The VEGFs/VEGFRs system in Alzheimer's and Parkinson's diseases: Pathophysiological roles and therapeutic implications.

The vascular endothelial growth factors (VEGFs) and their cognate receptors (VEGFRs), besides their well-known involvement in physiological angiogenesis/lymphangiogenesis and in diseases associated to pathological vessel formation, play multifaceted functions in the central nervous system (CNS). In addition to shaping brain development, by controlling cerebral vasculogenesis and regulating neurogenesis as well as astrocyte differentiation, the VEGFs/VEGFRs axis exerts essential functions in the adult brain both in physiological and pathological contexts. In this article, after describing the physiological VEGFs/VEGFRs functions in the CNS, we focus on the VEGFs/VEGFRs involvement in neurodegenerative diseases by reviewing the current literature on the rather complex VEGFs/VEGFRs contribution to the pathogenic mechanisms of Alzheimer's (AD) and Parkinson's (PD) diseases. Thereafter, based on the outcome of VEGFs/VEGFRs targeting in animal models of AD and PD, we discuss the factual relevance of pharmacological VEGFs/VEGFRs modulation as a novel and potential disease-modifying approach for these neurodegenerative pathologies. Specific VEGFRs targeting, aimed at selective VEGFR-1 inhibition, while preserving VEGFR-2 signal transduction, appears as a promising strategy to hit the molecular mechanisms underlying AD pathology. Moreover, therapeutic VEGFs-based approaches can be proposed for PD treatment, with the aim of fine-tuning their brain levels to amplify neurotrophic/neuroprotective effects while limiting an excessive impact on vascular permeability.

Impact of Physical Exercise on Melanoma Hallmarks: Current Status of Preclinical and Clinical Research.

In recent years, accumulating evidence from preclinical and clinical studies consistently indicated that physical activity/exercise plays a crucial role in reducing the incidence and recurrence of various malignancies, by exerting a beneficial modulation of cancer hallmarks. Moreover, physical activity is suggested to attenuate certain adverse effects of anticancer therapy, including the reduction of cardiovascular toxicity and symptoms related to depression and anxiety, among others, while preserving muscular strength. In the case of melanoma, the relationship with physical activity has been critically debated. Historically, several cohort studies and meta-analyses reported a positive association between physical activity/exercise and melanoma risk. This association was primarily attributed to outdoor activities that may expose the skin to UV radiation, a well-known risk factor for melanocyte transformation. However, more recent evidence does not support such association and recognizes physical activity/exercise role in both melanoma prevention and progression. Nevertheless, sun protection is recommended during outdoor training to minimize UV radiation exposure. This narrative review summarizes preclinical and clinical data about physical activity effects on melanoma hallmarks. Specifically, experimental evidence is reported concerning (i) invasion and metastasis, (ii) reprogramming of energy metabolism, (iii) angiogenesis, (iv) resistance to cell death, (v) evasion from immune destruction, and (vi) tumor-promoting inflammation.

Novel frontiers in neuroprotective therapies in glaucoma: Molecular and clinical aspects.

In the last years, neuroprotective therapies have attracted the researcher interests as modern and challenging approach for the treatment of neurodegenerative diseases, aimed at protecting the nervous system from injuries. Glaucoma is a neurodegenerative disease characterized by progressive excavation of the optic nerve head, retinal axonal injury and corresponding vision loss that affects millions of people on a global scale. The molecular basis of the pathology is largely uncharacterized yet, and the therapeutic approaches available do not change the natural course of the disease. Therefore, in accordance with the therapeutic regimens proposed for other neurodegenerative diseases, a modern strategy to treat glaucoma includes prescription of drugs with neuroprotective activities. With respect to this, several preclinical and clinical investigations on a plethora of different drugs are currently ongoing. In this review, first, the conceptualization of the rationale for the adoption of neuroprotective strategies for retina is summarized. Second, the molecular aspects highlighting glaucoma as a neurodegenerative disease are reported. In conclusion, the molecular and pharmacological properties of most promising direct neuroprotective drugs used to delay glaucoma progression are examined, including: neurotrophic factors, NMDA receptor antagonists, the α2-adrenergic agonist, brimonidine, calcium channel blockers, antioxidant agents, nicotinamide and statins.

