Electrical excitability of cancer cells-CELEX model updated.

The normal functioning of every cell in the body depends on its bioelectric properties and many diseases are caused by genetic and/or epigenetic dysregulation of the underlying ion channels. Metastasis, the main cause of death from cancer, is a complex multi-stage process in which cells break away from a primary tumour, invade the surrounding tissues, enter the circulation by encountering a blood vessel and spread around the body, ultimately lodging in distant organs and reproliferating to form secondary tumours leading to devastating organ failure. Such cellular behaviours are well known to involve ion channels. The CELEX model offers a novel insight to metastasis where it is the electrical excitation of the cancer cells that is responsible for their aggressive and invasive behaviour. In turn, the hyperexcitability is underpinned by concomitant upregulation of functional voltage-gated sodium channels and downregulation of voltage-gated potassium channels. Here, we update the in vitro and in vivo evidence in favour of the CELEX model for carcinomas. The results are unequivocal for the sodium channel. The potassium channel arm is also broadly supported by existing evidence although these data are complicated by the impact of the channels on the membrane potential and consequent secondary effects. Finally, consistent with the CELEX model, we show (i) that carcinomas are indeed electrically excitable and capable of generating action potentials and (ii) that combination of a sodium channel inhibitor and a potassium channel opener can produce a strong, additive anti-invasive effect. We discuss the possible clinical implications of the CELEX model in managing cancer.

Ranolazine: a potential anti-metastatic drug targeting voltage-gated sodium channels.

BACKGROUND: Multi-faceted evidence from a range of cancers suggests strongly that de novo expression of voltage-gated sodium channels (VGSCs) plays a significant role in driving cancer cell invasiveness. Under hypoxic conditions, common to growing tumours, VGSCs develop a persistent current (INaP) which can be blocked selectively by ranolazine. METHODS: Several different carcinomas were examined. We used data from a range of experimental approaches relating to cellular invasiveness and metastasis. These were supplemented by survival data mined from cancer patients. RESULTS: In vitro, ranolazine inhibited invasiveness of cancer cells especially under hypoxia. In vivo, ranolazine suppressed the metastatic abilities of breast and prostate cancers and melanoma. These data were supported by a major retrospective epidemiological study on breast, colon and prostate cancer patients. This showed that risk of dying from cancer was reduced by ca.60% among those taking ranolazine, even if this started 4 years after the diagnosis. Ranolazine was also shown to reduce the adverse effects of chemotherapy on heart and brain. Furthermore, its anti-cancer effectiveness could be boosted by co-administration with other drugs. CONCLUSIONS: Ranolazine, alone or in combination with appropriate therapies, could be reformulated as a safe anti-metastatic drug offering many potential advantages over current systemic treatment modalities.

Ion Transporting Proteins and Cancer: Progress and Perspectives.

Ion transporting proteins (ITPs) comprise a wide range of ion channels, exchangers, pumps and ionotropic receptors many of which are expressed in tumours and contribute dynamically to the different components and stages of the complex cancer process, from initiation to metastasis. In this promising major field of biomedical research, several candidate ITPs have emerged as clinically viable. Here, we consider a series of general issues concerning the oncological potential of ITPs focusing on voltage-gated sodium channels as a 'case study'. First, we outline some key properties of 'cancer' as a whole. These include epigenetics, stemness, metastasis, heterogeneity, neuronal characteristics and bioelectricity. Cancer specificity of ITP expression is evaluated in relation to tissue restriction, splice variance, functional specificity and macro-molecular complexing. As regards clinical potential, diagnostics is covered with emphasis on enabling early detection. For therapeutics, we deal with molecular approaches, drug repurposing and combinations. Importantly, we emphasise the need for carefully designed clinical trials. We highlight also the area of 'social responsibility' and the need to involve the public (cancer patients and healthy individuals) in the work of cancer research professionals as well as clinicians. In advising patients how best to manage cancer, and live with it, we offer the following four principles: Awareness and prevention, early detection, specialist, integrated care, and psychological support. Finally, we highlight four key prerequisites for commercialisation of ITP-based technologies against cancer. We conclude that ITPs offer significant potential as regards both understanding the intricacies of the complex process of cancer and for developing much needed novel therapies.

