Electrophysiological Impact of SARS-CoV-2 Envelope Protein in U251 Human Glioblastoma Cells: Possible Implications in Gliomagenesis?

SARS-CoV-2 is the causative agent of the COVID-19 pandemic, the acute respiratory disease which, so far, has led to over 7 million deaths. There are several symptoms associated with SARS-CoV-2 infections which include neurological and psychiatric disorders, at least in the case of pre-Omicron variants. SARS-CoV-2 infection can also promote the onset of glioblastoma in patients without prior malignancies. In this study, we focused on the Envelope protein codified by the virus genome, which acts as viroporin and that is reported to be central for virus propagation. In particular, we characterized the electrophysiological profile of E-protein transfected U251 and HEK293 cells through the patch-clamp technique and FURA-2 measurements. Specifically, we observed an increase in the voltage-dependent (Kv) and calcium-dependent (KCa) potassium currents in HEK293 and U251 cell lines, respectively. Interestingly, in both cellular models, we observed a depolarization of the mitochondrial membrane potential in accordance with an alteration of U251 cell growth. We, therefore, investigated the transcriptional effect of E protein on the signaling pathways and found several gene alterations associated with apoptosis, cytokines and WNT pathways. The electrophysiological and transcriptional changes observed after E protein expression could explain the impact of SARS-CoV-2 infection on gliomagenesis.

Localization of Catecholaminergic Neurofibers in Pregnant Cervix as a Possible Myometrial Pacemaker.

In eutocic labor, the autonomic nervous system is dominated by the parasympathetic system, which ensures optimal blood flow to the uterus and placenta. This study is focused on the detection of the quantitative presence of catecholamine (C) neurofibers in the internal uterine orifice (IUO) and in the lower uterine segment (LUS) of the pregnant uterus, which could play a role in labor and delivery. A total of 102 women were enrolled before their submission to a scheduled cesarean section (CS); patients showed a singleton fetus in a cephalic presentation outside labor. During CS, surgeons sampled two serial consecutive full-thickness sections 5 mm in depth (including the myometrial layer) on the LUS and two randomly selected samples of 5 mm depth from the IUO of the cervix. All histological samples were studied to quantify the distribution of A nerve fibers. The authors demonstrated a significant and notably higher concentration of A fibers in the IUO (46 ± 4.8) than in the LUS (21 ± 2.6), showing that the pregnant cervix has a greater concentration of A neurofibers than the at-term LUS. Pregnant women's mechanosensitive pacemakers can operate normally when the body is in a physiological state, which permits normal uterine contractions and eutocic delivery. The increased frequency of C neurofibers in the cervix may influence the smooth muscle cell bundles' activation, which could cause an aberrant mechano-sensitive pacemaker activation-deactivation cycle. Stressful circumstances (anxiety, tension, fetal head position) cause the sympathetic nervous system to become more active, working through these nerve fibers in the gravid cervix. They might interfere with the mechano-sensitive pacemakers, slowing down the uterine contractions and cervix ripening, which could result in dystocic labor.

Electro-Metabolic Coupling of Cumulus-Oocyte Complex.

Oocyte-cumulus cell interaction is essential for oocyte maturation and competence. The bidirectional crosstalk network mediated by gap junctions is fundamental for the metabolic cooperation between these cells. As cumulus cells exhibit a more glycolytic phenotype, they can provide metabolic substrates that the oocyte can use to produce ATP via oxidative phosphorylation. The impairment of mitochondrial activity plays a crucial role in ovarian aging and, thus, in fertility, determining the success or failure of assisted reproductive techniques. This review aims to deepen the knowledge about the electro-metabolic coupling of the cumulus-oocyte complex and to hypothesize a putative role of potassium channel modulators in order to improve fertility, promote intracellular Ca2+ influx, and increase the mitochondrial biogenesis and resulting ATP levels in cumulus cells.

Focus on the Use of Resveratrol in Bladder Cancer.