The Insulin-Degrading Enzyme from Structure to Allosteric Modulation: New Perspectives for Drug Design.

The insulin-degrading enzyme (IDE) is a Zn2+ peptidase originally discovered as the main enzyme involved in the degradation of insulin and other amyloidogenic peptides, such as the ß-amyloid (Aß) peptide. Therefore, a role for the IDE in the cure of diabetes and Alzheimer's disease (AD) has been long envisaged. Anyway, its role in degrading amyloidogenic proteins remains not clearly defined and, more recently, novel non-proteolytic functions of the IDE have been proposed. From a structural point of view, the IDE presents an atypical clamshell structure, underscoring unique enigmatic enzymological properties. A better understanding of the structure-function relationship may contribute to solving some existing paradoxes of IDE biology and, in light of its multifunctional activity, might lead to novel therapeutic approaches.

Targeting of PDGF-C/NRP-1 autocrine loop as a new strategy for counteracting the invasiveness of melanoma resistant to braf inhibitors.

Melanoma resistance to BRAF inhibitors (BRAFi) is often accompanied by a switch from a proliferative to an invasive phenotype. Therefore, the identification of signaling molecules involved in the development of metastatic properties by resistant melanoma cells is of primary importance. We have previously demonstrated that activation of neuropilin-1 (NRP-1) by platelet-derived growth factor (PDGF)-C confers melanoma cells with an invasive behavior similar to that of BRAFi resistant tumors. Aims of the present study were to evaluate the role of PDGF-C/NRP-1 autocrine loop in the acquisition of an invasive and BRAFi-resistant phenotype by melanoma cells and the effect of its inhibition on drug resistance and extracellular matrix (ECM) invasion. Furthermore, we investigated whether PDGF-C serum levels were differentially modulated by drug treatment in metastatic melanoma patients responsive or refractory to BRAFi as single agents or in combination with MEK inhibitors (MEKi). The results indicated that human melanoma cells resistant to BRAFi express higher levels of PDGF-C and NRP-1 as compared to their susceptible counterparts. Overexpression occurs early during development of drug resistance and contributes to the invasive properties of resistant cells. Accordingly, silencing of NRP-1 or PDGF-C reduces tumor cell invasiveness. Analysis of PDGF-C in the serum collected from patients treated with BRAFi or BRAFi+MEKi, showed that in responders PDGF-C levels decrease after treatment and raise again at tumor progression. Conversely, in non-responders treatment does not affect PDGF-C serum levels. Thus, blockade of NRP-1 activation by PDGF-C might represent a new therapeutic approach to counteract the invasiveness of BRAFi-resistant melanoma.

Physical Exercise and the Hallmarks of Breast Cancer: A Narrative Review.

Growing evidence suggests that, among the different molecular/cellular pathophysiological mechanisms associated with cancer, there are 14 hallmarks that play a major role, including: (i) sustaining proliferative signaling, (ii) evading growth suppressors, (iii) activating invasion and metastasis, (iv) enabling replicative immortality, (v) inducing angiogenesis, (vi) resisting cell death, (vii) reprogramming energy metabolism, (viii) evading immune destruction, (ix) genome instability and mutations, (x) tumor-promoting inflammation, (xi) unlocking phenotypic plasticity, (xii) nonmutational epigenetic reprogramming, (xiii) polymorphic microbiomes, and (xiv) senescent cells. These hallmarks are also associated with the development of breast cancer, which represents the most prevalent tumor type in the world. The present narrative review aims to describe, for the first time, the effects of physical activity/exercise on these hallmarks. In summary, an active lifestyle, and particularly regular physical exercise, provides beneficial effects on all major hallmarks associated with breast cancer, and might therefore help to counteract the progression of the disease or its associated burden.

