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.

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.

A step forward on site-specific environmental risk assessment and insight into the main influencing factors of CECs removal from wastewater.

The presence of Contaminants of Emerging Concern (CECs) in water systems has been recognized as a potential source of risk for human health and the ecosystem. The present paper aims at evaluating the effects of different characteristics of full-scale Wastewater Treatment Plants (WWTPs) on the removal of 14 selected CECs belonging to the classes of caffeine, illicit drugs and pharmaceuticals. Particularly, the investigated plants differed because of the treatment lay-out, the type of biological process, the value of the operating parameters, the fate of the treated effluent (i.e. release into surface water or reuse), and the treatment capacity. The activity consisted of measuring concentrations of the selected CECs and also traditional water quality parameters (i.e. COD, phosphorous, nitrogen species and TSS) in the influent and effluent of 8 plants. The study highlights that biodegradable CECs (cocaine, methamphetamine, amphetamine, benzoylecgonine, 11-nor-9carboxy-Δ9-THC, lincomycin, trimethoprim, sulfamethoxazole, sulfadiazine, sulfadimethoxine, carbamazepine, ketoprofen, warfarin and caffeine) were well removed by all the WWTPs, with the best performance achieved by the MBR for antibiotics. Carbamazepine was removed at the lowest extent by all the WWTPs. The environmental risk assessed by using the site-specific value of the dilution factor resulted to be high in 3 out of 8 WWTPs for carbamazepine and less frequently for caffeine. However, the risk was reduced when the dilution factor was assumed equal to the default value of 10 as proposed by EU guidelines. Therefore, a specific determination of this factor is needed taking into account the hydraulic characteristics of the receiving water body.

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.

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.

Impact of COVID19 restrictions on organic micropollutants in wastewater treatment plants and human consumption rates.

COVID19 pandemic and the consequent restrictions to constrain SARS-CoV-2 spreading produced several impacts on the worldwide population. The present study focused on 10 Organic Micropollutants (illicit drugs, pharmaceuticals including some antibiotics and caffeine) and aimed to assess: (1) if COVID19 pandemic restrictions affected the load of those contaminants released into the sewage network and consequently the removal achieved by the Wastewater Treatment Plants; (2) if pursuant to the COVID19 pandemic, there was a change in population consumption rates of the same compounds through the wastewater-based epidemiology (WBE) approach. Two full-scale wastewater treatment plants (WWTPs) located in Central Italy were chosen as case studies, which are distinguished by different characteristics of the catchment area and water treatment layouts. The study was based on a 2-years monitoring activity of the concentration of the above organic micropollutants, traditional water quality parameters (COD, TSS, nitrogen compounds, total phosphorous) and flow rate in the influent and effluent. The statistical analysis of the monitoring data showed an increase of the influent load of most of the organic micropollutants. A decrease from 22% to -18% of the median removal efficiency was observed for carbamazepine in the WWTP with the lower treatment capacity only. The other compounds were removed roughly at the same rate. The application of the WBE approach demonstrated an increase in the consumption rate of cocaine, trimethoprim, sulfamethoxazole, sulfadiazine, carbamazepine and above all caffeine during the COVID19 restrictions period. These results highlight that COVID19 pandemic affected people's lifestyle and habits also as far as drugs consumption is concerned, which in turn might have an impact on the treatment efficacy of plants and finally on the receiving water body quality. Therefore, it is mandatory to keep monitoring to improve knowledge and eventually to implement the required measures to address this new problem.

Targeting Tumor-Associated Macrophages to Increase the Efficacy of Immune Checkpoint Inhibitors: A Glimpse into Novel Therapeutic Approaches for Metastatic Melanoma.

Immune checkpoint inhibitors (ICIs) represent a promising therapeutic intervention for a variety of advanced/metastatic solid tumors, including melanoma, but in a large number of cases, patients fail to establish a sustained anti-tumor immunity and to achieve a long-lasting clinical benefit. Cells of the tumor micro-environment such as tumor-associated M2 macrophages (M2-TAMs) have been reported to limit the efficacy of immunotherapy, promoting tumor immune evasion and progression. Thus, strategies targeting M2-TAMs have been suggested to synergize with immune checkpoint blockade. This review recapitulates the molecular mechanisms by which M2-TAMs promote cancer immune evasion, with focus on the potential cross-talk between pharmacological interventions targeting M2-TAMs and ICIs for melanoma treatment.

