Optimizing adjuvant inhaled chemotherapy: Synergistic enhancement in paclitaxel cytotoxicity by flubendazole nanocrystals in a cycle model approach.

Lung cancer is the leading cause of cancer-related death. In addition to new innovative approaches, practical strategies that improve the efficacy of already available drugs are urgently needed. In this study, an inhalable dry powder formulation is used to repurpose flubendazole, a poorly soluble anthelmintic drug with potential against a variety of cancer lineages. Flubendazole nanocrystals were obtained through nanoprecipitation, and dry powder was produced by spray drying. Through fractional factorial design, the spray drying parameters were optimized and the impact of formulation on aerolization properties was clarified. The loading limitations were clarified through response surface methodology, and a 15% flubendazole loading was feasible through the addition of 20% L-leucine, leading to a flubendazole particle size of 388.6 nm, median mass aerodynamic diameter of 2.9 µm, 50.3% FPF, emitted dose of 83.2% and triple the initial solubility. Although the cytotoxicity of this formulation in A549 cells was limited, the formulation showed a synergistic effect when associated with paclitaxel, leading to a surprising 1000-fold reduction in the IC50. Compared to 3 cycles of paclitaxel alone, a 3-cycle model combined treatment increased the threshold of cytotoxicity by 25% for the same dose. Our study suggests, for the first time, that orally inhaled flubendazole nanocrystals show high potential as adjuvants to increase cytotoxic agents' potency and reduce adverse effects.

Bj-PRO-10c, as an allosteric regulator of argininosuccinate synthase, is a potential therapy for neuroblastoma metastasis.

Cancer is a global public health issue. Neuroblastoma (NB) originates from any tissue of the sympathetic nervous system, and the most affected site is the abdomen. The adrenal gland is the primary site in 38% of cases. Approximately 50% of patients have metastatic disease at diagnosis, and bone marrow is often affected. Metastatic disease is characterized by the spreading of cancer cells that are frequently resistant to chemotherapy and radiotherapy from the primary tumor to other specific parts of the body and is responsible for 90% of cancer-related deaths. Increasing evidence has indicated that nitric oxide (NO) signaling is implicated in the pathophysiology of many types of cancer, particularly in tumorigenesis and cancer progression. However, the effect of NO on metastasis cannot be easily classified as prometastatic or antimetastatic. An understanding at the molecular level of the role of NO in cancer will have profound therapeutic implications for the diagnosis and treatment of disease. Here, the proline-rich decapeptide isolated from Bothrops jararaca venom (Bj-PRO-10c) that enhances and sustains the generation of NO was used to unravel the role of metabolic NO in steps of metastasis. Bj-PRO-10c showed an antimetastatic effect, mainly by interfering with actin cytoskeleton rearrangement, controlling cell proliferation, and decreasing the seeding efficiency of NB in metastatic niches. Therefore, we proposed that an approach for controlled NO induction with the right molecular strategies can hopefully inhibit metastasis and increase the lifespan of NB patients.

Eicosapentaenoic acid-rich oil supplementation activates PPAR-γ and delays skin wound healing in type 1 diabetic mice.

Delayed wound healing is a devastating complication of diabetes and supplementation with fish oil, a source of anti-inflammatory omega-3 (ω-3) fatty acids including eicosapentaenoic acid (EPA), seems an appealing treatment strategy. However, some studies have shown that ω-3 fatty acids may have a deleterious effect on skin repair and the effects of oral administration of EPA on wound healing in diabetes are unclear. We used streptozotocin-induced diabetes as a mouse model to investigate the effects of oral administration of an EPA-rich oil on wound closure and quality of new tissue formed. Gas chromatography analysis of serum and skin showed that EPA-rich oil increased the incorporation of ω-3 and decreased ω-6 fatty acids, resulting in reduction of the ω-6/ω-3 ratio. On the tenth day after wounding, EPA increased production of IL-10 by neutrophils in the wound, reduced collagen deposition, and ultimately delayed wound closure and impaired quality of the healed tissue. This effect was PPAR-γ-dependent. EPA and IL-10 reduced collagen production by fibroblasts in vitro. In vivo, topical PPAR-γ-blockade reversed the deleterious effects of EPA on wound closure and on collagen organization in diabetic mice. We also observed a reduction in IL-10 production by neutrophils in diabetic mice treated topically with the PPAR-γ blocker. These results show that oral supplementation with EPA-rich oil impairs skin wound healing in diabetes, acting on inflammatory and non-inflammatory cells.

