GBM progression and aggressiveness by direct cell-cell interaction and via secretome, also facing the perspectives and challenges in treatment strategies.

MCM2 and Carbonic Anhydrase 9 Are Novel Potential Targets for Neuroblastoma Pharmacological Treatment.

To overcome the lack of effective pharmacological treatments for high-risk neuroblastoma (HR-NB), the development of novel in vitro and in vivo models that better recapitulate the disease is required. Here, we used an in vitro multiclonal cell model encompassing NB cell differentiation stages, to identify potential novel pharmacological targets. This model allowed us to identify, by low-density RT-PCR arrays, two gene sets, one over-expressed during NB cell differentiation, and the other up-regulated in more malignant cells. Challenging two HR-NB gene expression datasets, we found that these two gene sets are related to high and low survival, respectively. Using mouse NB cisplatin-treated xenografts, we identified two genes within the list associated to the malignant stage (MCM2 and carbonic anhydrase 9), whose expression is positively correlated with tumor growth. Thus, we tested their pharmacological targeting as potential therapeutic strategy. We measured mice survival and tumor growth rate after xenografts of human NB treated with cisplatin in the presence of MCM2/carbonic anhydrase 9 inhibitors (ciprofloxacin and acetazolamide). MCM2 or carbonic anhydrase 9 inhibition significantly increased cisplatin activity, supporting their possible testing for NB therapy.

Emerging Role of Cellular Prion Protein in the Maintenance and Expansion of Glioma Stem Cells.

Cellular prion protein (PrPC) is a membrane-anchored glycoprotein representing the physiological counterpart of PrP scrapie (PrPSc), which plays a pathogenetic role in prion diseases. Relatively little information is however available about physiological role of PrPC. Although PrPC ablation in mice does not induce lethal phenotypes, impairment of neuronal and bone marrow plasticity was reported in embryos and adult animals. In neurons, PrPC stimulates neurite growth, prevents oxidative stress-dependent cell death, and favors antiapoptotic signaling. However, PrPC activity is not restricted to post-mitotic neurons, but promotes cell proliferation and migration during embryogenesis and tissue regeneration in adult. PrPC acts as scaffold to stabilize the binding between different membrane receptors, growth factors, and basement proteins, contributing to tumorigenesis. Indeed, ablation of PrPC expression reduces cancer cell proliferation and migration and restores cell sensitivity to chemotherapy. Conversely, PrPC overexpression in cancer stem cells (CSCs) from different tumors, including gliomas-the most malignant brain tumors-is predictive for poor prognosis, and correlates with relapses. The mechanisms of the PrPC role in tumorigenesis and its molecular partners in this activity are the topic of the present review, with a particular focus on PrPC contribution to glioma CSCs multipotency, invasiveness, and tumorigenicity.

Multiple biochemical similarities between infectious and non-infectious aggregates of a prion protein carrying an octapeptide insertion.

A nine-octapeptide insertion in the prion protein (PrP) gene is associated with an inherited form of human prion disease. Transgenic (Tg) mice that express the mouse homolog of this mutation (designated PG14) spontaneously accumulate in their brains an insoluble and weakly protease-resistant form of the mutant protein. This form (designated PG14(Spon)) is highly neurotoxic, but is not infectious in animal bioassays. In contrast, when Tg(PG14) mice are inoculated with the Rocky Mountain Laboratory (RML) strain of prions, they accumulate a different form of PG14 PrP (designated PG14(RML)) that is highly protease resistant and infectious in animal transmission experiments. We have been interested in characterizing the molecular properties of PG14(Spon) and PG14(RML), with a view to identifying features that determine two, apparently distinct properties of PrP aggregates: their infectivity and their pathogenicity. In this paper, we have subjected PG14(Spon) and PG14(RML) to a panel of assays commonly used to distinguish infectious PrP (PrP(Sc)) from cellular PrP (PrP(C)), including immobilized metal affinity chromatography, precipitation with sodium phosphotungstate, and immunoprecipitation with PrP(C)- and PrP(Sc)-specific antibodies. Surprisingly, we found that aggregates of PG14(Spon) and PG14(RML) behave identically to each other, and to authentic PrP(Sc), in each of these biochemical assays. PG14(Spon) however, in contrast to PG14(RML) and PrP(Sc), was unable to seed the misfolding of PrP(C) in an in vitro protein misfolding cyclic amplification reaction. Collectively, these results suggest that infectious and non-infectious aggregates of PrP share common structural features accounting for their toxicity, and that self-propagation of PrP involves more subtle molecular differences.