Novel Acetamide-Based HO-1 Inhibitor Counteracts Glioblastoma Progression by Interfering with the Hypoxic-Angiogenic Pathway.

Glioblastoma multiforme (GBM) represents the deadliest tumor among brain cancers. It is a solid tumor characterized by uncontrolled cell proliferation generating the hypoxic niches in the cancer core. By inducing the transcription of hypoxic inducible factor (HIF), hypoxia triggers many signaling cascades responsible for cancer progression and aggressiveness, including enhanced expression of vascular endothelial growth factor (VEGF) or antioxidant enzymes, such as heme oxygenase-1 (HO-1). The present work aimed to investigate the link between HO-1 expression and the hypoxic microenvironment of GBM by culturing two human glioblastoma cell lines (U87MG and A172) in the presence of a hypoxic mimetic agent, deferoxamine (DFX). By targeting hypoxia-induced HO-1, we have tested the effect of a novel acetamide-based HO-1 inhibitor (VP18/58) on GBM progression. Results have demonstrated that hypoxic conditions induced upregulation and nuclear expression of HO-1 in a cell-dependent manner related to malignant phenotype. Moreover, our data demonstrated that the HO-1 inhibitor counteracted GBM progression by modulating the HIFα/HO-1/VEGF signaling cascade in cancer cells bearing more malignant phenotypes.

Pleural mesothelioma from fluoro-edenite exposure: PACAP and PAC1 receptor. A preliminary report.

Pleural mesothelioma is a devastating malignancy primarily associated with asbestos exposure. However, emerging evidence suggests that exposure to fluoro-edenite fibers, a naturally occurring mineral fiber, can also lead to the development of pleural mesothelioma. In this study, based on the hypothesis that pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP-preferring receptor (PAC1R) expressions could be dysregulated in pleural mesothelioma samples and that they could potentially act as diagnostic or prognostic biomarkers, we aimed to investigate the immunohistochemical expression of PACAP and PAC1R in pleural biopsies from patients with pleural mesothelioma exposed to fluoro-edenite fibers. A total of 12 patients were included in this study, and their biopsies were processed for immunohistochemical analysis to evaluate the expression of PACAP and its receptor. The study revealed a correlation between the overexpression of PACAP and PAC1R and shorter overall survival in patients with malignant mesothelioma. These findings suggest that PACAP and PAC1R expression levels could serve as potential prognostic biomarkers for malignant mesothelioma. Furthermore, the immunohistochemical analysis of PACAP and PAC1R may provide valuable information for clinicians to guide therapeutic decisions and identify patients with poorer prognosis.

Moderate Physical Activity Increases the Expression of ADNP in Rat Brain.

Activity-dependent neuroprotective protein (ADNP) is a neuroprotective protein essential for embryonic development, proper brain development, and neuronal plasticity. Its mutation causes the autism-like ADNP syndrome (also called the Helsmoortel-Van der Aa syndrome), characterized by neural developmental disorders and motor dysfunctions. Similar to the ADNP syndrome, the ADNP haploinsufficient mouse shows low synapse density, leading to motor and cognitive ability delays. Moderate physical activity (PA) has several neuroprotective and cognitive benefits, promoting neuronal survival, differentiation, neurogenesis, and plasticity. Until now, no study has investigated the effect of moderate exercise on ADNP expression and distribution in the rat brain. The aim of the current investigation was to study the effects of moderate exercise on the ADNP expression and neuronal activation measured by the microtubule protein ß-Tubulin III. In pursuit of this objective, twenty-four rats were selected and evenly distributed into two categories: sedentary control rats and rats exposed to moderate physical activity on a treadmill over a span of 12 weeks. Our results showed that moderate PA increases the expression of ADNP and ß-Tubulin III in the dentate gyrus (DG) hippocampal region and cerebellum. Moreover, we found a co-localization of ADNP and ß-Tubulin III in both DG and cerebellum, suggesting a direct association of ADNP with adult neuronal activation induced by moderate PA.

The Interplay between Glioblastoma Cells and Tumor Microenvironment: New Perspectives for Early Diagnosis and Targeted Cancer Therapy.