Purinergic signaling in cognitive impairment and neuropsychiatric symptoms of Alzheimer's disease.

About 10 million new cases of dementia develop worldwide each year, of which up to 70% are attributable to Alzheimer's disease (AD). In addition to the widely known symptoms of memory loss and cognitive impairment, AD patients frequently develop non-cognitive symptoms, referred to as behavioral and psychological symptoms of dementia (BPSDs). Sleep disorders are often associated with AD, but mood alterations, notably depression and apathy, comprise the most frequent class of BPSDs. BPSDs negatively affect the lives of AD patients and their caregivers, and have a significant impact on public health systems and the economy. Because treatments currently available for AD are not disease-modifying and mainly aim to ameliorate some of the cognitive symptoms, elucidating the mechanisms underlying mood alterations and other BPSDs in AD may reveal novel avenues for progress in AD therapy. Purinergic signaling is implicated in the pathophysiology of several central nervous system (CNS) disorders, such as AD, depression and sleep disorders. Here, we review recent findings indicating that purinergic receptors, mainly the A1, A2A, and P2X7 subtypes, are associated with the development/progression of AD. Current evidence suggests that targeting purinergic signaling may represent a promising therapeutic approach in AD and related conditions. This article is part of the Special Issue on "Purinergic Signaling: 50 years".

Impairment of adenosine signaling disrupts early embryo development: unveiling the underlying mechanisms.

Purinergic signaling has been implicated in many biological functions, including development. In this study, we investigate the functions of extracellular adenosine and adenosine receptors using a mouse embryonic stem cell (ESC) line and morula stages isolated from mouse embryos. Feeder-free mouse ESC was investigated in the absence and presence of the leukemia inhibitory factor (LIF), configuring undifferentiated cells and cells undergoing spontaneous differentiation. High alkaline phosphatase (ALPL) and low CD73 levels resulting in low adenosine (eADO) levels were characteristic for pluripotent cells in the presence of the LIF, while LIF deprivation resulted in augmented adenosine levels and reduced pluripotency marker expression, which indicated differentiation. Tracing ESC proliferation by BrdU labeling revealed that the inhibition of ALPL by levamisole resulted in a decrease in proliferation due to less eADO accumulation. Furthermore, caffeine and levamisole treatment, inhibiting adenosine receptor and eADO accumulation, respectively, reduced ESC migration, similar to that observed in the absence of the LIF. Pharmacological approaches of selective adenosine receptor subtype inhibition triggered specific adenosine receptor activities, thus triggering calcium or MAP kinase pathways leading to differentiation. In line with the in vitro data, mouse embryos at the morula stage were sensitive to treatments with A1 and A3 receptor antagonists, leading to the conclusion that A1 receptor and A3 receptor inhibition impairs proliferation and self-renewal and triggers inappropriate differentiation, respectively. The findings herein define the functions of eADO signaling in early development with implications for developmental disorders, in which adenosine receptors or ectonucleotidase dysfunctions are involved, and which could lead to malformations and miscarriages, due to exposure to caffeine.

P2X7 receptor isoform B is a key drug resistance mediator for neuroblastoma.

Drug resistance is a major challenge for all oncological treatments that involve the use of cytotoxic agents. Recent therapeutic alternatives cannot circumvent the ability of cancer cells to adapt or alter the natural selection of resistant cells, so the problem persists. In neuroblastoma, recurrence can occur in up to 50% of high-risk patients. Therefore, the identification of novel therapeutic targets capable of modulating survival or death following classical antitumor interventions is crucial to address this problem. In this study, we investigated the role of the P2X7 receptor in chemoresistance. Here, we elucidated the contributions of P2X7 receptor A and B isoforms to neuroblastoma chemoresistance, demonstrating that the B isoform favors resistance through a combination of mechanisms involving drug efflux via MRP-type transporters, resistance to retinoids, retaining cells in a stem-like phenotype, suppression of autophagy, and EMT induction, while the A isoform has opposite and complementary roles.