Targeting immunoproteasome in neurodegeneration: A glance to the future.

The immunoproteasome is a specialized form of proteasome equipped with modified catalytic subunits that was initially discovered to play a pivotal role in MHC class I antigen processing and immune system modulation. However, over the last years, this proteolytic complex has been uncovered to serve additional functions unrelated to antigen presentation. Accordingly, it has been proposed that immunoproteasome synergizes with canonical proteasome in different cell types of the nervous system, regulating neurotransmission, metabolic pathways and adaptation of the cells to redox or inflammatory insults. Hence, studying the alterations of immunoproteasome expression and activity is gaining research interest to define the dynamics of neuroinflammation as well as the early and late molecular events that are likely involved in the pathogenesis of a variety of neurological disorders. Furthermore, these novel functions foster the perspective of immunoproteasome as a potential therapeutic target for neurodegeneration. In this review, we provide a brain and retina-wide overview, trying to correlate present knowledge on structure-function relationships of immunoproteasome with the variety of observed neuro-modulatory functions.

The Anti-Vascular Endothelial Growth Factor Receptor 1 (VEGFR-1) D16F7 Monoclonal Antibody Inhibits Melanoma Adhesion to Soluble VEGFR-1 and Tissue Invasion in Response to Placenta Growth Factor.

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family involved in tumor-associated angiogenesis and melanoma invasion of the extra-cellular matrix (ECM) through activation of membrane VEGF receptor 1 (VEGFR-1). A soluble VEGFR-1 (sVEGFR-1) form is released in the ECM, where it sequesters proangiogenic factors and stimulates endothelial or tumor cell adhesion and chemotaxis through interaction with α5ß1 integrin. The anti-VEGFR-1 monoclonal antibody (D16F7 mAb) inhibits VEGF-A or PlGF-mediated signal transduction without affecting ligand interaction, thus preserving sVEGFR-1 decoy function. The aim of this study was to investigate whether D16F7 mAb hampers melanoma spread by in vitro analysis of cell adhesion to sVEGFR-1, ECM invasion, transmigration through an endothelial cell monolayer and in vivo evaluation of tumor infiltrative potential in a syngeneic murine model. Results indicate that D16F7 mAb significantly inhibits melanoma adhesion to sVEGFR-1 and ECM invasion, as well as transmigration in response to PlGF. Moreover, treatment of melanoma-bearing mice with the anti-VEGFR-1 mAb not only inhibits tumor growth but also induces a significant reduction in bone infiltration associated with a decrease in PlGF-positive melanoma cells. Furthermore, D16F7 mAb reduces PlGF production by melanoma cells. Therefore, blockade of PLGF/VEGFR-1 signaling represents a suitable strategy to counteract the metastatic potential of melanoma.

Role of PARP Inhibitors in Cancer Immunotherapy: Potential Friends to Immune Activating Molecules and Foes to Immune Checkpoints.

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) induce cytotoxic effects as single agents in tumors characterized by defective repair of DNA double-strand breaks deriving from BRCA1/2 mutations or other abnormalities in genes associated with homologous recombination. Preclinical studies have shown that PARPi-induced DNA damage may affect the tumor immune microenvironment and immune-mediated anti-tumor response through several mechanisms. In particular, increased DNA damage has been shown to induce the activation of type I interferon pathway and up-regulation of PD-L1 expression in cancer cells, which can both enhance sensitivity to Immune Checkpoint Inhibitors (ICIs). Despite the recent approval of ICIs for a number of advanced cancer types based on their ability to reinvigorate T-cell-mediated antitumor immune responses, a consistent percentage of treated patients fail to respond, strongly encouraging the identification of combination therapies to overcome resistance. In the present review, we analyzed both established and unexplored mechanisms that may be elicited by PARPi, supporting immune reactivation and their potential synergism with currently used ICIs. This analysis may indicate novel and possibly patient-specific immune features that might represent new pharmacological targets of PARPi, potentially leading to the identification of predictive biomarkers of response to their combination with ICIs.

