M2 muscarinic receptors negatively modulate cell migration in human glioblastoma cells.

Glioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies. The aim of the present study was to investigate the ability of M2 mAChR to modulate cell migration in two different GB cell lines: U87 and U251. By wound healing assay and single cell migration analysis performed by time-lapse microscopy, we demonstrated the ability of M2 mAChRs to negatively modulate cell migration in U251 but not in the U87 cell line. In order to explain the different effects observed in the two cell lines we have evaluated the possible involvement of the intermediate conductance calcium-activated potassium (IKCa) channel. IKCa channel is present in the GB cells, and it has been demonstrated to modulate cell migration. Using the perforated patch-clamp technique we have found that selective activation of M2 mAChR significantly reduced functional density of the IKCa current in U251 but not in U87 cells. To understand whether the M2 mAChR mediated reduction of ion channel density in the U251 cell line was relevant for the cell migration impairment, we tested the effects of TRAM-34, a selective inhibitor of the IKCa channel, in wound healing assay. We found that it was able to markedly reduce U251 cell migration and significantly decrease the number of invadopodia-like structure formations. These results suggest that only in U251 cells the reduced cell migration M2 mAChR-mediated might involve, at least in part, the IKCa channel.

Hypoxia, Ion Channels and Glioblastoma Malignancy.

The malignancy of glioblastoma (GBM), the most aggressive type of human brain tumor, strongly correlates with the presence of hypoxic areas within the tumor mass. Oxygen levels have been shown to control several critical aspects of tumor aggressiveness, such as migration/invasion and cell death resistance, but the underlying mechanisms are still unclear. GBM cells express abundant K+ and Cl- channels, whose activity supports cell volume and membrane potential changes, critical for cell proliferation, migration and death. Volume-regulated anion channels (VRAC), which mediate the swelling-activated Cl- current, and the large-conductance Ca2+-activated K+ channels (BK) are both functionally upregulated in GBM cells, where they control different aspects underlying GBM malignancy/aggressiveness. The functional expression/activity of both VRAC and BK channels are under the control of the oxygen levels, and these regulations are involved in the hypoxia-induced GBM cell aggressiveness. The present review will provide a comprehensive overview of the literature supporting the role of these two channels in the hypoxia-mediated GBM malignancy, suggesting them as potential therapeutic targets in the treatment of GBM.

Ca2+ -activated K+ channels regulate cell volume in human glioblastoma cells.

Glioblastoma (GBM), the most lethal form of brain tumors, bases its malignancy on the strong ability of its cells to migrate and invade the narrow spaces of healthy brain parenchyma. Cell migration and invasion are both critically dependent on changes in cell volume and shape driven by the transmembrane transport of osmotically important ions such as K+ and Cl- . However, while the Cl- channels participating in cell volume regulation have been clearly identified, the precise nature of the K+ channels involved is still uncertain. Using a combination of electrophysiological and imaging approaches in GBM U87-MG cells, we found that hypotonic-induced cell swelling triggered the opening of Ca2+ -activated K+ (KCa ) channels of large and intermediate conductance (BKCa and IKCa , respectively), both highly expressed in GBM cells. The influx of Ca2+ mediated by the hypotonic-induced activation of mechanosensitive channels was found to be a key step for opening both the BKCa and the IKCa channels. Finally, the activation of both KCa channels mediated by mechanosensitive channels was found to be essential for the development of the regulatory volume decrease following hypotonic shock. Taken together, these data indicate that KCa channels are the main K+ channels responsible for the volume regulation in U87-MG cells.

The CaMKII/MLC1 Axis Confers Ca2+-Dependence to Volume-Regulated Anion Channels (VRAC) in Astrocytes.

Astrocytes, the main glial cells of the central nervous system, play a key role in brain volume control due to their intimate contacts with cerebral blood vessels and the expression of a distinctive equipment of proteins involved in solute/water transport. Among these is MLC1, a protein highly expressed in perivascular astrocytes and whose mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, chronic brain edema, cysts, myelin vacuolation, and astrocyte swelling. Although, in astrocytes, MLC1 mutations are known to affect the swelling-activated chloride currents (ICl,swell) mediated by the volume-regulated anion channel (VRAC), and the regulatory volume decrease, MLC1's proper function is still unknown. By combining molecular, biochemical, proteomic, electrophysiological, and imaging techniques, we here show that MLC1 is a Ca2+/Calmodulin-dependent protein kinase II (CaMKII) target protein, whose phosphorylation, occurring in response to intracellular Ca2+ release, potentiates VRAC-mediated ICl,swell. Overall, these findings reveal that MLC1 is a Ca2+-regulated protein, linking volume regulation to Ca2+ signaling in astrocytes. This knowledge provides new insight into the MLC1 protein function and into the mechanisms controlling ion/water exchanges in the brain, which may help identify possible molecular targets for the treatment of MLC and other pathological conditions caused by astrocyte swelling and brain edema.