Glioblastoma multiforme (GBM) stands out as the most tremendous brain tumor, constituting 60% of primary brain cancers, accompanied by dismal survival rates. Despite advancements in research, therapeutic options remain limited to chemotherapy and surgery. GBM molecular heterogeneity, the intricate interaction with the tumor microenvironment (TME), and non-selective treatments contribute to the neoplastic relapse. Diagnostic challenges arise from GBM advanced-stage detection, necessitating the exploration of novel biomarkers for early diagnosis. Using data from the literature and a bioinformatic tool, the current manuscript delineates the molecular interplay between human GBM, astrocytes, and myeloid cells, underscoring selected protein pathways belonging to astroglia and myeloid lineage, which can be considered for targeted therapies. Moreover, the pivotal role of extracellular vesicles (EVs) in orchestrating a favorable microenvironment for cancer progression is highlighted, suggesting their utility in identifying biomarkers for GBM early diagnosis.

Formulating Resveratrol and Melatonin Self-Nanoemulsifying Drug Delivery Systems (SNEDDS) for Ocular Administration Using Design of Experiments.

Recent studies have demonstrated that Sirtuin-1 (SIRT-1)-activating molecules exert a protective role in degenerative ocular diseases. However, these molecules hardly reach the back of the eye due to poor solubility in aqueous environments and low bioavailability after topical application on the eye's surface. Such hindrances, combined with stability issues, call for the need for innovative delivery strategies. Within this context, the development of self-nanoemulsifying drug delivery systems (SNEDDS) for SIRT-1 delivery can represent a promising approach. The aim of the work was to design and optimize SNEDDS for the ocular delivery of two natural SIRT-1 agonists, resveratrol (RSV) and melatonin (MEL), with potential implications for treating diabetic retinopathy. Pre-formulation studies were performed by a Design of Experiment (DoE) approach to construct the ternary phase diagram. The optimization phase was carried out using Response Surface Methodology (RSM). Four types of SNEDDS consisting of different surfactants (Tween® 80, Tween® 20, Solutol® HS15, and Cremophor® EL) were optimized to achieve the best physico-chemical parameters for ocular application. Stability tests indicated that SNEDDS produced with Tween® 80 was the formulation that best preserved the stability of molecules, and so it was, therefore, selected for further technological studies. The optimized formulation was prepared with Capryol® PGMC, Tween® 80, and Transcutol® P and loaded with RSV or MEL. The SNEDDS were evaluated for other parameters, such as the mean size (found to be ˂50 nm), size homogeneity (PDI < 0.2), emulsion time (around 40 s), transparency, drug content (>90%), mucoadhesion strength, in vitro drug release, pH and osmolarity, stability to dilution, and cloud point. Finally, an in vitro evaluation was performed on a rabbit corneal epithelial cell line (SIRC) to assess their cytocompatibility. The overall results suggest that SNEDDS can be used as promising nanocarriers for the ocular drug delivery of RSV and MEL.

Neuroprotective effect of the PACAP-ADNP axis on SOD1G93A mutant motor neuron death induced by trophic factors deprivation.

Amyotrophic lateral Sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motor neurons in the central nervous system. Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) account for approximately in 20% of familial ALS cases. The pathological mechanisms underlying the toxicity induced by mutated SOD1 are still unknown. However, it has been hypothesized that oxidative stress (OS) has a crucial role in motor neuron degeneration in ALS patients. Moreover, it has been described that SOD1 mutation interferes expression of nuclear factor erythroid 2-related factor 2 (Nrf2), a protective key modulator against OS and reactive oxygen species (ROS) formation. The protective effect of pituitary adenylate cyclase-activating peptide (PACAP) has been demonstrated in various neurological disorders, including ALS. Some of its effects are mediated by the stimulation of an intracellular factor known as activity-dependent protein (ADNP). The role of PACAP-ADNP axis on mutated SOD1 motor neuron degeneration has not been explored, yet. The present study aimed to investigate whether PACAP prevented apoptotic cell death induced by growth factor deprivation through ADNP activation and whether the peptidergic axis can counteract the OS insult. By using an in vitro model of ALS, we demonstrated that PACAP by binding to PAC1 receptor (PAC1R) prevented motor neuron death induced by serum deprivation through induction of the ADNP expression via PKC stimulation. Furthermore, we have also demonstrated that the PACAP/ADNP axis counteracted ROS formation by inducing translocation of the Nfr2 from the cytoplasm to the nucleus. In conclusion, our study provides new insights regarding the protective role of PACAP-ADNP in ALS.