Cancer Metabostemness and Metabolic Reprogramming via P2X7 Receptor.

The heterogeneity of tumor cell mass and the plasticity of cancer cell phenotypes in solid tumors allow for the insurgence of resistant and metastatic cells, responsible for cancer patients' clinical management's main challenges. Among several factors that are responsible for increased cancer aggression, metabolic reprogramming is recently emerging as an ultimate cancer hallmark, as it is central for cancer cell survival and self-renewal, metastasis and chemoresistance. The P2X7 receptor, whose expression is upregulated in many solid and hematological malignancies, is also emerging as a good candidate in cancer metabolic reprogramming and the regulation of stem cell proliferation and differentiation. Metabostemness refers to the metabolic reprogramming of cancer cells toward less differentiated (CSCs) cellular states, and we believe that there is a strong correlation between metabostemness and P2X7 receptor functions in oncogenic processes. Here, we summarize important aspects of P2X7 receptor functions in normal and tumor tissues as well as essential aspects of its structure, regulation, pharmacology and its clinical use. Finally, we review current knowledge implicating P2X7 receptor functions in cancer-related molecular pathways, in metabolic reprogramming and in metabostemness.

Implications of SARS-Cov-2 infection on eNOS and iNOS activity: Consequences for the respiratory and vascular systems.

Symptoms of COVID-19 range from asymptomatic/mild symptoms to severe illness and death, consequence of an excessive inflammatory process triggered by SARS-CoV-2 infection. The diffuse inflammation leads to endothelium dysfunction in pulmonary blood vessels, uncoupling eNOS activity, lowering NO production, causing pulmonary physiological alterations and coagulopathy. On the other hand, iNOS activity is increased, which may be advantageous for host defense, once NO plays antiviral effects. However, overproduction of NO may be deleterious, generating a pro-inflammatory effect. In this review, we discussed the role of endogenous NO as a protective or deleterious agent of the respiratory and vascular systems, the most affected in COVID-19 patients, focusing on eNOS and iNOS roles. We also reviewed the currently available NO therapies and pointed out possible alternative treatments targeting NO metabolism, which could help mitigate health crises in the present and future CoV's spillovers.

Hyperactivation of P2X7 receptors as a culprit of COVID-19 neuropathology.

Scientists and health professionals are exhaustively trying to contain the coronavirus disease 2019 (COVID-19) pandemic by elucidating viral invasion mechanisms, possible drugs to prevent viral infection/replication, and health cares to minimize individual exposure. Although neurological symptoms are being reported worldwide, neural acute and long-term consequences of SARS-CoV-2 are still unknown. COVID-19 complications are associated with exacerbated immunoinflammatory responses to SARS-CoV-2 invasion. In this scenario, pro-inflammatory factors are intensely released into the bloodstream, causing the so-called "cytokine storm". Both pro-inflammatory factors and viruses may cross the blood-brain barrier and enter the central nervous system, activating neuroinflammatory responses accompanied by hemorrhagic lesions and neuronal impairment, which are largely described processes in psychiatric disorders and neurodegenerative diseases. Therefore, SARS-CoV-2 infection could trigger and/or worse brain diseases. Moreover, patients with central nervous system disorders associated to neuroimmune activation (e.g. depression, Parkinson's and Alzheimer's disease) may present increased susceptibility to SARS-CoV-2 infection and/or achieve severe conditions. Elevated levels of extracellular ATP induced by SARS-CoV-2 infection may trigger hyperactivation of P2X7 receptors leading to NLRP3 inflammasome stimulation as a key mediator of neuroinvasion and consequent neuroinflammatory processes, as observed in psychiatric disorders and neurodegenerative diseases. In this context, P2X7 receptor antagonism could be a promising strategy to prevent or treat neurological complications in COVID-19 patients.

ATP and spontaneous calcium oscillations control neural stem cell fate determination in Huntington's disease: a novel approach for cell clock research.