Anakinra restores cellular proteostasis by coupling mitochondrial redox balance to autophagy.

Autophagy selectively degrades aggregation-prone misfolded proteins caused by defective cellular proteostasis. However, the complexity of autophagy may prevent the full appreciation of how its modulation could be used as a therapeutic strategy in disease management. Here, we define a molecular pathway through which recombinant IL-1 receptor antagonist (IL-1Ra, anakinra) affects cellular proteostasis independently from the IL-1 receptor (IL-1R1). Anakinra promoted H2O2-driven autophagy through a xenobiotic sensing pathway involving the aryl hydrocarbon receptor that, activated through the indoleamine 2,3-dioxygenase 1-kynurenine pathway, transcriptionally activated NADPH oxidase 4 independent of the IL-1R1. By coupling the mitochondrial redox balance to autophagy, anakinra improved the dysregulated proteostasis network in murine and human cystic fibrosis. We anticipate that anakinra may represent a therapeutic option in addition to its IL-1R1-dependent antiinflammatory properties by acting at the intersection of mitochondrial oxidative stress and autophagy with the capacity to restore conditions in which defective proteostasis leads to human disease.

Ion Channels in Glioma Malignancy.

Brain tumors come in many types and differ greatly in outcome. They are classified by the cell of origin (astrocytoma, ependymoma, meningioma, medulloblastoma, glioma), although more recently molecular markers are used in addition to histology. Brain tumors are graded (from I to IV) to measure their malignancy. Glioblastoma, one of the most common adult primary brain tumors, displays the highest malignancy (grade IV), and median survival of about 15 months. Main reasons for poor outcome are incomplete surgical resection, due to the highly invasive potential of glioblastoma cells, and chemoresistance that commonly develops during drug treatment. An important role in brain tumor malignancy is played by ion channels. The Ca2+-activated K+ channels of large and intermediate conductance, KCa3.1 and KCa1.1, and the volume-regulated anion channel, whose combined activity results in the extrusion of KCl and osmotic water, control cell volume, and in turn migration, invasion, and apoptotic cell death. The transient receptor potential (TRP) channels and low threshold-activated Ca (T-type) channels have equally critical role in brain tumor malignancy, as dysregulated Ca2+ signals heavily impact on glioma cell proliferation, migration, invasion. The review provides an overview of the current evidence involving these channels in brain tumor malignancy, and the application of these insights in the light of future prospects for experimental and clinical practice.

Dexamethasone in Glioblastoma Multiforme Therapy: Mechanisms and Controversies.

Glioblastoma multiforme (GBM) is the most common and malignant of the glial tumors. The world-wide estimates of new cases and deaths annually are remarkable, making GBM a crucial public health issue. Despite the combination of radical surgery, radio and chemotherapy prognosis is extremely poor (median survival is approximately 1 year). Thus, current therapeutic interventions are highly unsatisfactory. For many years, GBM-induced brain oedema and inflammation have been widely treated with dexamethasone (DEX), a synthetic glucocorticoid (GC). A number of studies have reported that DEX also inhibits GBM cell proliferation and migration. Nevertheless, recent controversial results provided by different laboratories have challenged the widely accepted dogma concerning DEX therapy for GBM. Here, we have reviewed the main clinical features and genetic and epigenetic abnormalities underlying GBM. Finally, we analyzed current notions and concerns related to DEX effects on cerebral oedema, cancer cell proliferation and migration and clinical outcome.

The Volume-Regulated Anion Channel in Glioblastoma.

Malignancy of glioblastoma multiforme (GBM), the most common and aggressive form of human brain tumor, strongly depends on its enhanced cell invasion and death evasion which make surgery and accompanying therapies highly ineffective. Several ion channels that regulate membrane potential, cytosolic Ca2+ concentration and cell volume in GBM cells play significant roles in sustaining these processes. Among them, the volume-regulated anion channel (VRAC), which mediates the swelling-activated chloride current (IClswell) and is highly expressed in GBM cells, arguably plays a major role. VRAC is primarily involved in reestablishing the original cell volume that may be lost under several physiopathological conditions, but also in sustaining the shape and cell volume changes needed for cell migration and proliferation. While experimentally VRAC is activated by exposing cells to hypotonic solutions that cause the increase of cell volume, in vivo it is thought to be controlled by several different stimuli and modulators. In this review we focus on our recent work showing that two conditions normally occurring in pathological GBM tissues, namely high serum levels and severe hypoxia, were both able to activate VRAC, and their activation was found to promote cell migration and resistance to cell death, both features enhancing GBM malignancy. Also, the fact that the signal transduction pathway leading to VRAC activation appears to involve GBM specific intracellular components, such as diacylglicerol kinase and phosphatidic acid, reportedly not involved in the activation of VRAC in healthy tissues, is a relevant finding. Based on these observations and the impact of VRAC in the physiopathology of GBM, targeting this channel or its intracellular regulators may represent an effective strategy to contrast this lethal tumor.