Strategies to improve ellagic acid bioavailability: from natural or semisynthetic derivatives to nanotechnological approaches based on innovative carriers.

Ellagic acid (EA) is a polyphenolic compound whose dietary consumption is mainly associated with the intake of red fruits, including pomegranates, strawberries, blackberries, blackcurrants, raspberries, grapes or dried fruits, like walnuts and almonds. A number of studies indicate that EA exerts health-beneficial effects against several chronic pathologies associated with oxidative damage, including different kinds of cancer, cardiovascular and neurodegenerative diseases. Furthermore, EA possesses wound-healing properties, antibacterial and antiviral effects, and acts as a systemic antioxidant. However, clinical applications of this polyphenol have been hampered and prevented by its poor water solubility (9.7 ± 3.2 µg ml-1 in water) and pharmacokinetic profile (limited absorption rate and plasma half-life <1 h after ingestion of pomegranate juice), properties due to the chemical nature of the organic heterotetracyclic compound. Little has been reported on efficient strategies to enhance EA poor oral bioavailability, including chemical structure modifications, encapsulation within nano-microspheres to be used as carriers, and molecular dispersion in polymer matrices. In this review we summarize the experimental approaches investigated so far in order to improve EA pharmacokinetics, supporting the hypothesis that enhancement in EA solubility is a feasible route for increasing its oral absorption.

Role of VEGFs/VEGFR-1 Signaling and its Inhibition in Modulating Tumor Invasion: Experimental Evidence in Different Metastatic Cancer Models.

The vascular endothelial growth factor (VEGF) family members, VEGF-A, placenta growth factor (PlGF), and to a lesser extent VEGF-B, play an essential role in tumor-associated angiogenesis, tissue infiltration, and metastasis formation. Although VEGF-A can activate both VEGFR-1 and VEGFR-2 membrane receptors, PlGF and VEGF-B exclusively interact with VEGFR-1. Differently from VEGFR-2, which is involved both in physiological and pathological angiogenesis, in the adult VEGFR-1 is required only for pathological angiogenesis. Besides this role in tumor endothelium, ligand-mediated stimulation of VEGFR-1 expressed in tumor cells may directly induce cell chemotaxis and extracellular matrix invasion. Furthermore, VEGFR-1 activation in myeloid progenitors and tumor-associated macrophages favors cancer immune escape through the release of immunosuppressive cytokines. These properties have prompted a number of preclinical and clinical studies to analyze VEGFR-1 involvement in the metastatic process. The aim of the present review is to highlight the contribution of VEGFs/VEGFR-1 signaling in the progression of different tumor types and to provide an overview of the therapeutic approaches targeting VEGFR-1 currently under investigation.

Role of VEGFR-1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib.

The vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase receptor frequently expressed in melanoma. Its activation by VEGF-A or placental growth factor (PlGF) promotes tumour cell survival, migration and invasiveness. Moreover, VEGFR-1 stimulation contributes to pathological angiogenesis and induces recruitment of tumour-associated macrophages. Since melanoma acquired resistance to BRAF inhibitors (BRAFi) has been associated with activation of pro-angiogenic pathways, we have investigated VEGFR-1 involvement in vemurafenib resistance. Results indicate that human melanoma cells rendered resistant to vemurafenib secrete greater amounts of VEGF-A and express higher VEGFR-1 levels compared with their BRAFi-sensitive counterparts. Transient VEGFR-1 silencing in susceptible melanoma cells delays resistance development, whereas in resistant cells it increases sensitivity to the BRAFi. Consistently, enforced VEGFR-1 expression, by stable gene transfection in receptor-negative melanoma cells, markedly reduces sensitivity to vemurafenib. Moreover, melanoma cells expressing VEGFR-1 are more invasive than VEGFR-1 deficient cells and receptor blockade by a specific monoclonal antibody (D16F7 mAb) reduces extracellular matrix invasion triggered by VEGF-A and PlGF. These data suggest that VEGFR-1 up-regulation might contribute to melanoma progression and spreading after acquisition of a drug-resistant phenotype. Thus, VEGFR-1 inhibition with D16F7 mAb might be a suitable adjunct therapy for VEGFR-1 positive tumours with acquired resistance to vemurafenib.