Lidocaine Inhibits Rat Prostate Cancer Cell Invasiveness and Voltage-Gated Sodium Channel Expression in Plasma Membrane.

There is increasing evidence, mostly from breast cancer, that use of local anaesthetics during surgery can inhibit disease recurrence by suppressing the motility of the cancer cells dependent on inherent voltage-gated sodium channels (VGSCs). Here, the possibility that lidocaine could affect cellular behaviours associated with metastasis was tested using the Dunning cell model of rat prostate cancer. Mostly, the strongly metastatic (VGSC-expressing) Mat-LyLu cells were used under both normoxic and hypoxic conditions. The weakly metastatic AT-2 cells served for comparison in some experiments. Lidocaine (1-500 µM) had no effect on cell viability or growth but suppressed Matrigel invasion dose dependently in both normoxia and hypoxia. Used as a control, tetrodotoxin produced similar effects. Exposure to hypoxia increased Nav1.7 mRNA expression but VGSCα protein level in plasma membrane was reduced. Lidocaine under both normoxia and hypoxia had no effect on Nav1.7 mRNA expression. VGSCα protein expression was suppressed by lidocaine under normoxia but no effect was seen in hypoxia. It is concluded that lidocaine can suppress prostate cancer invasiveness without effecting cellular growth or viability. Extended to the clinic, the results would suggest that use of lidocaine, and possibly other local anaesthetics, during surgery can suppress any tendency for post-operative progression of prostate cancer.

PREDICTION OF IRON DEFICIENCY IN CHILDREN USING EASY LABORATORY TOOLS.

Iron deficiency anemia is a common condition in children that can impair growth and development. This study aimed to evaluate the utility of easy, routinely available laboratory tests in predicting iron deficiency anemia. Medical records of 55 children (mean age 4.9±3.2 years) with laboratory evaluation of anemia were analyzed retrospectively. Parameters included complete blood count, serum iron, total iron binding capacity (TIBC), serum ferritin and transferrin saturation. Based on reference ranges, hemoglobin, hematocrit, serum ferritin and transferrin saturation were reduced below normal, indicating clear iron deficiency. Although within normal limits, red blood cell count, mean corpuscular volume, mean corpuscular hemoglobin and serum iron were close to lower limits, suggesting subtle deficiency. In contrast, red cell distribution width, platelet count, white blood cell count, TIBC and unsaturated iron binding capacity were unaffected. In conclusion, hemoglobin, and hematocrit from complete blood count, along with subtle changes in some of the red cell indices, can strongly suggest, iron deficiency anemia in children, Taking into consideration the increased body demand of iron in this age group with follow-up to ensure a desirable response, with less need for more advanced iron studies. These widely available, inexpensive tests may facilitate early detection and treatment of this common pediatric condition.

Integrative Management of Pancreatic Cancer (PDAC): Emerging Complementary Agents and Modalities.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. The standard first-line treatment for PDAC is gemcitabine chemotherapy which, unfortunately, offers only limited chance of a lasting cure. This review further evaluates the hypothesis that the effectiveness of gemcitabine can be improved by combining it with evidence-based complementary measures. Previously, supported by clinical trial data, we suggested that a number of dietary factors and nutraceuticals can be integrated with gemcitabine therapy. Here, we evaluate a further 10 agents for which no clinical trials have (yet) been carried out but there are promising data from in vivo and/or in vitro studies including experiments involving combined treatments with gemcitabine. Two groups of complementary agents are considered: Dietary factors (resveratrol, epigallocatechin gallate, vitamin B9, capsaicin, quercetin and sulforaphane) and nutraceutical agents (artemisinin, garcinol, thymoquinone and emodin). In addition, we identified seven promising agents for which there is currently only basic (mostly in vitro) data. Finally, as a special case of combination therapy, we highlighted synergistic drug combinations involving gemcitabine with "repurposed" aspirin or metformin. We conclude overall that integrated management of PDAC currently is likely to produce the best outcome for patients and for this a wide range of complementary measures is available.

A Predictive Test in The Screening of HIV Positive Cases; Interferon Gamma Inducible Protein 10 (IP-10).