Calcium, the most versatile second messenger, regulates essential biology including crucial cellular events in embryogenesis. We investigated impacts of calcium channels and purinoceptors on neuronal differentiation of normal mouse embryonic stem cells (ESCs), with outcomes being compared to those of in vitro models of Huntington's disease (HD). Intracellular calcium oscillations tracked via real-time fluorescence and luminescence microscopy revealed a significant correlation between calcium transient activity and rhythmic proneuronal transcription factor expression in ESCs stably expressing ASCL-1 or neurogenin-2 promoters fused to luciferase reporter genes. We uncovered that pharmacological manipulation of L-type voltage-gated calcium channels (VGCCs) and purinoceptors induced a two-step process of neuronal differentiation. Specifically, L-type calcium channel-mediated augmentation of spike-like calcium oscillations first promoted stable expression of ASCL-1 in differentiating ESCs, which following P2Y2 purinoceptor activation matured into GABAergic neurons. By contrast, there was neither spike-like calcium oscillations nor responsive P2Y2 receptors in HD-modeling stem cells in vitro. The data shed new light on mechanisms underlying neurogenesis of inhibitory neurons. Moreover, our approach may be tailored to identify pathogenic triggers of other developmental neurological disorders for devising targeted therapies.

The P2X7 Receptor: Central Hub of Brain Diseases.

The P2X7 receptor is a cation channel activated by high concentrations of adenosine triphosphate (ATP). Upon long-term activation, it complexes with membrane proteins forming a wide pore that leads to cell death and increased release of ATP into the extracellular milieu. The P2X7 receptor is widely expressed in the CNS, such as frontal cortex, hippocampus, amygdala and striatum, regions involved in neurodegenerative diseases and psychiatric disorders. Despite P2X7 receptor functions in glial cells have been extensively studied, the existence and roles of this receptor in neurons are still controversially discussed. Regardless, P2X7 receptors mediate several processes observed in neuropsychiatric disorders and brain tumors, such as activation of neuroinflammatory response, stimulation of glutamate release and neuroplasticity impairment. Moreover, P2X7 receptor gene polymorphisms have been associated to depression, and isoforms of P2X7 receptors are implicated in neuropsychiatric diseases. In view of that, the P2X7 receptor has been proposed to be a potential target for therapeutic intervention in brain diseases. This review discusses the molecular mechanisms underlying P2X7 receptor-mediated signaling in neurodegenerative diseases, psychiatric disorders, and brain tumors. In addition, it highlights the recent advances in the development of P2X7 receptor antagonists that are able of penetrating the central nervous system.

Insights in Chloroquine Action: Perspectives and Implications in Malaria and COVID-19.

Malaria is a threat to human mankind and kills about half a million people every year. On the other hand, COVID-19 resulted in several hundred thousand deaths since December 2019 and remains without an efficient and safe treatment. The antimalarials chloroquine (CQ) and its analog, hydroxychloroquine (HCQ), have been tested for COVID-19 treatment, and several conflicting evidence has been obtained. Therefore, the aim of this review was to summarize the evidence regarding action mechanisms of these compounds against Plasmodium and SARS-CoV-2 infection, together with cytometry applications. CQ and HCQ act on the renin angiotensin system, with possible implications on the cardiorespiratory system. In this context, flow and image cytometry emerge as powerful technologies to investigate the mechanism of therapeutic candidates, as well as for the identification of the immune response and prognostics of disease severity. Data from the large randomized trials support the conclusion that CQ and HCQ do not provide any clinical improvements in disease severity and progression of SARS-CoV-2 patients, as well as they do not present any solid evidence of increased serious side effects. These drugs are safe and effective antimalarials agents, but in SARS-CoV-2 patients, they need further studies in the context of clinical trials. © 2020 International Society for Advancement of Cytometry.

Using Cytometry for Investigation of Purinergic Signaling in Tumor-Associated Macrophages.

Tumor-associated macrophages are widely recognized for their importance in guiding pro-tumoral or antitumoral responses. Mediating inflammation or immunosuppression, these cells support many key events in cancer progression: cell growth, chemotaxis, invasiveness, angiogenesis and cell death. The communication between cells in the tumor microenvironment strongly relies on the secretion and recognition of several molecules, including damage-associated molecular patterns (DAMPs), such as adenosine triphosphate (ATP). Extracellular ATP (eATP) and its degradation products act as signaling molecules and have extensively described roles in immune response and inflammation, as well as in cancer biology. These multiple functions highlight the purinergic system as a promising target to investigate the interplay between macrophages and cancer cells. Here, we reviewed purinergic signaling pathways connecting cancer cells and macrophages, a yet poorly investigated field. Finally, we present a new tool for the characterization of macrophage phenotype within the tumor. Image cytometry emerges as a cutting-edge tool, capable of providing a broad set of information on cell morphology, expression of specific markers, and its cellular or subcellular localization, preserving cell-cell interactions within the tumor section and providing high statistical strength in small-sized experiments. Thus, image cytometry allows deeper investigation of tumor heterogeneity and interactions between these cells. © 2020 International Society for Advancement of Cytometry.