Experimental Evidence of the Antitumor, Antimetastatic and Antiangiogenic Activity of Ellagic Acid.

Ellagic acid (EA) is a naturally occurring polyphenolic compound endowed with strong antioxidant and anticancer properties that is present in high quantity in a variety of berries, pomegranates, and dried fruits. The antitumor activity of EA has been mostly attributed to direct antiproliferative and apoptotic effects. Moreover, EA can inhibit tumour cell migration, extra-cellular matrix invasion and angiogenesis, all processes that are crucial for tumour infiltrative behaviour and the metastatic process. In addition, EA may increase tumour sensitivity to chemotherapy and radiotherapy. The aim of this review is to summarize the in vitro and in vivo experimental evidence supporting the anticancer activity of pure EA, its metabolites, and EA-containing fruit juice or extracts in a variety of solid tumour models. The EA oral administration as supportive therapy to standard chemotherapy has been recently evaluated in small clinical studies with colorectal or prostate cancer patients. Novel formulations with improved solubility and bioavailability are expected to fully develop the therapeutic potential of EA derivatives in the near future.

The Anti-Vascular Endothelial Growth Factor Receptor-1 Monoclonal Antibody D16F7 Inhibits Glioma Growth and Angiogenesis In Vivo.

The vascular endothelial growth factor (VEGF) receptor-1 (VEGFR-1) is a tyrosine kinase receptor that does not play a relevant role in physiologic angiogenesis in adults, whereas it is important in tumor angiogenesis. In high-grade glioma VEGFR-1 expression by tumor endothelium and neoplastic cells contributes to the aggressive phenotype. We recently generated an anti-VEGFR-1 monoclonal antibody (D16F7 mAb) characterized by a novel mechanism of action, since it hampers receptor activation without interfering with ligand binding. The mAb is able to inhibit chemotaxis and extracellular matrix invasion of glioma cells in vitro stimulated by VEGF-A and by the VEGFR-1-selective ligand placental growth factor (PlGF). In this study, we have investigated the influence of D16F7 on glioma growth and angiogenesis in vivo using C6 glioma cells transfected with the human VEGFR-1. D16F7 was able to inhibit receptor activation and migration and extracellular matrix invasion of C6 cells overexpressing the receptor in response to PlGF and VEGF-A. In nude mice, treatment with 10 and 20 mg/kg D16F7 as a single agent was well tolerated and significantly inhibited glioma growth (P < 0.001). Strikingly, in an intracranial orthotopic model, mice dosed with 20 mg/kg D16F7 demonstrated a 65% increase in median survival time compared with vehicle-treated controls (P < 0.001) with a high percentage of long-term survivors (46%). These effects were associated with glioma cell apoptosis and decreased tumor-associated vessel formation. Overall, these results highlight the therapeutic potential of D16F7 in glioma treatment, deserving further investigation after a humanization process as single agent or in combination therapies.

Neuro-differentiated Ntera2 cancer stem cells encapsulated in alginate beads: First evidence of biological functionality.