Cell Cycle Reactivation, at the Start of Neurodegeneration, Induced by Forskolin and Aniline in Differentiated Neuroblastoma Cells.

A characteristic hallmark of Alzheimer's disease (AD) is the intracellular accumulation of hyperphosphorylated tau protein, a phenomenon that appears to have associations with oxidative stress, double-stranded DNA breakage, and the de-condensation of heterochromatin. Re-entry into the cell division cycle appears to be involved in the onset of this neurodegenerative process. Indeed, the cell cycle cannot proceed regularly in the differentiated neurons leading to cell death. Here, we induced cell cycle reactivation in neuronal-like cells, obtained by neuroblastoma cells treated with retinoic acid, by exposure to forskolin or aniline. These compounds determine tau hyperphosphorylation or oxidative stress, respectively, resulting in the appearance of features resembling the start of neuronal degeneration typical of AD, such as tau hyperphosphorylation and re-entry into the cell cycle. Indeed, we detected an increased transcriptional level of cyclins and the appearance of a high number of mitotic cells. We also observed a delay in the initiation of the cell cycle when forskolin was co-administered with pituitary adenylate cyclase-activating polypeptide (PACAP). This delay was not observed when PACAP was co-administered with aniline. Our data demonstrate the relevance of tau hyperphosphorylation in initiating an ectopic cell cycle in differentiated neuronal cells, a condition that can lead to neurodegeneration. Moreover, we highlight the utility of neuroblastoma cell lines as an in vitro cellular model to test the possible neuroprotective effects of natural molecules.

Protective effect of pituitary adenylate cyclase activating polypeptide in diabetic keratopathy.

Diabetic keratopathy (DK) is the major complication of the cornea characterizing diabetes-affected patients. This ocular pathology is correlated with the hyperglycemic state leading to delayed corneal wound healing and recurrent corneal ulcers. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread distribution throughout the body, and exerting cytoprotective effects in the neural and non-neuronal parts of the eye, including the cornea. The purpose of the present study was to investigate whether changes in PACAP expression can concur for delayed epithelial wound healing in diabetic cornea and whether the protective effect of the peptide could be mediated through the activation of the EGFR signaling pathway, which has been reported to be impaired in DK. Expression and distribution of PACAP, PAC1R, and EGFR were investigated through immunohistochemistry analysis in the cornea of normal and diabetic rats. The role of the peptide on wound healing during DK was evaluated in an in vitro model represented by rabbit corneal epithelial cells grown in high glucose conditions. Western blotting and immunofluorescence analysis were used to examine the ability of PACAP to induce the activation of the EGFR/ERK1/2 signaling pathway. Our results showed that in diabetic cornea the expression of PACAP, PAC1R, and EGFR is drastically reduced. The treatment with PACAP via PAC1R activation enhanced cell viability and corneal epithelium wound healing in cells grown under high glucose conditions. Furthermore, both EGFR and ERK1/2 signaling was induced upon the peptide treatment. Overall, our results showed the trophic efficiency of PACAP for enhancing the corneal epithelium re-epithelialization suggesting that the peptide could be beneficially valuable as a treatment for DK.