A novel and atypical NF-KB pro-inflammatory program regulated by a CamKII-proteasome axis is involved in the early activation of Muller glia by high glucose.

BACKGROUND: Diabetic retinopathy (DR) is a microvascular complication of diabetes with a heavy impact on the quality of life of subjects and with a dramatic burden for health and economic systems on a global scale. Although the pathogenesis of DR is largely unknown, several preclinical data have pointed out to a main role of Muller glia (MG), a cell type which spans across the retina layers providing nourishment and support for Retina Ganglion Cells (RGCs), in sensing hyper-glycemia and in acquiring a pro-inflammatory polarization in response to this insult. RESULTS: By using a validated experimental model of DR in vitro, rMC1 cells challenged with high glucose, we uncovered the induction of an early (within minutes) and atypical Nuclear Factor-kB (NF-kB) signalling pathway regulated by a calcium-dependent calmodulin kinase II (CamKII)-proteasome axis. Phosphorylation of proteasome subunit Rpt6 (at Serine 120) by CamKII stimulated the accelerated turnover of IkBα (i.e., the natural inhibitor of p65-50 transcription factor), regardless of the phosphorylation at Serine 32 which labels canonical NF-kB signalling. This event allowed the p65-p50 heterodimer to migrate into the nucleus and to induce transcription of IL-8, Il-1ß and MCP-1. Pharmacological inhibition of CamKII as well as proteasome inhibition stopped this pro-inflammatory program, whereas introduction of a Rpt6 phospho-dead mutant (Rpt6-S120A) stimulated a paradoxical effect on NF-kB probably through the activation of a compensatory mechanism which may involve phosphorylation of 20S α4 subunit. CONCLUSIONS: This study introduces a novel pathway of MG activation by high glucose and casts some light on the biological relevance of proteasome post-translational modifications in modulating pathways regulated through targeted proteolysis.

Monoclonal Antibodies to CTLA-4 with Focus on Ipilimumab.

The immune checkpoint cytotoxic T lymphocyte-associated antigen 4 (CTLA-4 or CD152) is a negative regulator of T-cell-mediated immune responses which plays a critical role in suppressing autoimmunity and maintaining immune homeostasis. Because of its inhibitory activity on T cells, CTLA-4 has been investigated as a drug target to induce immunostimulation, blocking the interaction with its ligands. The antitumor effects mediated by CTLA-4 blockade have been attributed to a sustained active immune response against cancer cells, due to the release of a brake on T cell activation. Ipilimumab (Yervoy, Bristol-Myers Squibb) is a fully human anti-CTLA-4 IgG1κ monoclonal antibody (mAb) that represents the first immune checkpoint inhibitor approved as monotherapy by FDA and EMA in 2011 for the treatment of unresectable/metastatic melanoma. In 2015, FDA also granted approval to ipilimumab monotherapy as adjuvant treatment of stage III melanoma to reduce the risk of tumour recurrence. The subsequent approved indications of ipilimumab for metastatic melanoma, regardless of BRAF mutational status, and other advanced/metastatic solid tumours always involve its use in association with the anti-programmed cell death protein 1 (PD-1) mAb nivolumab. Currently, ipilimumab is evaluated in ongoing clinical trials for refractory/advanced solid tumours mainly in combination with additional immunostimulating agents.

Insulin-Degrading Enzyme Is a Non Proteasomal Target of Carfilzomib and Affects the 20S Proteasome Inhibition by the Drug.