BACKGROUND: HIV (human immunodeficiency virus), causing acquired immunodeficiency syndrome (AIDS), is one of the most important health problems in the world. Certain cytokines produced during the cytokine storm in an acute infection can be biomarker candidates. The strong association of IFN-γ inducible protein 10 (IP-10) with low CD4 cell counts suggests that it can be an acute phase biomarker. METHODS: In this study, IP-10 was monitored at routine controls during pre-treatment and/or in subsequent phases of treatment, and its correlation with CD4 cell count and viral load was assessed. Venous blood samples, taken from 30 patients (at 0 - 3 - 6 months), and 20 healthy volunteers, were sent to the Laboratory for flow cytometry, nucleic acid tests (NAT) and ELISA tests. RESULTS: The mean IP-10 concentration of patients was 344 pg/mL, and these values for the untreated, treated and control groups were 422 pg/mL and 210 pg/mL and 68 pg/mL respectively. A statistically significant difference was found between the IP-10 values of the patient and control groups (p = 0.006). There was a significant, positive and moderate relation between IP-10 and viral load values (r = 0.59, p < 0.001), while there was a significant, negative and moderate relation between IP-10 and CD4 cell count (r = -0.51, p < 0.001). CONCLUSIONS: IP-10 levels in early HIV-1 patients, which are shown to be closely related to CD4 cell levels and viral replication, may be an alternative or support marker compared to the more expensive viral load tests in monitor-ing viremia changes and response to antiretroviral treatment.

Protective effect of liraglutide on experimental testicular ischaemia reperfusion in rats.

This study was performed to evaluate the effect of liraglutide on experimental testicular ischaemia reperfusion in rats in terms of biochemistry, histopathology and immunohistochemistry. A total of 28 male Wistar-Albino rats were divided randomly into 4 groups: control (7), sham (7), ischaemia-reperfusion (7) and ischaemia-reperfusion + liraglutide (7). Biochemically, Nitric Oxide, Malondialdehyde, Superoxide dismutase, Glutathione peroxidase and Catalase levels were measured in the testis. Apoptosis protease activating factor-1 and inducible nitric oxide synthase activity were evaluated immunohistochemically as well. Statistical analyses were made via the Kruskal-Wallis and Mann-Whitney U tests. In the reperfusion group, CAT and SOD values were increased (p > .05), NO and MDA values were decreased (p < .05) after administration of liraglutide. In addition, GPx values were significantly increased in ischaemia reperfusion + liraglutide administered group compared to reperfusion group (p < .05). Apaf-1 and iNOS activity were significantly decreased with the addition of liraglutide treatment to the ischaemia-reperfusion group (p < .05). First of all, we would like to say that liraglutide treatment is moderately preventive against I/R injury in testicular torsion. The anti-inflammatory, antioxidant and antiapoptotic properties of liraglutide are create a moderately protective effect as we show in this study.

Why Do Muse Stem Cells Present an Enduring Stress Capacity? Hints from a Comparative Proteome Analysis.

Muse cells are adult stem cells that are present in the stroma of several organs and possess an enduring capacity to cope with endogenous and exogenous genotoxic stress. In cell therapy, the peculiar biological properties of Muse cells render them a possible natural alternative to mesenchymal stromal cells (MSCs) or to in vitro-generated pluripotent stem cells (iPSCs). Indeed, some studies have proved that Muse cells can survive in adverse microenvironments, such as those present in damaged/injured tissues. We performed an evaluation of Muse cells' proteome under basic conditions and followed oxidative stress treatment in order to identify ontologies, pathways, and networks that can be related to their enduring stress capacity. We executed the same analysis on iPSCs and MSCs, as a comparison. The Muse cells are enriched in several ontologies and pathways, such as endosomal vacuolar trafficking related to stress response, ubiquitin and proteasome degradation, and reactive oxygen scavenging. In Muse cells, the protein-protein interacting network has two key nodes with a high connectivity degree and betweenness: NFKB and CRKL. The protein NFKB is an almost-ubiquitous transcription factor related to many biological processes and can also have a role in protecting cells from apoptosis during exposure to a variety of stressors. CRKL is an adaptor protein and constitutes an integral part of the stress-activated protein kinase (SAPK) pathway. The identified pathways and networks are all involved in the quality control of cell components and may explain the stress resistance of Muse cells.

Overview of the oncogenic signaling pathways in colorectal cancer: Mechanistic insights.