The ε-Isozyme of Protein Kinase C (PKCε) Is Impaired in ALS Motor Cortex and Its Pulse Activation by Bryostatin-1 Produces Long Term Survival in Degenerating SOD1-G93A Motor Neuron-like Cells.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disease, characterized by a progressive depletion of upper and lower motor neurons (MNs) in the brain and spinal cord. The aberrant regulation of several PKC-mediated signal transduction pathways in ALS has been characterized so far, describing either impaired expression or altered activity of single PKC isozymes (α, ß, ζ and δ). Here, we detailed the distribution and cellular localization of the ε-isozyme of protein kinase C (PKCε) in human postmortem motor cortex specimens and reported a significant decrease in both PKCε mRNA (PRKCE) and protein immunoreactivity in a subset of sporadic ALS patients. We furthermore investigated the steady-state levels of both pan and phosphorylated PKCε in doxycycline-activated NSC-34 cell lines carrying the human wild-type (WT) or mutant G93A SOD1 and the biological long-term effect of its transient agonism by Bryostatin-1. The G93A-SOD1 cells showed a significant reduction of the phosphoPKCε/panPKCε ratio compared to the WT. Moreover, a brief pulse activation of PKCε by Bryostatin-1 produced long-term survival in activated G93A-SOD1 degenerating cells in two different cell death paradigms (serum starvation and chemokines-induced toxicity). Altogether, the data support the implication of PKCε in ALS pathophysiology and suggests its pharmacological modulation as a potential neuroprotective strategy, at least in a subgroup of sporadic ALS patients.

Drug-Repurposing Strategy for Dimethyl Fumarate.

In the area of drug discovery, repurposing strategies represent an approach to discover new uses of approved drugs besides their original indications. We used this approach to investigate the effects of dimethyl fumarate (DMF), a drug approved for relapsing-remitting multiple sclerosis and psoriasis treatment, on early injury associated with diabetic retinopathy (DR). We used an in vivo streptozotocin (STZ)-induced diabetic rat model. Diabetes was induced by a single injection of STZ in rats, and after 1 week, a group of animals was treated with a daily intraperitoneal injection of DMF or a vehicle. Three weeks after diabetes induction, the retinal expression levels of key enzymes involved in DR were evaluated. In particular, the biomarkers COX-2, iNOS, and HO-1 were assessed via Western blot and immunohistochemistry analysis. Diabetic rats showed a significant retinal upregulation of COX-2 and iNOS compared to the retina of normal rats (non-diabetic), and an increase in HO-1 was also observed in the STZ group. This latter result was due to a mechanism of protection elicited by the pathological condition. DMF treatment significantly induced the retinal expression of HO-1 in STZ-induced diabetic animals with a reduction in iNOS and COX-2 retinal levels. Taken together, these results suggested that DMF might be useful to counteract the inflammatory process and the oxidative response in DR. In conclusion, we believe that DMF represents a potential candidate to treat diabetic retinopathy and warrants further in vivo and clinical evaluation.

CXCR2 Is Deregulated in ALS Spinal Cord and Its Activation Triggers Apoptosis in Motor Neuron-Like Cells Overexpressing hSOD1-G93A.

Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterized by progressive depletion of motor neurons (MNs). Recent evidence suggests a role in ALS pathology for the C-X-C motif chemokine receptor 2 (CXCR2), whose expression was found increased at both mRNA and protein level in cortical neurons of sporadic ALS patients. Previous findings also showed that the receptor inhibition is able to prevent iPSC-derived MNs degeneration in vitro and improve neuromuscular function in SOD1-G93A mice. Here, by performing transcriptional analysis and immunofluorescence studies, we detailed the increased expression and localization of CXCR2 and its main ligand CXCL8 in the human lumbar spinal cord of sporadic ALS patients. We further investigated the functional role of CXCR2/ligands axis in NSC-34 motor neuron-like cells expressing human wild-type (WT) or mutant (G93A) SOD1. A significant expression of CXCR2 was found in doxycycline-induced G93A-SOD1-expressing cells, but not in WT cells. In vitro assays showed CXCR2 activation by GROα and MIP2α, two murine endogenous ligands and functional homologs of CXCL8, reduces cellular viability and triggers apoptosis in a dose dependent manner, while treatment with reparixin, a non-competitive allosteric CXCR2 inhibitor, effectively counteracts GROα and MIP2α toxicity, significantly inhibiting the chemokine-induced cell death. Altogether, data further support a role of CXCR2 axis in ALS etiopathogenesis and confirm its pharmacological modulation as a candidate therapeutic strategy.