Carfilzomib is a last generation proteasome inhibitor (PI) with proven clinical efficacy in the treatment of relapsed/refractory multiple myeloma. This drug is considered to be extremely specific in inhibiting the chymotrypsin-like activity of the 20S proteasome, encoded by the ß5 subunit, overcoming some bortezomib limitations, the first PI approved for multiple myeloma therapy which is however burdened by a significant toxicity profile, due also to its off-target effects. Here, molecular approaches coupled with molecular docking studies have been used to unveil that the Insulin-Degrading Enzyme, a ubiquitous and highly conserved Zn2+ peptidase, often found to associate with proteasome in cell-based models, is targeted by carfilzomib in vitro. The drug behaves as a modulator of IDE activity, displaying an inhibitory effect over 10-fold lower than for the 20S. Notably, the interaction of IDE with the 20S enhances in vitro the inhibitory power of carfilzomib on proteasome, so that the IDE-20S complex is an even better target of carfilzomib than the 20S alone. Furthermore, IDE gene silencing after delivery of antisense oligonucleotides (siRNA) significantly reduced carfilzomib cytotoxicity in rMC1 cells, a validated model of Muller glia, suggesting that, in cells, the inhibitory activity of this drug on cell proliferation is somewhat linked to IDE and, possibly, also to its interaction with proteasome.

Antibody-drug conjugates: Resurgent anticancer agents with multi-targeted therapeutic potential.

Antibody-drug conjugates (ADCs) constitute a relatively new group of anticancer agents, whose first appearance took place about two decades ago, but a renewed interest occurred in recent years, following the success of anti-cancer immunotherapy with monoclonal antibodies. Indeed, an ADC combines the selectivity of a monoclonal antibody with the cell killing properties of a chemotherapeutic agent (payload), joined together through an appropriate linker. The antibody moiety targets a specific cell surface antigen expressed by tumor cells and/or cells of the tumor microenvironment and acts as a carrier that delivers the cytotoxic payload within the tumor mass. Despite advantages in terms of selectivity and potency, the development of ADCs is not devoid of challenges, due to: i) low tumor selectivity when the target antigens are not exclusively expressed by cancer cells; ii) premature release of the cytotoxic drug into the bloodstream as a consequence of linker instability; iii) development of tumor resistance mechanisms to the payload. All these factors may result in lack of efficacy and/or in no safety improvement compared to unconjugated cytotoxic agents. Nevertheless, the development of antibodies engineered to remain inert until activated in the tumor (e.g., antibodies activated proteolytically after internalization or by the acidic conditions of the tumor microenvironment) together with the discovery of innovative targets and cytotoxic or immunomodulatory payloads, have allowed the design of next-generation ADCs that are expected to possess improved therapeutic properties. This review provides an overview of approved ADCs, with related advantages and limitations, and of novel targets exploited by ADCs that are presently under clinical investigation.

Clinical experience with CTLA-4 blockade for cancer immunotherapy: From the monospecific monoclonal antibody ipilimumab to probodies and bispecific molecules targeting the tumor microenvironment.

The immune checkpoint cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is an inhibitory regulator of T-cell mediated responses that has been investigated as target of monoclonal antibodies (mAbs) for cancer immunotherapy. The anti-CTLA-4 mAb ipilimumab represents the first immune checkpoint inhibitor that significantly improved overall survival in patients with unresectable/metastatic melanoma. The subsequent approved indications (often in the first-line setting) for melanoma and other advanced/metastatic solid tumors always require ipilimumab combination with nivolumab, an anti-programmed cell death protein 1 (PD-1) mAb. However, the improved clinical efficacy of the mAb combination is associated with increased immune-related adverse events, which might require treatment discontinuation even in responding patients. This drawback is expected to be overcome by the recent development of anti-CTLA-4 probodies proteolitycally activated in the tumor microenvironment and bispecific molecules targeting both CTLA-4 and PD-1, whose co-expression is characteristic of tumor-infiltrating T cells. These molecules would preferentially stimulate immune responses against the tumor, reducing toxicity toward normal tissues.

At the Cutting Edge against Cancer: A Perspective on Immunoproteasome and Immune Checkpoints Modulation as a Potential Therapeutic Intervention.