Bladder cancer is the most common tumor of the urinary system, with a high incidence in the male population. Surgery and intravesical instillations can eradicate it, although recurrences are very common, with possible progression. For this reason, adjuvant therapy should be considered in all patients. Resveratrol displays a biphasic dose response both in vitro and in vivo (intravesical application) with an antiproliferative effect at high concentrations and antiangiogenic action in vivo (intraperitoneal application) at a low concentration, suggesting a potential role for it in clinical management as an adjuvant to conventional therapy. In this review, we examine the standard therapeutical approach to bladder cancer and the preclinical studies that have investigated resveratrol in xenotransplantation models of bladder cancer. Molecular signals are also discussed, with a particular focus on the STAT3 pathway and angiogenic growth factor modulation.

Neutral Sphingomyelinase Behaviour in Hippocampus Neuroinflammation of MPTP-Induced Mouse Model of Parkinson's Disease and in Embryonic Hippocampal Cells.

Neutral sphingomyelinase is known to be implicated in growth arrest, differentiation, proliferation, and apoptosis. Although previous studies have reported the involvement of neutral sphingomyelinase in hippocampus physiopathology, its behavior in the hippocampus during Parkinson's disease remains undetected. In this study, we show an upregulation of inducible nitric oxide synthase and a downregulation of neutral sphingomyelinase in the hippocampus of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP-) induced mouse model of Parkinson's disease. Moreover, the stimulation of neutral sphingomyelinase activity with vitamin 1,25-dihydroxyvitamin D3 reduces specifically saturated fatty acid sphingomyelin by making sphingomyelin a less rigid molecule that might influence neurite plasticity. The possible biological relevance of the increase of neutral sphingomyelinase in Parkinson's disease is discussed.

Acid sphingomyelinase as target of Lycium Chinense: promising new action for cell health.

BACKGROUND: Sphingomyelin plays very important roles in cell function under physiological and pathological conditions. Physical and chemical stimuli produce reactive oxygen species that stimulate acid sphingomyelinase to induce apoptosis. Antioxidant plants of the traditional Chinese Pharmacopoeia, such as Lycium Barbarum and Lycium Chinense, have become increasingly popular in Western countries. We investigated the effects of Lycium Chinense on acid sphingomyelinase and sphingomyelin species in relation to gene expression. METHODS: We prepared Lycium Chinense berry extracts and evaluated their antioxidant properties. Increasing amount of extracts was used to test cytotoxic and genotoxic effect on HepG2 cells. Gene expression, protein amount and enzyme activity of acid sphingomyelinase were tested by RT-PCR, immunoblotting and enzymatic activity assay, respectively. Sphingomyelin species were analyzed by UFLC MS/MS. A panel of 96 genes involved in oxidative stress, proliferation, apoptosis and cancer was used to test the effect of LC on gene expression. GLRX2, RNF7, and PTGS1 proteins were analyzed by immunoblotting. RESULTS: We showed that Lycium Chinense berries have high antioxidant properties, have an IC50value of 9.55 mg/mL, do not induce genotoxic effect and maintain high level of cell viability. The berry extracts inhibit acid sphingomyelinase activity and increase both very long fatty acid sphingomyelin species and unsaturated fatty acid sphingomyelin species. Among 96 genes, Lycium Chinense berries up-regulate Glutaredoxin 2 and Ring Finger Protein 7 genes and proteins, able to protect cells from apoptosis. Intrigantly, Lycium Chinense berries down-regulates Prostaglandin H synthase 1 gene but the protein is not expressed in HepG2 cells. CONCLUSION: The results identify acid sphingomyelinase as a novel target of Lycium Chinense berries to decrease saturated/unsaturated fatty acid sphingomyelin ratio, known to be useful for cell health. Consistent with these data, the berries regulate specifically gene expression to protect cells from apoptosis.

M2 receptors exert analgesic action on DRG sensory neurons by negatively modulating VR1 activity.

The peripheral application of the M2 cholinergic agonist arecaidine on sensory nerve endings shows anti-nociceptive properties. In this work, we analyze in vitro, the mechanisms downstream M2 receptor activation causing the analgesic effects, and in vivo the effects produced by M2 agonist arecaidine administration on nociceptive responses in a murine model of nerve growth factor (NGF)-induced pain. Cultured DRG neurons treated with arecaidine showed a decreased level of VR1 and SP transcripts. Conversely, we found an increased expression of VR1 and SP transcripts in DRG from M2/M4(-/-) mice compared to WT and M1(-/-) mice, confirming the inhibitory effect in particular of M2 receptors on SP and VR1 expression. Patch-clamp experiments in the whole-cell configuration showed that arecaidine treatment caused a reduction of the fraction of capsaicin-responsive cells, without altering the mean capsaicin-activated current in responsive cells. We also demonstrated that arecaidine prevents PKCϵ translocation to the plasma membrane after inflammatory agent stimulation, mainly in medium-small sensory neurons. Finally, in mice, we have observed that intraperitoneal injection of arecaidine reduces VR1 expression blocking hyperalgesia and allodynia caused by NGF intraplantar administration. In conclusion, our data demonstrate that in vivo M2 receptor activation induces desensitization to mechanical and heat stimuli by a down-regulation of VR1 expression and by the inhibition of PKCϵ activity hindering its translocation to the plasma membrane, as suggested by in vitro experiments.