Identification of Molecular Targets and Anti-Cancer Agents in GBM: New Perspectives for Cancer Therapy.

The authors of the present literature piece were invited to participate in the present Special Issue at the beginning of 2022, and we were all very enthusiastic at the prospect of assembling a series of articles on new molecular targets and anti-cancer agents in glioblastoma multiforme (GBM) [...].

Evaluation of the Antioxidant and Antiangiogenic Activity of a Pomegranate Extract in BPH-1 Prostate Epithelial Cells.

Benign prostatic hypertrophy (BPH) is a noncancerous enlargement of the prostate gland that develops from hyper-proliferation of the stromal and epithelium region. Activation of pathways involving inflammation and oxidative stress can contribute to cell proliferation in BPH and tumorigenesis. Agricultural-waste-derived extracts have drawn the attention of researchers as they represent a valid and sustainable way to exploit waste production. Indeed, such extracts are rich in bioactive compounds and can provide health-promoting effects. In particular, extracts obtained from pomegranate wastes and by-products have been shown to exert antioxidant and anti-inflammatory effects. This study focused on the evaluation of the anti-angiogenic effects and chemopreventive action of a pomegranate extract (PWE) in cellular models of BPH. In our experimental conditions, we observed that PWE was able to significantly (p < 0.001) reduce the proliferation and migration rates (up to 60%), together with the clonogenic capacity of BPH-1 cells concomitantly with the reduction in inflammatory cytokines (e.g., IL-6, PGE2) and pro-angiogenic factor (VEGF-ADMA) release. Additionally, we demonstrated the ability of PWE in reducing angiogenesis in an in vitro model of BPH consisting in transferring BPH-1-cell-conditioned media to human endothelial H5V cells. Indeed, PWE was able to reduce tube formation in H5V cells through VEGF level reduction even at low concentrations. Overall, we confirmed that inhibition of angiogenesis may be an alternative therapeutic option to prevent neovascularization in prostate tissue with BPH and its transformation into malignant prostate cancer.

PACAP-ADNP axis prevents outer retinal barrier breakdown and choroidal neovascularization by interfering with VEGF secreted from retinal pigmented epitelium cells.

During diabetic retinopathy (DR) progression, the retina undergoes various metabolic changes, including hypoxia-signalling cascade induction in the cells of retinal pigmented epithelium (RPE). The overexpression of hypoxic inducible factors causes transcription of many target genes including vascular endothelial growth factor (VEGF). The RPE cells form the outer blood retinal barrier (oBRB), a specialized structure that regulates ions and metabolites flux into the retina to maintain a suitable quality of its extracellular microenvironment. VEGF worsens retinal condition since its secretion from the basolateral compartment of RPE cells compromises the barrier's integrity and induces choroidal neovascularization. In this work, we hypothesized that PACAP prevents the damage to oBRB and controls choroidal neovascularization through the induction of ADNP. Firstly, we demonstrated that ADNP is expressed in Streptozotocin (STZ)-induced diabetic animals. To validate our hypothesis, we cultured endothelial cells (H5V) forming vessels-like structures, in a conditioned medium (CM) derived from ARPE-19 cells exposed to hyperglycaemic/hypoxic insult, containing a known VEGF concentration. The involvement of PACAP-ADNP axis on oBRB integrity was evaluated through the measurement of trans-epithelial-electrical resistance and permeability assay performed on ARPE cell monolayer cultured in CM and by analysing the expression of two tight junction forming proteins, ZO1 and occludin. By culturing H5V in CM, we demonstrated that PACAP-ADNP axis counteracted vessels-like structures formation promoted by VEGF. In conclusion, the results suggested a primary role of PACAP/ADNP axis in preventing oBRB damage and in controlling aberrant choroidal neovascularization induced by VEGF secreted from RPE cells exposed to hyperglycaemia/hypoxic insult in DR.

Regulation of UV-B-Induced Inflammatory Mediators by Activity-Dependent Neuroprotective Protein (ADNP)-Derived Peptide (NAP) in Corneal Epithelium.