The present communication investigates an application of alginate encapsulation technology to the differentiation of the embryonic cancer stem NTera2 cells (NT2) into dopamine-producing cells. The encapsulation of cells in polymeric beads allows their immune isolation and makes them eligible for transplantation, thus representing a promising biotech tool for the delivery of biologically active compounds to the brain. The polysaccharide alginate is one of the most commonly used material for this procedure since it is well tolerated by various tissues, including the brain. Two different initial cell concentrations (i.e. 0.5∗106/ml and 1.0∗106/ml) were tested, in order to identify which one could better reflect the homogeneous cell distribution into the alginate beads and guarantee a good cell viability at different times of culture. As evidenced, the higher number of cells promoted the formation of clusters resulting in a better interaction among encapsulated cells and the subsequent promotion of mitotic activity. The distribution of alive/dead cells into the alginate beads was verified and followed at different time points through the fluorescein diacetate/propidium iodide (FDA/PI) staining, confirming the presence of living neuronal positive cells, as determined from fluorescence microscopy imaging. The functionality of the encapsulated NT2 cells was confirmed by their dopamine production capability as assessed by UV-Vis spectrophotometric analysis and by liquid chromatography-mass spectrometry (LC-MS). The NT2/microspheres system can be considered a groundbreaking experimental procedure, a functionally active platform, able to produce and release dopamine, and thus potentially exploitable for therapy in Parkinson's disease.

The anti-vascular endothelial growth factor receptor-1 monoclonal antibody D16F7 inhibits invasiveness of human glioblastoma and glioblastoma stem cells.

BACKGROUND: Glioblastoma (GBM) is a highly migratory, invasive, and angiogenic brain tumor. Like vascular endothelial growth factor-A (VEGF-A), placental growth factor (PlGF) promotes GBM angiogenesis. VEGF-A is a ligand for both VEGF receptor-1 (VEGFR-1) and VEGFR-2, while PlGF interacts exclusively with VEGFR-1. We recently generated the novel anti-VEGFR-1 monoclonal antibody (mAb) D16F7 that diminishes VEGFR-1 homodimerization/activation without affecting VEGF-A and PlGF binding. METHODS: In the present study, we evaluated the expression of VEGFR-1 in human GBM tissue samples (n = 42) by immunohistochemistry, in cell lines (n = 6) and GBM stem cells (GSCs) (n = 18) by qRT-PCR and/or western blot analysis. In VEGFR-1 positive GBM or GSCs we also analyzed the ability of D16F7 to inhibit GBM invasiveness in response to VEGF-A and PlGF. RESULTS: Most of GBM specimens stained positively for VEGFR-1 and all but one GBM cell lines expressed VEGFR-1. On the other hand, in GSCs the expression of the receptor was heterogeneous. D16F7 reduced migration and invasion of VEGFR-1 positive GBM cell lines and patient-derived GSCs in response to VEGF-A and PlGF. Interestingly, this effect was also observed in VEGFR-1 positive GSCs transfected to over-express wild-type EGFR (EGFRwt+) or mutant EGFR (ligand binding domain-deficient EGFRvIII+). Furthermore, D16F7 suppressed intracellular signal transduction in VEGFR-1 over-expressing GBM cells by reducing receptor auto-phosphorylation at tyrosine 1213 and downstream Erk1/2 activation induced by receptor ligands. CONCLUSION: The results from this study suggest that VEGFR-1 is a relevant target for GBM therapy and that D16F7-derived humanized mAbs warrant further investigation.

Ellagic Acid Inhibits Bladder Cancer Invasiveness and In Vivo Tumor Growth.

Ellagic acid (EA) is a polyphenolic compound that can be found as a naturally occurring hydrolysis product of ellagitannins in pomegranates, berries, grapes, green tea and nuts. Previous studies have reported the antitumor properties of EA mainly using in vitro models. No data are available about EA influence on bladder cancer cell invasion of the extracellular matrix triggered by vascular endothelial growth factor-A (VEGF-A), an angiogenic factor associated with disease progression and recurrence, and tumor growth in vivo. In this study, we have investigated EA activity against four different human bladder cancer cell lines (i.e., T24, UM-UC-3, 5637 and HT-1376) by in vitro proliferation tests (measuring metabolic and foci forming activity), invasion and chemotactic assays in response to VEGF-A and in vivo preclinical models in nude mice. Results indicate that EA exerts anti-proliferative effects as a single agent and enhances the antitumor activity of mitomycin C, which is commonly used for the treatment of bladder cancer. EA also inhibits tumor invasion and chemotaxis, specifically induced by VEGF-A, and reduces VEGFR-2 expression. Moreover, EA down-regulates the expression of programmed cell death ligand 1 (PD-L1), an immune checkpoint involved in immune escape. EA in vitro activity was confirmed by the results of in vivo studies showing a significant reduction of the growth rate, infiltrative behavior and tumor-associated angiogenesis of human bladder cancer xenografts. In conclusion, these results suggest that EA may have a potential role as an adjunct therapy for bladder cancer.

Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies.

Apoptosis is a form of programmed cell death that results in the orderly and efficient removal of damaged cells, such as those resulting from DNA damage or during development. Apoptosis can be triggered by signals from within the cell, such as genotoxic stress, or by extrinsic signals, such as the binding of ligands to cell surface death receptors. Deregulation in apoptotic cell death machinery is an hallmark of cancer. Apoptosis alteration is responsible not only for tumor development and progression but also for tumor resistance to therapies. Most anticancer drugs currently used in clinical oncology exploit the intact apoptotic signaling pathways to trigger cancer cell death. Thus, defects in the death pathways may result in drug resistance so limiting the efficacy of therapies. Therefore, a better understanding of the apoptotic cell death signaling pathways may improve the efficacy of cancer therapy and bypass resistance. This review will highlight the role of the fundamental regulators of apoptosis and how their deregulation, including activation of anti-apoptotic factors (i.e., Bcl-2, Bcl-xL, etc) or inactivation of pro-apoptotic factors (i.e., p53 pathway) ends up in cancer cell resistance to therapies. In addition, therapeutic strategies aimed at modulating apoptotic activity are briefly discussed.

A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib.

Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib. To this aim we generated a subline of A375 melanoma resistant in vitro and in vivo to vemurafenib (A375-VR8) and characterized by NRAS G13R mutation, high basal levels of CRAF protein and phospho-activation of AKT. In these cells ERK phosphorylation was not significantly down-modulated by vemurafenib concentrations capable of abrogating ERK phosphorylation in sensitive A375 cells. Both NBDHEX and MC3181 induced marked antiproliferative and apoptotic effects in A375-VR8 cells and, at equitoxic concentrations, caused a strong phosphorylation of JNK, p38, and of the downstream mediators of apoptosis ATF2 and p53. Drug treatment further increased ERK phosphorylation, which was required for the cellular response to the NBD derivatives, as apoptosis was antagonized by the ERK inhibitor FR180204. Finally, in vivo administration of MC3181 provoked JNK activation at the tumor site and markedly reduced A375-VR8 growth. These evidences strongly suggest that the activation of multiple pro-apoptotic MAPK pathways by MC3181 might represent a new strategy for the treatment of melanoma resistant to BRAF inhibitors.

A not cytotoxic nickel concentration alters the expression of neuronal differentiation markers in NT2 cells.

Nickel, a known occupational/environmental hazard, may cross the placenta and reach appreciable concentrations in various fetal organs, including the brain. The aim of this study was to investigate whether nickel interferes with the process of neuronal differentiation. Following a 4 week treatment with retinoic acid (10µM), the human teratocarcinoma-derived NTera2/D1 cell line (NT2 cells) terminally differentiate into neurons which recapitulate many features of human fetal neurons. The continuous exposure of the differentiating NT2 cells to a not cytotoxic nickel concentration (10µM) increased the expression of specific neuronal differentiation markers such as neural cell adhesion molecule (NCAM) and microtubule associated protein 2 (MAP2). Furthermore, nickel exposure increased the expression of hypoxia-inducible-factor-1α (HIF-1α) and induced the activation of the AKT/PKB kinase pathway, as shown by the increase of P(Ser-9)-GSK-3ß, the inactive form of glycogen synthase kinase-3ß (GSK-3ß). Intriguingly, by the end of the fourth week the expression of tyrosine hydroxylase (TH) protein, a marker of dopaminergic neurons, was lower in nickel-treated than in control cultures. Thus, likely by partially mimicking hypoxic conditions, a not-cytotoxic nickel concentration appears to alter the process of neuronal differentiation and hinder the expression of the dopaminergic neuronal phenotype. Taken together, these results suggest that nickel, by altering normal brain development, may increase susceptibility to neuro-psychopathology later in life.