Identification of key signaling molecules involved in the activation of the swelling-activated chloride current in human glioblastoma cells.

The swelling-activated chloride current (I Cl,Vol) is abundantly expressed in glioblastoma (GBM) cells, where it controls cell volume and invasive migration. The transduction pathway mediating I Cl,Vol activation in GBM cells is, however, poorly understood. By means of pharmacological and electrophysiological approaches, on GL-15 human GBM cells we found that I Cl,Vol activation by hypotonic swelling required the activity of a U73122-sensitive phospholipase C (PLC). I Cl,Vol activation could also be induced by the membrane-permeable diacylglycerol (DAG) analog OAG. In contrast, neither calcium (Ca(2+)) chelation by BAPTA-AM nor changes in PKC activity were able to affect I Cl,Vol activation by hypotonic swelling. We further found that R59022, an inhibitor of diacylglycerol kinase (DGK), reverted I Cl,Vol activation, suggesting the involvement of phosphatidic acid. In addition, I Cl,Vol activation required the activity of a EHT1864-sensitive Rac1 small GTPase and the resulting actin polymerization, as I Cl,Vol activation was prevented by cytochalasin B. We finally show that I Cl,Vol can be activated by the promigratory fetal calf serum in a PLC- and DGK-dependent manner. This observation is potentially relevant because blood serum can likely come in contact with glioblastoma cells in vivo as a result of the tumor-related partial breakdown of the blood-brain barrier. Given the relevance of I Cl,Vol in GBM cell volume regulation and invasiveness, the several key signaling molecules found in this study to be involved in the activation of the I Cl,Vol may represent potential therapeutic targets against this lethal cancer.

Synthesis and Characterization of ZIF-90 Nanoparticles as Potential Brain Cancer Therapy.

Human glioblastoma is probably the most malignant and aggressive among cerebral tumors, of which it represents approximately 80% of the reported cases, with an overall survival rate that is quite low. Current therapies include surgery, chemotherapy, and radiotherapy, with associated consistent side effects and low efficacy. The hardness in reaching the site of action, and overcoming the blood-brain barrier, is a major limitation of pharmacological treatments. In this paper, we report the synthesis and characterization of ZIF-90 (ZIF, Zeolitic Imidazolate Framework) nanoparticles as putative carriers of anticancer drugs to the brain. In particular, we successfully evaluated the biocompatibility of these nanoparticles, their stability in body fluids, and their ability to uptake in U251 human glioblastoma cell lines. Furthermore, we managed to synthesize ZIF-90 particles loaded with berberine, an alkaloid reported as a possible effective adjuvant in the treatment of glioblastoma. These findings could suggest ZIF-90 as a possible new strategy for brain cancer therapy and to study the physiological processes present in the central nervous system.

Oxytocin augmentation and neurotransmitters in prolonged delivery: An experimental appraisal.

The uterus is a highly innervated organ, and during labor, this innervation is at its highest level. Oxytocinergic fibers play an important role in labor and delivery and, in particular, the Lower Uterine Segment, cervix, and fundus are all controlled by motor neurofibers. Oxytocin is a neurohormone that acts on receptors located on the membrane of the smooth cells of the myometrium. During the stages of labor and delivery, its binding causes myofibers to contract, which enables the fundus of the uterus to act as a mediator. The aim of this study was to investigate the presence of oxytocinergic fibers in prolonged and non-prolonged dystocic delivery in a cohort of 90 patients, evaluated during the first and second stages of labor. Myometrial tissue samples were collected and evaluated by electron microscopy, in order to quantify differences in neurofibers concentrations between the investigated and control cohorts of patients. The authors of this experiment showed that the concentration of oxytocinergic fibers differs between non-prolonged and prolonged dystocic delivery. In particular, in prolonged dystocic delivery, compared to non-prolonged dystocic delivery, there is a lower amount of oxytocin fiber. The increase in oxytocin appeared to be ineffective in patients who experienced prolonged dystocic delivery, since the dystocic labor ended as a result of the altered presence of oxytocinergic fibers detected in this group of patients.