The corneal epithelium, representing the outermost layer of the cornea, acts as a barrier to protect the eye against external insults such as ultraviolet B (UV-B) radiations. The inflammatory response induced by these adverse events can alter the corneal structure, leading to visual impairment. In a previous study, we demonstrated the positive effects of NAP, the active fragment of activity-dependent protein (ADNP), against oxidative stress induced by UV-B radiations. Here, we investigated its role to counteract the inflammatory event triggered by this insult contributing to the disruption of the corneal epithelial barrier. The results indicated that NAP treatment prevents UV-B-induced inflammatory processes by affecting IL-1ß cytokine expression and NF-κB activation, as well as maintaining corneal epithelial barrier integrity. These findings may be useful for the future development of an NAP-based therapy for corneal disease.

Carnosol attenuates high glucose damage in human retinal endothelial cells through regulation of ERK/Nrf2/HO-1 pathway.

Carnosol is a natural compound with antioxidant properties. Based on this evidence, in the present study we investigated whether this compound can protect retinal vascular endothelium from hyperglycemic insult responsible for diabetic retinopathy development. We performed in vitro study on human retinal endothelial cells (HREC) cultured both in normal and high glucose conditions to assess the effects of carnosol on cell viability, Nrf2 expression, HO-1 activity, and ERK1/2 expression. HREC exposed to high glucose insult were treated with carnosol. Data indicated that carnosol treatment is able to induce HO-1 expression via Nrf2 activation and counteracts the damage elicited by high glucose. Further, carnosol activation of Nrf2/HO-1 signaling axis involves ERK1/2 pathway. These data confirm the therapeutic value of carnosol by suggesting its use to treat diabetic retinopathy.

Modulatory activity of ADNP on the hypoxia­induced angiogenic process in glioblastoma.

Glioblastoma multiforme (GBM) is a brain cancer with a poor prognosis that affects adults. This is a solid tumor characterized by a high rate of cell migration and invasion. The uncontrolled cell proliferation creates hypoxic niches in the tumor mass, which leads to the overexpression of hypoxia­inducible factors (HIFs). This induces the activation of the vascular endothelial growth factor (VEGF), which is responsible for uncontrolled neoangiogenesis. Recent studies have demonstrated the anti­invasive effect of pituitary adenylate cyclase­activating peptide (PACAP) in GBM. PACAP effects on the central nervous system are also mediated through the activity­dependent neuroprotective protein (ADNP) activation. To date, no evidence exists regarding its role in GBM. Therefore, the ADNP involvement in GBM was investigated. By analyzing ADNP expression in a human GBM sample through confocal microscopy, a high ADNP immunoreactivity was detected in most glial cells and its predominant expression in hypoxic areas overexpressing HIF­1α was highlighted. To investigate the role of ADNP on the HIF­VEGF axis in GBM, a human U87MG GBM cell line was cultured with a hypoxic mimetic agent, deferoxamine, and cells were treated with the smallest active fragment of ADNP, known as NAP. The protein expression and distribution of HIF­1α and VEGF was detected using western blot analysis and immunofluorescence assay. Results demonstrated that ADNP modulates the hypoxic­angiogenic pathway in GBM cells by reducing VEGF secretion, detected through ELISA assay, as well as modulating their migration, assessed through wound healing assay. Although deeper investigation is necessary, the present study suggested that ADNP could be involved in PACAP anti­invasive effects in GBM.

Activity-Dependent Neuroprotective Protein (ADNP): An Overview of Its Role in the Eye.

Vision is one of the dominant senses in humans and eye health is essential to ensure a good quality of life. Therefore, there is an urgent necessity to identify effective therapeutic candidates to reverse the progression of different ocular pathologies. Activity-dependent neuroprotective protein (ADNP) is a protein involved in the physio-pathological processes of the eye. Noteworthy, is the small peptide derived from ADNP, known as NAP, which shows protective, antioxidant, and anti-apoptotic properties. Herein, we review the current state of knowledge concerning the role of ADNP in ocular pathologies, while providing an overview of eye anatomy.