The P2X7 Receptor in the Maintenance of Cancer Stem Cells, Chemoresistance and Metastasis.

Metastasis is the worst prognosis predictor in the clinical course of cancer development. Features of metastatic cancer cells include migratory ability, low degree of differentiation, self-renewal and proliferation potentials, as well as resistance to therapies. Metastatic cells do not present all of the necessary characteristics at once. Indeed, they have a unique phenotypic plasticity, allowing the acquisition of features that make them successful in all steps of metastasis. Cancer stem cells (CSC), the most undifferentiated cells in the tumor mass, display highest metastatic potential and resistance to radio- and chemotherapy. Growing tumors exhibit marked upregulation of P2X7 receptor expression and secrete ATP. Since the P2X7 receptor plays an important role in the maintenance of undifferentiated state of pluripotent cells, its importance on cell fate regulation in the tumor mass is suggested. Considering the extensive crosstalk between CSCs, epithelial-mesenchymal transition, drug resistance and metastasis, current knowledge implicating P2X7 receptor function in these phenomena and new avenues for therapeutic strategies to control metastasis are reviewed.

Publisher Correction: Combination of Chemical and Neurotrophin Stimulation Modulates Neurotransmitter Receptor Expression and Activity in Transdifferentiating Human Adipose Stromal Cells.

The original version of this article unfortunately contained mistakes. Several errors were introduced within Table 1.

Combination of Chemical and Neurotrophin Stimulation Modulates Neurotransmitter Receptor Expression and Activity in Transdifferentiating Human Adipose Stromal Cells.

Adipose stromal cells are promising tools for clinical applications in regeneration therapies, due to their ease of isolation from tissue and its high yield; however, their ability to transdifferentiate into neural phenotypes is still a matter of controversy. Here, we show that combined chemical and neurotrophin stimulation resulted in neuron-like morphology and regulated expression and activity of several genes involved in neurogenesis and neurotransmission as well as ion currents mediated by NMDA and GABA receptors. Among them, expression patterns of genes coding for kinin-B1 and B2, α7 nicotinic, M1, M3 and M4 muscarinic acetylcholine, glutamatergic (AMPA2 and mGlu2), purinergic P2Y1 and P2Y4 and GABAergic (GABA-A, ß3-subunit) receptors and neuronal nitric oxide synthase were up-regulated compared to levels of undifferentiated cells. Simultaneously, expression levels of P2X1, P2X4, P2X7 and P2Y6 purinergic and M5 muscarinic acetylcholine receptors were down-regulated. Agonist-induced activity levels of the studied receptor classes also augmented during neuronal transdifferentiation. Transdifferentiated cells expressed high levels of neuronal ß3-tubulin, NF-H, NeuN and MAP-2 proteins as well as increased ASCL1, MYT1 and POU3F2 gene expression known to drive neuronal fate determination. The presented work contributes to a better understanding of transdifferentiation induced by neurotrophins for a prospective broad spectrum of medical applications.

Kinin-B2 Receptor Activity in Skeletal Muscle Regeneration and Myoblast Differentiation.

The bioactive peptide bradykinin obtained from cleavage of precursor kininogens activates the kinin-B2 receptor functioning in induction of inflammation and vasodilatation. In addition, bradykinin participates in kidney and cardiovascular development and neuronal and muscle differentiation. Here we show that kinin-B2 receptors are expressed throughout differentiation of murine C2C12 myoblasts into myotubes. An autocrine loop between receptor activation and bradykinin secretion is suggested, since bradykinin secretion is significantly reduced in the presence of the kinin-B2 receptor antagonist HOE-140 during differentiation. Expression of skeletal muscle markers and regenerative capacity were decreased after pharmacological inhibition or genetic ablation of the B2 receptor, while its antagonism increased the number of myoblasts in culture. In summary, the present work reveals to date no functions described for the B2 receptor in muscle regeneration due to the control of proliferation and differentiation of muscle precursor cells.