Increased Vasoactive Intestinal Peptide (VIP) in polycystic ovary syndrome patients undergoing IVF.

Introduction: Polycystic ovary syndrome (PCOS) is a common multifactorial and polygenic disorder of the endocrine system, affecting up to 20% of women in reproductive age with a still unknown etiology. Follicular fluid (FF) represents an environment for the normal development of follicles rich in metabolites, hormones and neurotransmitters, but in some instances of PCOS the composition can be different. Vasoactive intestinal peptide (VIP) is an endogenous autonomic neuropeptide involved in follicular atresia, granulosa cell physiology and steroidogenesis. Methods: ELISA assays were performed to measure VIP and estradiol levels in human follicular fluids, while AMH, FSH, LH, estradiol and progesterone in the plasma were quantified by chemiluminescence. UHPLC/QTOF was used to perform the untargeted metabolomic analysis. Results: Our ELISA and metabolomic results show: i) an increased concentration of VIP in follicular fluid of PCOS patients (n=9) of about 30% with respect to control group (n=10) (132 ± 28 pg/ml versus 103 ± 26 pg/ml, p=0,03) in women undergoing in vitro fertilization (IVF), ii) a linear positive correlation (p=0.05, r=0.45) between VIP concentration and serum Anti-Müllerian Hormone (AMH) concentration and iii) a linear negative correlation between VIP and noradrenaline metabolism. No correlation between VIP and estradiol (E2) concentration in follicular fluid was found. A negative correlation was found between VIP and noradrenaline metabolite 3,4-dihydroxyphenylglycolaldehyde (DOPGAL) in follicular fluids. Conclusion: VIP concentration in follicular fluids was increased in PCOS patients and a correlation was found with noradrenaline metabolism indicating a possible dysregulation of the sympathetic reflex in the ovarian follicles. The functional role of VIP as noradrenergic modulator in ovarian physiology and PCOS pathophysiology was discussed.

Cranberry/Chondroitin Sulfate Co-precipitate as a New Method for Controlling Urinary Tract Infections.

Urinary tract infections (UTI), which are among the most frequent cases of infectious diseases, mainly affect women. The most common treatment approach involves the use of antibiotics, although this solution is not always the most suitable, mainly because of the resistance that bacterial strains develop. Proanthocyanidins are a class of polyphenols, abundantly contained in cranberry extracts, which have shown beneficial effects in the treatment of urinary tract infections, due to their anti-adhesive properties toward bacteria, with respect to the membranes of the cells of the urothelium and intestine, thus reducing their virulence. In this work, we demonstrate via microscopy and scattering measurements how a mixture of cranberry and chondroitin sulfate can form a crosslinked structure with barrier properties. By using a design of experiment (DOE), we optimized the mass ratio to obtain a precipitate between cranberry extract and chondroitin sulfate in the presence of N-acetylcysteine and hyaluronic acid. By using transepithelial electrical resistance (TEER) chambers, we confirmed the barrier properties of the best mixture obtained with the DOE. Lastly, the antibiofilm action was investigated against five strains of Escherichia coli with different antibiotic sensitivity. The precipitate displayed a variable inhibitory effect in biofilm formation with major effects in UTI with an antibiotic resistance profile.