Cellular proteostasis: a new twist in the action of thymosin α1.

INTRODUCTION: Thymosin alpha 1 (Tα1) is a naturally occurring polypeptide of 28 amino acids, whose mechanism of action is thought to be related to its ability to signal through innate immune receptors. Tα1 (ZADAXIN®) is used worldwide for treating viral infections, immunodeficiencies, and malignancies. Owing to its ability to activate the tolerogenic pathway of tryptophan catabolism - via the immunoregulatory enzyme indoleamine 2,3-dioxygenase - Tα1 potentiates immune tolerance mechanisms, breaking the vicious circle that perpetuates chronic inflammation in response to a variety of infectious noxae. AREAS COVERED: Tα1 has never been studied in Cystic fibrosis (CF) in which the hyperinflammatory state is associated with early and nonresolving activation of innate immunity, which impairs microbial clearance and promotes a self-sustaining condition of progressive lung damage. Optimal CF treatments should, indeed, not only rescue CF transmembrane conductance regulator protein localization and functionality but also alleviate the associated hyperinflammatory pathology. Because of the inherent complexity of the pathogenetic mechanisms, a multidrug approach is required. EXPERT OPINION: By providing a multipronged attack against CF, i.e. restraining inflammation and correcting the basic defect, Tα1 favorably opposed CF symptomatology in preclinical relevant disease settings, thus suggesting its possible exploitation for 'real-life' clinical efficacy in CF. This could represent a major conceptual advance in the CF field, namely the proposal of a drug with the unique activity to correct CFTR defects through regulation of inflammation.

Author Correction: Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis.

In the version of this article originally published, some labels in Fig. 1f are incorrect. The "ß-actin" labels on the second and fourth rows of blots should instead be "ß-tubulin". The error has been corrected in the HTML and PDF versions of this article.

Publisher Correction: Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis.

In the version of this article originally published, the amino acid sequence for Tα1 described in the Online Methods is incorrect. The sequence is described as "Ac-SDAAVDTSSEITTJDLKEKKEVVEEAEN-OH". It should be "Ac-SDAAVDTSSEITTKDLKEKKEVVEEAEN-OH". The error has been corrected in the HTML and PDF versions of this article.

Kv1.3 activity perturbs the homeostatic properties of astrocytes in glioma.

Glial cells actively maintain the homeostasis of brain parenchyma, regulating neuronal excitability and preserving the physiological composition of the extracellular milieu. Under pathological conditions, some functions of glial cells could be compromised, exacerbating the neurotoxic processes. We investigated if the homeostatic activities of astrocytes and microglia could be modulated by the voltage-gated K+ channel Kv1.3. To this end we used in vitro and in vivo systems to model cell-to-cell interactions in tumoral conditions, using a specific inhibitor of Kv1.3 channels, 5-(4-phenoxybutoxy) psoralen (PAP-1). We demonstrated that PAP-1 increases astrocytic glutamate uptake, reduces glioma-induced neurotoxicity, and decreases microglial migration and phagocytosis. We also found in a tumor blood brain barrier model that Kv1.3 activity is required for its integrity. The crucial role of Kv1.3 channels as modulators of glial cell activity was confirmed in a mouse model of glioma, where PAP-1 treatment reduces tumor volume only in the presence of active glutamate transporters GLT-1. In the same mouse model, PAP-1 reduces astrogliosis and microglial infiltration. PAP-1 also reduces tumor cell invasion. All these findings point to Kv1.3 channels as potential targets to re-instruct glial cells toward their homeostatic functions, in the context of brain tumors.

BK channels blockage inhibits hypoxia-induced migration and chemoresistance to cisplatin in human glioblastoma cells.