Colorectal cancer is a multifaceted disease which is therapeutically challenging. Based on insights gleaned from almost a quarter century of research, it is obvious that deregulation of spatio-temporally controlled signaling pathways play instrumental role in development and progression of colorectal cancer. High-throughput technologies have helped to develop a sharper and broader understanding of the wide ranging signal transduction cascades which also contribute to development of drug resistance, loss of apoptosis and, ultimately, of metastasis. In this review, we have set the spotlight on role of JAK/STAT, TGF/SMAD, Notch, WNT/ß-Catenin, SHH/GLI and p53 pathways in the development and progression of colorectal cancer. We have also highlighted recent reports on TRAIL-mediated pathways and molecularly distinct voltage-gated sodium channels in colorectal cancer.

A comparative study on normal and obese mice indicates that the secretome of mesenchymal stromal cells is influenced by tissue environment and physiopathological conditions.

BACKGROUND: The term mesenchymal stromal cells (MSCs) designates an assorted cell population comprised of stem cells, progenitor cells, fibroblasts, and stromal cells. MSCs contribute to the homeostatic maintenance of many organs through paracrine and long-distance signaling. Tissue environment, in both physiological and pathological conditions, may affect the intercellular communication of MSCs. METHODS: We performed a secretome analysis of MSCs isolated from subcutaneous adipose tissue (sWAT) and visceral adipose tissue (vWAT), and from bone marrow (BM), of normal and obese mice. RESULTS: The MSCs isolated from tissues of healthy mice share a common core of released factors: components of cytoskeletal and extracellular structures; regulators of basic cellular functions, such as protein synthesis and degradation; modulators of endoplasmic reticulum stress; and counteracting oxidative stress. It can be hypothesized that MSC secretome beneficially affects target cells by the horizontal transfer of many released factors. Each type of MSC may exert specific signaling functions, which could be determined by looking at the many factors that are exclusively released from every MSC type. The vWAT-MSCs release factors that play a role in detoxification activity in response to toxic substances and drugs. The sWAT-MSC secretome contains proteins involved in in chondrogenesis, osteogenesis, and angiogenesis. Analysis of BM-MSC secretome revealed that these cells exert a signaling function by remodeling extracellular matrix structures, such as those containing glycosaminoglycans. Obesity status profoundly modified the secretome content of MSCs, impairing the above-described activity and promoting the release of inflammatory factors. CONCLUSION: We demonstrated that the content of MSC secretomes depends on tissue microenvironment and that pathological condition may profoundly alter its composition. Video abstract.

The other side of the medallion in heart failure: Reverse metabolic syndrome.

BACKGROUND AND AIMS: Lower levels of cardiovascular risk factors are associated with an increase in mortality in H.F. To explain this paradox, the term reverse metabolic syndrome (RMetS) has recently been proposed. We suggest defining these patients with lower levels of three risk factors can be combined under the heading "RMetS." We aimed to investigate the effect of MetS and RMetS on hemodynamic parameters and prognosis in patients with H.F. and reduced ejection fraction (HFrEF). METHODS AND RESULTS: We included 304 patients who were performed right heart catheterization and followed up for a median of 16 (0-48) months. We first grouped patients according to the presence of MetS or not, then we added the RMetS category and stratified patients into three groups as MetS, RMetS, and metabolic healthy. Compared with not MetS group, Pulmonary arterial pressures and VO2 were higher in MetS group. In the second step, LVEF, CI, VO2I, O2 delivery, and LVSWI were lowest in RMetS, pulmonary artery pressures were higher in MetS group. In multivariate Cox regression analysis, being in RMetS group was associated with 2.4 times and 1.8 times increased risk for composite end point (CEP) and all-cause mortality, respectively. In Kaplan Meier analysis, RMetS had the highest all-cause mortality and CEP. CONCLUSIONS: We determined that RMetS patients had the worst prognosis with unfavorable hemodynamic profile. Hence, a better understanding of the pathophysiology of RMetS may help refine the treatment targets of CV risk factors, may yield new interventions targeting catabolic syndrome.

Effect of prolonged application of single-step self-etching primer and hydrofluoric acid on the surface roughness and shear bond strength of CAD/CAM materials.