Therapeutic Targeting Potential of Novel Silver Nanoparticles Coated with Anti-CD20 Antibody against Chronic Lymphocytic Leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is an incurable disorder associated with alterations in several pathways essential for survival and proliferation. Despite the advances made in CLL therapy with the new target agents, in some cases, relapses and resistance could occur, making the discovery of new alternatives to manage CLL refractoriness necessary. To provide new therapeutic strategies for CLL, we investigated the anti-leukemic activity of silver nanoparticles (AgNPs), whose impact on CLL cells has been poorly explored. METHODS: We studied the action mechanisms of AgNPs in vitro through flow cytometry and molecular analyses. To improve the bioavailability of AgNPs, we generated AgNPs coated with the anti-CD20 antibody Rituximab (AgNPs@Rituximab) and carried out imaging-based approaches and in vivo experiments to evaluate specificity, drug uptake, and efficacy. RESULTS: AgNPs reduced the viability of primary CLL cells and the HG-3 cell line by inducing an intrinsic apoptotic pathway characterized by Bax/Bcl-2 imbalance, caspase activation, and PARP degradation. Early apoptotic events triggered by AgNPs included enhanced Ca2+ influx and ROS overproduction. AgNPs synergistically potentiated the cytotoxicity of Venetoclax, Ibrutinib, and Bepridil. In vitro, the AgNPs@Rituximab conjugates were rapidly internalized within CLL cells and strongly prolonged the survival of CLL xenograft models compared to each unconjugated single agent. CONCLUSIONS: AgNPs showed strong anti-leukemic activity in CLL, with the potential for clinical translation in combination with agents used in CLL. The increased specificity of AgNPs@Rituximab toward CLL cells could be relevant for overcoming in vivo AgNPs' non-specific distribution and increasing their efficacy.

Goji Berry in the Diet of the Rabbit Buck: Effects on Semen Quality, Oxidative Status and Histological Features of the Reproductive Tract.

Goji berry (GB) shows beneficial effects on human health, although its effects on the male rabbit have been little investigated. This study examines the impact of GB dietary supplementation on the semen traits, antioxidant capacity of seminal plasma, and histological features of the reproductive tract of rabbit buck. Eighteen rabbits were distributed into two dietary groups: one receiving a commercial feed (Control), and the other a feed supplemented with 1% of GB (Goji). After a nutritional adaptation period of 60 days, the animals were subjected to semen collection every 15 days. The semen traits, libido, antioxidant, and inflammatory parameters were collected and analyzed. The rabbits were sacrificed after 60 days, and tissues of the genital tract were analyzed. Compared to the Control group, the Goji group showed higher spermatozoa concentration, motility, and vitality (p < 0.05), as well as fewer abnormal spermatozoa and a higher libido (p < 0.1). Histological features such as functional activity and hyperplasia were improved by GB and correlated with some semen traits (p < 0.05). Conversely, antioxidant and anti-inflammatory parameters were unaffected by the diet. These findings suggest that GB acts on the tissues of the reproductive tract positively influencing semen quality, although further studies are needed to understand the effect on oxidative stress.

A theoretical study on the role of Ca(2+)-activated K+ channels in the regulation of hormone-induced Ca2+ oscillations and their synchronization in adjacent cells.

BACKGROUND INFORMATION: In many non-excitable cells hormone stimulation triggers repetitive oscillations of the intracellular Ca(2+) concentration, thought to be important in several cell functions. Although most of these cells respond to an elevation of the intracellular Ca(2+) concentration with a membrane hyperpolarization, due to the activation of Ca(2+)-activated K(+) channels, theoretical models do not usually consider the contribution of the membrane potential dynamics in defining the properties of the intracellular Ca(2+) concentration oscillations and their synchronization in adjacent, coupled cells. RESULTS: We developed a theoretical model of intracellular Ca(2+) oscillations that includes the dynamics of the membrane potential controlled by the cyclic activation of Ca(2+)-activated K(+) channels. We found that membrane potential oscillations determine an in-phase oscillating Ca(2+) influx that significantly affects the amplitude, duration and oscillatory frequency of the intracellular Ca(2+) concentration oscillations. Under specific levels of hormone stimulation Ca(2+)-activated K(+) channels are essential for establishing or inhibiting the intracellular Ca(2+) concentration oscillatory activity, as also suggested by some experimental findings. We also found that in electrically coupled cells displaying Ca(2+)-activated K(+) channels-induced membrane potential oscillations, the synchronization of intracellular Ca(2+) concentration oscillations in adjacent cells can occur in the complete absence of gap junction Ca(2+) or inositol trisphosphate diffusion, the simple electrical coupling being sufficient for synchronization. Finally, electrical coupling between adjacent cells was found to work in synergy with gap junction Ca(2+) permeability in the synchronization of intracellular Ca(2+) concentration oscillations, making it to occur at lower gap junction Ca(2+) permeabilities. CONCLUSIONS: Data from our model indicate that Ca(2+)-activated K(+) channel activity may be critical to establish important properties of the intracellular Ca(2+) concentration oscillations, and may help synchronize intracellular Ca(2+) concentration oscillations in electrically coupled cells. The model we propose here thus represents a third model of synchronization of intracellular Ca(2+) concentration oscillations in adjacent cells, based exclusively on the gap junction electrical coupling between cells displaying Ca(2+)-activated K(+) channel-induced membrane potential oscillations.