Glioblastoma (GBM) cells express large-conductance, calcium-activated potassium (BK) channels, whose activity is important for several critical aspects of the tumor, such as migration/invasion and cell death. GBMs are also characterized by a heavy hypoxic microenvironment that exacerbates tumor aggressiveness. Since hypoxia modulates the activity of BK channels in many tissues, we hypothesized that a hypoxia-induced modulation of these channels may contribute to the hypoxia-induced GBM aggressiveness. In U87-MG cells, hypoxia induced a functional upregulation of BK channel activity, without interfering with their plasma membrane expression. Wound healing and transwell migration assays showed that hypoxia increased the migratory ability of U87-MG cells, an effect that could be prevented by BK channel inhibition. Toxicological experiments showed that hypoxia was able to induce chemoresistance to cisplatin in U87-MG cells and that the inhibition of BK channels prevented the hypoxia-induced chemoresistance. Clonogenic assays showed that BK channels are also used to increase the clonogenic ability of U87-MG GBM cells in presence, but not in absence, of cisplatin. BK channels were also found to be essential for the hypoxia-induced de-differentiation of GBM cells. Finally, using immunohistochemical analysis, we highlighted the presence of BK channels in hypoxic areas of human GBM tissues, suggesting that our findings may have physiopathological relevance in vivo. In conclusion, our data show that BK channels promote several aspects of the aggressive potential of GBM cells induced by hypoxia, such as migration and chemoresistance to cisplatin, suggesting it as a potential therapeutic target in the treatment of GBM.

Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that compromise its chloride channel activity. The most common mutation, p.Phe508del, results in the production of a misfolded CFTR protein, which has residual channel activity but is prematurely degraded. Because of the inherent complexity of the pathogenetic mechanisms involved in CF, which include impaired chloride permeability and persistent lung inflammation, a multidrug approach is required for efficacious CF therapy. To date, no individual drug with pleiotropic beneficial effects is available for CF. Here we report on the ability of thymosin alpha 1 (Tα1)-a naturally occurring polypeptide with an excellent safety profile in the clinic when used as an adjuvant or an immunotherapeutic agent-to rectify the multiple tissue defects in mice with CF as well as in cells from subjects with the p.Phe508del mutation. Tα1 displayed two combined properties that favorably opposed CF symptomatology: it reduced inflammation and increased CFTR maturation, stability and activity. By virtue of this two-pronged action, Tα1 has strong potential to be an efficacious single-molecule-based therapeutic agent for CF.

Hypoxia Modulates the Swelling-Activated Cl Current in Human Glioblastoma Cells: Role in Volume Regulation and Cell Survival.

The malignancy of glioblastoma multiforme (GBM), the most common human brain tumor, correlates with the presence of hypoxic areas, but the underlying mechanisms are unclear. GBM cells express abundant Cl channels whose activity supports cell volume and membrane potential changes, ultimately leading to cell proliferation, migration, and escaping death. In non-tumor tissues Cl channels are modulated by hypoxia, which prompted us to verify whether hypoxia would also modulate Cl channels in GBM cells. Our results show that in GBM cell lines, acute application of a hypoxic solution activates a Cl current displaying the biophysical and pharmacological features of the swelling-activated Cl current (ICl,swell ). We also found that acute hypoxia increased the cell volume by about 20%, and a 30% hypertonic solution partially inhibited the hypoxia-activated Cl current, suggesting that cell swelling and the activation of the Cl current are sequential events. Notably, the hypoxia-induced cell swelling was followed by a regulatory volume decrease (RVD) mediated mainly by ICl,swell . Since, a hypoxia-induced prolonged cell swelling is usually regarded as a death insult, we hypothesized that the hypoxia-activated Cl current could limit cell swelling and prevent necrotic death of GBM cells under hypoxic conditions. In accordance, we found that the ICl,swell inhibitor DCPIB hampered the RVD process, and more importantly it sensibly increased the hypoxia-induced necrotic death in these cells. Taken together, these results suggest that Cl channels are strongly involved in the survival of GBM cells in a hypoxic environment, and may thus represent a new therapeutic target for this malignant tumor. J. Cell. Physiol. 232: 91-100, 2017. © 2016 Wiley Periodicals, Inc.

Overexpression of Large-Conductance Calcium-Activated Potassium Channels in Human Glioblastoma Stem-Like Cells and Their Role in Cell Migration.