Immunoproteasome is a noncanonical form of proteasome with enzymological properties optimized for the generation of antigenic peptides presented in complex with class I MHC molecules. This enzymatic property makes the modulation of its activity a promising area of research. Nevertheless, immunotherapy has emerged as a front-line treatment of advanced/metastatic tumors providing outstanding improvement of life expectancy, even though not all patients achieve a long-lasting clinical benefit. To enhance the efficacy of the currently available immunotherapies and enable the development of new strategies, a broader knowledge of the dynamics of antigen repertoire processing by cancer cells is needed. Therefore, a better understanding of the role of immunoproteasome in antigen processing and of the therapeutic implication of its modulation is mandatory. Studies on the potential crosstalk between proteasome modulators and immune checkpoint inhibitors could provide novel perspectives and an unexplored treatment option for a variety of cancers.

VEGF-A/VEGFR-1 signalling and chemotherapy-induced neuropathic pain: therapeutic potential of a novel anti-VEGFR-1 monoclonal antibody.

BACKGROUND: Neuropathic pain is a clinically relevant adverse effect of several anticancer drugs that markedly impairs patients' quality of life and frequently leads to dose reduction or therapy discontinuation. The poor knowledge about the mechanisms involved in neuropathy development and pain chronicization, and the lack of effective therapies, make treatment of chemotherapy-induced neuropathic pain an unmet medical need. In this context, the vascular endothelial growth factor A (VEGF-A) has emerged as a candidate neuropathy hallmark and its decrease has been related to pain relief. In the present study, we have investigated the role of VEGF-A and its receptors, VEGFR-1 and VEGFR-2, in pain signalling and in chemotherapy-induced neuropathy establishment as well as the therapeutic potential of receptor blockade in the management of pain. METHODS: Behavioural and electrophysiological analyses were performed in an in vivo murine model, by using selective receptor agonists, blocking monoclonal antibodies or siRNA-mediated silencing of VEGF-A and VEGFRs. Expression of VEGF-A and VEGFR-1 in astrocytes and neurons was detected by immunofluorescence staining and confocal microscopy analysis. RESULTS: In mice, the intrathecal infusion of VEGF-A (VEGF165 isoforms) induced a dose-dependent noxious hypersensitivity and this effect was mediated by VEGFR-1. Consistently, electrophysiological studies indicated that VEGF-A strongly stimulated the spinal nociceptive neurons activity through VEGFR-1. In the dorsal horn of the spinal cord of animals affected by oxaliplatin-induced neuropathy, VEGF-A expression was increased in astrocytes while VEGFR-1 was mainly detected in neurons, suggesting a VEGF-A/VEGFR-1-mediated astrocyte-neuron cross-talk in neuropathic pain pathophysiology. Accordingly, the selective knockdown of astrocytic VEGF-A by intraspinal injection of shRNAmir blocked the development of oxaliplatin-induced neuropathic hyperalgesia and allodynia. Interestingly, both intrathecal and systemic administration of the novel anti-VEGFR-1 monoclonal antibody D16F7, endowed with anti-angiogenic and antitumor properties, reverted oxaliplatin-induced neuropathic pain. Besides, D16F7 effectively relieved hypersensitivity induced by other neurotoxic chemotherapeutic agents, such as paclitaxel and vincristine. CONCLUSIONS: These data strongly support the role of the VEGF-A/VEGFR-1 system in mediating chemotherapy-induced neuropathic pain at the central nervous system level. Thus, treatment with the anti-VEGFR-1 mAb D16F7, besides exerting antitumor activity, might result in the additional advantage of attenuating neuropathic pain when combined with neurotoxic anticancer agents.

Saffron and Its Major Ingredients' Effect on Colon Cancer Cells with Mismatch Repair Deficiency and Microsatellite Instability.