This in vitro study aimed to assess the influence of different concentrations and durations of hydrofluoric acid (HF) and Monobond Etch & Prime (MEP) etching on the surface roughness (Ra ) of different CAD/CAM materials and on the shear bond strength (SBS) of a self-adhesive resin bonded to the materials. Seventy specimens of hybrid ceramic, leucite-based glass-ceramic, lithium disilicate glass-ceramic, and zirconia-reinforced lithium silicate glass-ceramic were prepared and divided into seven groups according to the surface treatments: Control (C); MEP etching for 60 (MEP60 ) and 120 (MEP120 ) s; 5% HF etching for 60 (HF-5%60 ) and 120 (HF-5%120 ) s; 9.5% HF etching for 60 (HF-9.5%60 ) and 120 (HF-9.5%120 ) s. The Ra was measured using a 3D profilometer. All groups were treated with a universal primer except for the C, MEP60 , and MEP120 groups. A self-adhesive resin cement was bonded to all specimens, and the bond strengths were measured using a universal testing machine. All surface treatments increased both Ra and SBS values compared to the control in each material. Neither the duration of surface treatments nor the HF acid concentrations had a statistically significant effect on SBS. Within the limitations of this experimental study, it can be concluded that Monobond Etch & Prime may be a preferable method to achieve high bond strength values.

Propranolol inhibits neonatal Nav1.5 activity and invasiveness of MDA-MB-231 breast cancer cells: Effects of combination with ranolazine.

The MDA-MB-231 cell line was used as a model of triple negative breast cancer to investigate the interaction of ß-adrenergic receptor (ß-AR) and voltage-gated sodium channel (VGSC). There was significant (86%) overlap in their expression. Short-term (acute) application of the ß-AR antagonist propranolol (25 µM) led to reduction of peak current and quickening of current inactivation (the latter occurred only in 5% fetal bovine serum). Long-term (48 hr) incubation with propranolol (25 µM) resulted in several changes in VGSC characteristics: shifts in (a) current-voltage relationship and (b) steady-state inactivation, both to more negative potentials and (c) the slowing of recovery from inactivation. We then investigated the effects of propranolol and ranolazine, a blocker of VGSC activity, alone and in combination, on lateral motility and Matrigel invasion. These assays were carried out under hypoxic conditions more representative of tumor progression. Propranolol (2.5 and 25 µM) and ranolazine (5 µM), and their combination inhibited lateral motility. Also, propranolol (25 µM) and ranolazine (5 µM), and their combination inhibited invasion. However, no synergy was observed in the pharmacological combinations for both assays. Propranolol also significantly decreased total neonatal Nav1.5 protein expression, the predominant VGSC subtype expressed in these cells. We conclude (a) that ß-AR and VGSC are functionally coupled in MDA-MB-231 cells; (b) that propranolol has direct blocking action on the VGSC; (c) that the action of propranolol is modulated by serum; and (d) that the antimetastatic cellular effects of propranolol and ranolazine are not additive.

Colorectal cancer invasiveness in vitro: Predominant contribution of neonatal Nav1.5 under normoxia and hypoxia.

Functional expression of voltage-gated Na+ channels (VGSCs) occurs in human carcinomas and promotes invasiveness in vitro and metastasis in vivo. Both neonatal and adult forms of Nav1.5 (nNav1.5 and aNav1.5, respectively) have been reported to be expressed at messenger RNA (mRNA) level in colorectal cancer (CRCa) cells. Here, three CRCa cell lines (HT29, HCT116 and SW620) were studied and found to express nNav1.5 mRNA and protein. In SW620 cells, adopted as a model, effects of gene silencing (by several small interfering RNAs [siRNAs]) selectively targeting nNav1.5 or aNav1.5 were determined on (a) channel activity and (b) invasiveness in vitro. Silencing nNav1.5 made the currents more "adult-like" and suppressed invasion by up to 73%. Importantly, subsequent application of the highly specific, general VGSC blocker, tetrodotoxin (TTX), had no further effect. Conversely, silencing aNav1.5 made the currents more "neonatal-like" but suppressed invasion by only 17% and TTX still induced a significant effect. Hypoxia increased invasiveness and this was also blocked completely by siRNA targeting nNav1.5. The effect of hypoxia was suppressed dose dependently by ranolazine, but its effect was lost in cells pretreated with nNav1.5-siRNA. We conclude that (a) functional nNav1.5 expression is common to human CRCa cells, (b) hypoxia increases the invasiveness of SW620 cells, (c) the VGSC-dependent invasiveness is driven predominantly by nNav1.5 under both normoxic and hypoxic conditions and (d) the hypoxia-induced increase in invasiveness is likely to be mediated by the persistent current component of nNav1.5.