Glioblastomas (GBMs) are brain tumors characterized by diffuse invasion of cancer cells into the healthy brain parenchyma, and establishment of secondary foci. GBM cells abundantly express large-conductance, calcium-activated potassium (BK) channels that are thought to promote cell invasion. Recent evidence suggests that the GBM high invasive potential mainly originates from a pool of stem-like cells, but the expression and function of BK channels in this cell subpopulation have not been studied. We investigated the expression of BK channels in GBM stem-like cells using electrophysiological and immunochemical techniques, and assessed their involvement in the migratory process of this important cell subpopulation. In U87-MG cells, BK channel expression and function were markedly upregulated by growth conditions that enriched the culture in GBM stem-like cells (U87-NS). Cytofluorimetric analysis further confirmed the appearance of a cell subpopulation that co-expressed high levels of BK channels and CD133, as well as other stem cell markers. A similar association was also found in cells derived from freshly resected GBM biopsies. Finally, transwell migration tests showed that U87-NS cells migration was much more sensitive to BK channel block than U87-MG cells. Our data show that BK channels are highly expressed in GBM stem-like cells, and participate to their high migratory activity. J. Cell. Physiol. 232: 2478-2488, 2017. © 2016 Wiley Periodicals, Inc.

Gain-of-function defects of astrocytic Kir4.1 channels in children with autism spectrum disorders and epilepsy.

Dysfunction of the inwardly-rectifying potassium channels Kir4.1 (KCNJ10) represents a pathogenic mechanism contributing to Autism-Epilepsy comorbidity. To define the role of Kir4.1 variants in the disorder, we sequenced KCNJ10 in a sample of affected individuals, and performed genotype-phenotype correlations. The effects of mutations on channel activity, protein trafficking, and astrocyte function were investigated in Xenopus laevis oocytes, and in human astrocytoma cell lines. An in vivo model of the disorder was also explored through generation of kcnj10a morphant zebrafish overexpressing the mutated human KCNJ10. We detected germline heterozygous KCNJ10 variants in 19/175 affected children. Epileptic spasms with dysregulated sensory processing represented the main disease phenotype. When investigated on astrocyte-like cells, the p.R18Q mutation exerted a gain-of-function effect by enhancing Kir4.1 membrane expression and current density. Similarly, the p.R348H variant led to gain of channel function through hindrance of pH-dependent current inhibition. The frequent polymorphism p.R271C seemed, instead, to have no obvious functional effects. Our results confirm that variants in KCNJ10 deserve attention in autism-epilepsy, and provide insight into the molecular mechanisms of autism and seizures. Similar to neurons, astrocyte dysfunction may result in abnormal synaptic transmission and electrical discharge, and should be regarded as a possible pharmacological target in autism-epilepsy.

Reconciling the discrepancies on the involvement of large-conductance Ca(2+)-activated K channels in glioblastoma cell migration.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor, and is notable for spreading so effectively through the brain parenchyma to make complete surgical resection virtually impossible, and prospect of life dismal. Several ion channels have been involved in GBM migration and invasion, due to their critical role in supporting volume changes and Ca(2+) influx occuring during the process. The large-conductance, Ca(2+)-activated K (BK) channels, markedly overexpressed in biopsies of patients with GBMs and in GBM cell lines, have attracted much interest and have been suggested to play a central role in cell migration and invasion as candidate channels for providing the ion efflux and consequent water extrusion that allow cell shrinkage during migration. Available experimental data on the role of BK channel in migration and invasion are not consistent though. While BK channels block typically resulted in inhibition of cell migration or in no effect, their activation would either enhance or inhibit the process. This short review reexamines the relevant available data on the topic, and presents a unifying paradigm capable of reconciling present discrepancies. According to this paradigm, BK channels would not contribute to migration under conditions where the [Ca(2+)] i is too low for their activation. They will instead positively contribute to migration for intermediate [Ca(2+)] i , insufficient as such to activate BK channels, but capable of predisposing them to cyclic activation following oscillatory [Ca(2+)] i increases. Finally, steadily active BK channels because of prolonged high [Ca(2+)] i would inhibit migration as their steady activity would be unsuitable to match the cyclic cell volume changes needed for proper cell migration.

The role of ion channels in the hypoxia-induced aggressiveness of glioblastoma.

The malignancy of glioblastoma multiform (GBM), the most common and aggressive form of human brain tumors, strongly correlates with the presence of hypoxic areas, but the mechanisms controlling the hypoxia-induced aggressiveness are still unclear. GBM cells express a number of ion channels whose activity supports cell volume changes and increases in the cytosolic Ca(2+) concentration, ultimately leading to cell proliferation, migration or death. In several cell types it has previously been shown that low oxygen levels regulate the expression and activity of these channels, and more recent data indicate that this also occurs in GBM cells. Based on these findings, it may be hypothesized that the modulation of ion channel activity or expression by the hypoxic environment may participate in the acquisition of the aggressive phenotype observed in GBM cells residing in a hypoxic environment. If this hypothesis will be confirmed, the use of available ion channels modulators may be considered for implementing novel therapeutic strategies against these tumors.