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide. One of its subtypes is associated with defective mismatch repair (dMMR) genes. Saffron has many potentially protective roles against colon malignancy. However, these roles in the context of dMMR tumors have not been explored. In this study, we aimed to investigate the effects of saffron and its constituents in CRC cell lines with dMMR. METHODS: Saffron crude extracts and specific compounds (safranal and crocin) were used in the human colorectal cancer cell lines HCT116, HCT116+3 (inserted MLH1), HCT116+5 (inserted MSH3), and HCT116+3+5 (inserted MLH1 and MSH3). CDC25b, p-H2AX, TPDP1, and GAPDH were analyzed by Western blot. Proliferation and cytotoxicity were analyzed by MTT. The scratch wound assay was also performed. RESULTS: Saffron crude extracts restricted (up to 70%) the proliferation in colon cells with deficient MMR (HCT116) compared to proficient MMR. The wound healing assay indicates that deficient MMR cells are doing better (up to 90%) than proficient MMR cells when treated with saffron. CDC25b and TDP1 downregulated (up to 20-fold) in proficient MMR cells compared to deficient MMR cells, while p.H2AX was significantly upregulated in both cell types, particularly at >10 mg/mL saffron in a concentration-dependent manner. The reduction in cellular proliferation was accompanied with upregulation of caspase 3 and 7. The major active saffron compounds, safranal and crocin reproduced most of the saffron crude extracts' effects. CONCLUSIONS: Saffron's anti-proliferative effect is significant in cells with deficient MMR. This novel effect may have therapeutic implications and benefits for MSI CRC patients who are generally not recommended for the 5-fluorouracil-based treatment.

hTERT Transduction Extends the Lifespan of Primary Pediatric Low-Grade Glioma Cells While Preserving the Biological Response to NGF.

The neurotrophin nerve growth factor (NGF) modulates the growth of human gliomas and is able to induce cell differentiation through the engagement of tropomyosin receptor kinase A (TrkA) receptor, although the role played in controlling glioma survival has proved controversial. Unfortunately, the slow growth rate of low-grade gliomas (LGG) has made it difficult to investigate NGF effects on these tumors in preclinical models. In fact, patient-derived low-grade human astrocytoma cells duplicate only a limited number of times in culture before undergoing senescence. Nevertheless, replicative senescence can be counteracted by overexpression of hTERT, the catalytic subunit of telomerase, which potentially increases the proliferative potential of human cells without inducing cancer-associated changes. We have extended, by hTERT transduction, the proliferative in vitro potential of a human LGG cell line derived from a pediatric pilocytic astrocytoma (PA) surgical sample. Remarkably, the hTERT-transduced LGG cells showed a behavior similar to that of the parental line in terms of biological responses to NGF treatment, including molecular events associated with induction of NGF-related differentiation. Therefore, transduction of LGG cells with hTERT can provide a valid approach to increase the in vitro life-span of patient-derived astrocytoma primary cultures, characterized by a finite proliferative potential.

High-Dose Vitamin C: Preclinical Evidence for Tailoring Treatment in Cancer Patients.

High-dose vitamin C has been proposed as a potential therapeutic approach for patients with advanced tumors who failed previous treatment with chemotherapy. Due to vitamin C complex pharmacokinetics, only intravenous administration allows reaching sufficiently high plasma concentrations required for most of the antitumor effects observed in preclinical studies (>0.250 mM). Moreover, vitamin C entry into cells is tightly regulated by SVCT and GLUT transporters, and is cell type-dependent. Importantly, besides its well-recognized pro-oxidant effects, vitamin C modulates TET enzymes promoting DNA demethylation and acts as cofactor of HIF hydroxylases, whose activity is required for HIF-1α proteasomal degradation. Furthermore, at pharmacological concentrations lower than those required for its pro-oxidant activity (<1 mM), vitamin C in specific genetic contexts may alter the DNA damage response by increasing 5-hydroxymethylcytosine levels. These more recently described vitamin C mechanisms offer new treatment opportunities for tumors with specific molecular defects (e.g., HIF-1α over-expression or TET2, IDH1/2, and WT1 alterations). Moreover, vitamin C action at DNA levels may provide the rationale basis for combination therapies with PARP inhibitors and hypomethylating agents. This review outlines the pharmacokinetic and pharmacodynamic properties of vitamin C to be taken into account in designing clinical studies that evaluate its potential use as anticancer agent.