Effect of monolithic CAD-CAM ceramic thickness on resin cement polymerization: An in-vitro study.

PURPOSE: To evaluate the effect of different thicknesses of CAD-CAM ceramic sections on the polymerization of two different resin cements. METHODS: Three CAD-CAM all-ceramic restorative materials were sectioned with four different thicknesses. A total of 240 resin cement specimens were prepared from light cured and dual cure resin cements and absorption peaks were recorded. 10 samples of each resin cement were examined before and after polymerization and served as the control group. Data were analyzed using one-way ANOVA, independent t- and Tukey HSD tests (P< 0.05). RESULTS: Control group showed the highest DOC values while samples cured under Vita Enamic section with a thickness of 2 mm presented the lowest values (P< 0.05). Polymerization performed under sections of 0.5 and 1 mm thicknesses provided statistically higher values. Dual cured resin cement samples showed higher DOC values compared to light cured resin cement samples. IPS Empress CAD sections with 0.5 and 1 mm thickness exhibited statistically higher values than other ceramics of the same thickness for light cured resin cement samples. A significant difference was observed between IPS Empress CAD and Vita Enamic while comparing ceramic sections of the same thickness (P< 0.05). There was no difference for sections of 1.5 and 2 mm (P>> 0.05). CLINICAL SIGNIFICANCE: Thickness of the restorative material for an indirect restoration is a key element to determine the type of resin cement.

Mesenchymal stem cell differentiation: Control by calcium-activated potassium channels.

Mesenchymal stem cells (MSCs) are widely used in modern medicine for which understanding the mechanisms controlling their differentiation is fundamental. Ion channels offer novel insights to this process because of their role in modulating membrane potential and intracellular milieu. Here, we evaluate the contribution of calcium-activated potassium (KCa ) channels to the three main components of MSC differentiation: initiation, proliferation, and migration. First, we demonstrate the importance of the membrane potential (Vm ) and the apparent association of hyperpolarization with differentiation. Of KCa subtypes, most evidence points to activity of big-conductance channels in inducing initiation. On the other hand, intermediate-conductance currents have been shown to promote progression through the cell cycle. While there is no information on the role of KCa channels in migration of MSCs, work from other stem cells and cancer cells suggest that intermediate-conductance and to a lesser extent big-conductance channels drive migration. In all cases, these effects depend on species, tissue origin and lineage. Finally, we present a conceptual model that demonstrates how KCa activity could influence differentiation by regulating Vm and intracellular Ca2+ oscillations. We conclude that KCa channels have significant involvement in MSC differentiation and could potentially enable novel tissue engineering approaches and therapies.

The Arabidopsis cellulose synthase complex: a proposed hexamer of CESA trimers in an equimolar stoichiometry.

Cellulose is the most abundant renewable polymer on Earth and a major component of the plant cell wall. In vascular plants, cellulose synthesis is catalyzed by a large, plasma membrane-localized cellulose synthase complex (CSC), visualized as a hexameric rosette structure. Three unique cellulose synthase (CESA) isoforms are required for CSC assembly and function. However, elucidation of either the number or stoichiometry of CESAs within the CSC has remained elusive. In this study, we show a 1:1:1 stoichiometry between the three Arabidopsis thaliana secondary cell wall isozymes: CESA4, CESA7, and CESA8. This ratio was determined utilizing a simple but elegant method of quantitative immunoblotting using isoform-specific antibodies and (35)S-labeled protein standards for each CESA. Additionally, the observed equimolar stoichiometry was found to be fixed along the axis of the stem, which represents a developmental gradient. Our results complement recent spectroscopic analyses pointing toward an 18-chain cellulose microfibril. Taken together, we propose that the CSC is composed of a hexamer of catalytically active CESA trimers, with each CESA in equimolar amounts. This finding is a crucial advance in understanding how CESAs integrate to form higher order complexes, which is a key determinate of cellulose microfibril and cell wall properties.