Biological and clinical effects of a resveratrol-based multivitamin supplement on intracytoplasmic sperm injection cycles: a single-center, randomized controlled trial.

BACKGROUND: Resveratrol display's positive effects on follicle growth and development in preclinical studies while there is scantly information from clinical trials. The aim of this study was to evaluate the biological and clinical impact of a resveratrol-based multivitamin supplement on intracytoplasmatic sperm injection (ICSI) cycles. METHODS: A randomized, single-center controlled trial conducted at the University Center of Assisted Reproductive Technologies involving 101 women infertile women undergoing ICSI cycles was conducted. A pretreatment with a daily resveratrol based nutraceutical was administered to the Study Group; Control Group received folic acid. The primary outcomes were the number of developed mature follicles (>16 mm), total oocytes and MII oocytes recovered, the fertilization rate and the number of cleavage embryos/blastocysts obtained. Secondary endpoints were the duration and dosage of gonadotropins, the number of embryos for transfer, implantation, biochemical, clinical pregnancy rates, live birth and miscarriage rates. RESULTS: A significantly higher number of oocytes and MII oocytes were retrieved in the Study Group than in Control Group (p = .03 and p = .04, respectively). A higher fertilization rate (p = .004), more cleavage embryos/patient (p = .01), blastocytes/patients (p = .01) and cryopreserved embryos (p = .03) were obtained in the Study Group. No significant differences in biochemical or clinical pregnancy, live birth, and miscarriage rates were revealed, but a trend to a higher live birth rate was revealed in the Study Group. CONCLUSIONS: A 3 months period of dietary supplementation with a resveratrol-based multivitamin nutraceutical leads to better biological effects on ICSI cycles. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov registration identifier: NCT04386499.

Resveratrol Treatment Induces Mito-miRNome Modification in Follicular Fluid from Aged Women with a Poor Prognosis for In Vitro Fertilization Cycles.

Advanced maternal age impairs reproductive performance, influencing the quantity and the quality of oocytes. Mitochondria dysfunction seems to play a decisive role in conditioning the quality of the female gamete. Different in vitro and in vivo studies, demonstrated the antioxidant and anti-inflammatory activities of Resveratrol and its ability to improve mitochondria function even if the exact mechanism of action has not yet been demonstrated in human oocytes. In this paper, by retrospective analysis, we evaluated follicular fluid (FF) miRNome modification in aged women with a poor ovarian reserve receiving a resveratrol-based supplement the three months before the in vitro Fertilization (IVF) cycle. We found 13 differentially expressed microRNAs (miRNAs) in women treated with resveratrol and specifically miR-125b-5p, miR-132-3p, miR-19a-3p, miR-30a-5p and miR-660-5p, regulating mitochondrial proteins, are able to control metabolism and mitochondrial biogenesis. MiRNA expression differences, observed after resveratrol treatment in FF from women with a poor prognosis for IVF, demonstrated that resveratrol may act on mitomiRNAs to improve follicular microenvironment by transcriptomic and proteomic modifications in granulosa cells.

Pharmacological targeting of the mitochondrial calcium-dependent potassium channel KCa3.1 triggers cell death and reduces tumor growth and metastasis in vivo.