Calcium signalling: A common target in neurological disorders and neurogenesis.

Calcium is an ubiquitous second messenger used by any living cell. The fine-tuning of intracellular free calcium concentration ([Ca2+]i) homeostasis and signalling pathways is crucial for the maintenance of the healthy organism. Many alterations in the homeostasis can be compensated by robust mechanisms; however, cells that already present some debility in those mechanisms, or that are over stimulated cannot compensate the stress and die. Many neurological diseases show [Ca2+] disbalance as trigger of apoptotic response resulting in massive neuronal loss and the neurodegeneration. In this review, we focus on presenting similarities and differences of neurological disorders like Huntington's, Parkinson's, Alzheimer's disease and schizophrenia and the current clinical trial status. Moreover, we describe the importance of Ca2+ signalling in neurogenesis, showing that interference of this signalling could go along with stem cell therapy in the central nervous system.

Kinin and Purine Signaling Contributes to Neuroblastoma Metastasis.

Bone marrow metastasis occurs in approximately 350,000 patients that annually die in the U.S. alone. In view of the importance of tumor cell migration into the bone marrow, we have here investigated effects of various concentrations of stromal cell-derived factor-1 (SDF-1), bradykinin- and ATP on bone marrow metastasis. We show for first time that bradykinin augmented chemotactic responsiveness of neuroblastoma cells to SDF-1 and ATP concentrations, encountered under physiological conditions. Bradykinin upregulated VEGF expression, increased metalloproteinase activity and induced adhesion of neuroblastoma cells. Bradykinin augmented SDF-1-induced intracellular Ca2+ mobilization as well as resensitization and expression of ATP-sensing P2X7 receptors. Bradykinin treatment resulted in higher gene expression levels of the truncated P2X7B receptor compared to those of the P2X7A full-length isoform. Bradykinin as pro-metastatic factor induced tumor proliferation that was significantly decreased by P2X7 receptor antagonists; however, the peptide did not enhance cell death nor P2X7A receptor-related pore activity, promoting neuroblastoma growth. Furthermore, immunodeficient nude/nude mice transplanted with bradykinin-pretreated neuroblastoma cells revealed significantly higher metastasis rates compared to animals injected with untreated cells. In contrast, animals receiving Brilliant Blue G, a P2X7 receptor antagonist, did not show any specific dissemination of neuroblastoma cells to the bone marrow and liver, and metastasis rates were drastically reduced. Our data suggests correlated actions of kinins and purines in neuroblastoma dissemination, providing novel avenues for clinic research in preventing metastasis.

Aptamers: novelty tools for cancer biology.

Although the term 'cancer' was still over two thousand years away of being coined, the first known cases of the disease date back to about 3000BC, in ancient Egypt. Five thousand years later, still lacking a cure, it has become one of the leading causes of death, killing over half a dozen million people yearly. So far, monoclonal antibodies are the most successful immune-therapy tools when it comes to fighting cancer. The number of clinical trials that use them has been increasing steadily during the past few years, especially since the Food and Drug Administration greenlit the use of the first immune-checkpoint blockade antibodies. However, albeit successful, this approach does come with the cost of auto-inflammatory toxicity. Taking this into account, the development of new therapeutic reagents with low toxicity becomes evident, particularly ones acting in tandem with the tools currently at our disposal. Ever since its discovery in the early nineties, aptamer technology has been used for a wide range of diagnostic and therapeutic applications. With similar properties to those of monoclonal antibodies, such as high-specificity of recognition and high-affinity binding, and the advantages of being developed using in vitro selection procedures, aptamers quickly became convenient building blocks for the generation of multifunctional constructs. In this review, we discuss the steps involved in the in vitro selection process that leads to functional aptamers - known as Systematic Evolution of Ligands by Exponential Enrichment - as well as the most recent applications of this technology in diagnostic and treatment of oncological illnesses. Moreover, we also suggest ways to improve such use.