Ion channels are non-conventional, druggable oncological targets. The intermediate-conductance calcium-dependent potassium channel (KCa3.1) is highly expressed in the plasma membrane and in the inner mitochondrial membrane (mitoKCa3.1) of various cancer cell lines. The role mitoKCa3.1 plays in cancer cells is still undefined. Here we report the synthesis and characterization of two mitochondria-targeted novel derivatives of a high-affinity KCa3.1 antagonist, TRAM-34, which retain the ability to block channel activity. The effects of these drugs were tested in melanoma, pancreatic ductal adenocarcinoma and breast cancer lines, as well as in vivo in two orthotopic models. We show that the mitochondria-targeted TRAM-34 derivatives induce release of mitochondrial reactive oxygen species, rapid depolarization of the mitochondrial membrane, fragmentation of the mitochondrial network. They trigger cancer cell death with an EC50 in the µM range, depending on channel expression. In contrast, inhibition of the plasma membrane KCa3.1 by membrane-impermeant Maurotoxin is without effect, indicating a specific role of mitoKCa3.1 in determining cell fate. At sub-lethal concentrations, pharmacological targeting of mitoKCa3.1 significantly reduced cancer cell migration by enhancing production of mitochondrial reactive oxygen species and nuclear factor-κB (NF-κB) activation, and by downregulating expression of Bcl-2 Nineteen kD-Interacting Protein (BNIP-3) and of Rho GTPase CDC-42. This signaling cascade finally leads to cytoskeletal reorganization and impaired migration. Overexpression of BNIP-3 or pharmacological modulation of NF-κB and CDC-42 prevented the migration-reducing effect of mitoTRAM-34. In orthotopic models of melanoma and pancreatic ductal adenocarcinoma, the tumors at sacrifice were 60% smaller in treated versus untreated animals. Metastasis of melanoma cells to lymph nodes was also drastically reduced. No signs of toxicity were observed. In summary, our results identify mitochondrial KCa3.1 as an unexpected player in cancer cell migration and show that its pharmacological targeting is efficient against both tumor growth and metastatic spread in vivo.

An investigation of the occurrence and properties of the mitochondrial intermediate-conductance Ca2+-activated K+ channel mtKCa3.1.

The mitochondrial intermediate-conductance Ca2+-activated K+ channel mtKCa3.1 has recently been discovered in the HCT116 colon tumor-derived cell line, which expresses relatively high levels of this protein also in the plasma membrane. Electrophysiological recordings revealed that the channel can exhibit different conductance states and kinetic modes, which we tentatively ascribe to post-translational modifications. To verify whether the localization of this channel in mitochondria might be a peculiarity of these cells or a more widespread feature we have checked for the presence of mtKCa3.1 in a few other cell lines using biochemical and electrophysiological approaches. It turned out to be present at least in some of the cells investigated. Functional assays explored the possibility that mtKCa3.1 might be involved in cell proliferation or play a role similar to that of the Shaker-type KV1.3 channel in lymphocytes, which interacts with outer mitochondrial membrane-inserted Bax thereby promoting apoptosis (Szabò, I. et al., Proc. Natl. Acad Sci. USA 105 (2008) 14861-14866). A specific KCa3.1 inhibitor however did not have any detectable effect on cell proliferation or death.

Serum-activated K and Cl currents underlay U87-MG glioblastoma cell migration.

Glioblastoma cells in vivo are exposed to a variety of promigratory signals, including undefined serum components that infiltrate into high grade gliomas as result of blood-brain barrier breakdown. Glioblastoma cell migration has been further shown to depend heavily on ion channels activity. We have then investigated the modulatory effects of fetal calf serum (FCS) on ion channels, and their involvement in U87-MG cells migration. Using the perforated patch-clamp technique we have found that, in a subpopulation of cells (42%), FCS induced: (1) an oscillatory activity of TRAM-34 sensitive, intermediate-conductance calcium-activated K (IK(Ca) ) channels, mediated by calcium oscillations previously shown to be induced by FCS in this cell line; (2) a stable activation of a DIDS- and NPPB-sensitive Cl current displaying an outward rectifying instantaneous current-voltage relationship and a slow, voltage-dependent inactivation. By contrast, in another subpopulation of cells (32%) FCS induced a single, transient IK(Ca) current activation, always accompanied by a stable activation of the Cl current. The remaining cells did not respond to FCS. In order to understand whether the FCS-induced ion channel activities are instrumental to promoting cell migration, we tested the effects of TRAM-34 and DIDS on the FCS-induced U87-MG cell migration using transwell migration assays. We found that these inhibitors were able to markedly reduce U87-MG cell migration in the presence of FCS, and that their co-application resulted in an almost complete arrest of migration. It is concluded that the modulation of K and Cl ion fluxes is essential for the FCS-induced glioblastoma cell migration.