Effect of Glycine on BV-2 Microglial Cells Treated with Interferon-γ and Lipopolysaccharide.

Microglia are first-line defense antigen-presenting phagocytes in the central nervous system. Activated microglial cells release pro-inflammatory cytokines and can trigger an oxidative burst. The amino acid glycine exerts anti-inflammatory, immunomodulatory and cytoprotective effects and influences cell volume regulation. This study aimed to investigate the role of glycine in the modulation of inflammatory processes in mouse BV-2 microglial cells. Inflammatory stress was induced by lipopolysaccharide/interferon-γ (LPS/IFN-γ) treatment for 24 h in the absence or presence of 1 or 5 mM glycine. Cells were analyzed by flow cytometry for cell volume, side scatter, apoptosis/necrosis and expression of activation-specific surface markers. Apoptosis progression was monitored by life cell imaging. Reduced glutathione/oxidized glutathione (GSH/GSSG) ratios and release of the pro-inflammatory cytokines IL-6 and TNF-α were measured using luminescence-based assays and ELISA, respectively. We found that LPS/IFN-γ-induced apoptosis was decreased and the fraction of living cells was increased by glycine. Expression of the surface markers CD11b, CD54 and CD80 was dose-dependently increased, while IL-6 and TNF-α release was not altered compared to LPS/IFN-γ-treated cells. We showed that in BV-2 microglial cells glycine improves viability and counteracts deleterious responses to LPS/IFN-γ, which might be relevant in neurodegenerative processes associated with inflammation, like Alzheimer's or Parkinson's disease.

A Swelling-Activated Chloride Current in Microglial Cells is Suppressed by Epac and Facilitated by PKA - Impact on Phagocytosis.

BACKGROUND/AIMS: Volume-regulated anion channels (VRACs) are of particular importance in regulating the cell volume (CV) and give rise to the swelling-activated Cl- current (ICl,swell), a main component driving global regulatory volume decrease (RVD) during cell swelling. Because ICl,swell affects numerous CV-regulated processes like migration, we assume that its role is also indispensable for phagocytosis which requires local cell swelling. Noradrenaline (NA) modulates phagocytosis in macrophages and microglial cells, macrophage-related cells in the central nervous system. Therefore we evaluated whether NA modulates ICl,swell and phagocytosis in microglia. METHODS: Experiments were performed in murine microglial BV-2 and primary mouse microglial cells. Patch clamp experiments were performed in BV-2 cells using the amphotericin-perforated method to minimize cytosolic disturbances. Phagocytosis was quantified by scanning electron microscopy. RESULTS: Following activation of ICl,swell by a hypotonic bath solution, noradrenaline, as well as the ß-adrenergic agonist isoproterenol, evoked a transient decrease of ICl,swell. Repeated application of adrenergic agonists caused a decline of this electrical response. Application of the agonist of exchange protein directly activated by cAMP (Epac), 8-pCPT-2-O-Me-cAMP, or the protein kinase A inhibitor H89 caused a persistent suppression of ICl,swell. When isoproterenol was added concomitantly with the hypotonic saline, ICl,swell developed more rapidly compared to control conditions. Uptake of IgG-coated beads was suppressed by NA or H89 when quantified after 15 min of exposure. CONCLUSION: The activation of ß-adrenergic receptors in microglial cells triggers a cAMP-Epac-dependent and a cAMP-PKA-dependent cascade which affects phagocytosis via modulation of the swelling-activated Cl- current ICl,swell.

Acid- and Volume-Sensitive Chloride Currents in Microglial Cells.

Many cell types express an acid-sensitive outwardly rectifying (ASOR) anion current of an unknown function. We characterized such a current in BV-2 microglial cells and then studied its interrelation with the volume-sensitive outwardly rectifying (VSOR) Cl- current and the effect of acidosis on cell volume regulation. We used patch clamp, the Coulter method, and the pH-sensitive dye BCECF to measure Cl- currents and cell membrane potentials, mean cell volume, and intracellular pH, respectively. The ASOR current activated at pH ≤ 5.0 and displayed an I- > Cl- > gluconate- permeability sequence. When compared to the VSOR current, it was similarly sensitive to DIDS, but less sensitive to DCPIB, and insensitive to tamoxifen. Under acidic conditions, the ASOR current was the dominating Cl- conductance, while the VSOR current was apparently inactivated. Acidification caused cell swelling under isotonic conditions and prevented the regulatory volume decrease under hypotonicity. We conclude that acidification, associated with activation of the ASOR- and inactivation of the VSOR current, massively impairs cell volume homeostasis. ASOR current activation could affect microglial function under acidotoxic conditions, since acidosis is a hallmark of pathophysiological events like inflammation, stroke or ischemia and migration and phagocytosis in microglial cells are closely related to cell volume regulation.

Glycine Induces Migration of Microglial BV-2 Cells via SNAT-Mediated Cell Swelling.

BACKGROUND/AIMS: The neutral, non-essential amino acid glycine has manifold functions and effects under physiological and pathophysiological conditions. Besides its function as a neurotransmitter in the central nervous system, glycine also exerts immunomodulatory effects and as an osmolyte it participates in cell volume regulation. During phagocytosis, glycine contributes to (local) cell volume-dependent processes like lamellipodium formation. Similar to the expansion of the lamellipodium we assume that glycine also affects the migration of microglial cells in a cell volume-dependent manner. METHODS: Mean cell volume (MCV) and cell migration were determined using flow cytometry and trans-well migration assays, respectively. Electrophysiological recordings of the cell membrane potential (Vmem) and swelling-dependent chloride (Cl-) currents (IClswell, VSOR, VRAC) were performed using the whole-cell patch clamp technique. RESULTS: In the murine microglial cell line BV-2, flow cytometry analysis revealed that glycine (5 mM) increases the MCV by ∼9%. The glycine-dependent increase in MCV was suppressed by the partial sodium-dependent neutral amino acid transporter (SNAT) antagonist MeAIB and augmented by the Cl- current blocker DCPIB. Electrophysiological recordings showed that addition of glycine activates a Cl- current under isotonic conditions resembling features of the swelling-activated Cl- current (IClswell). The cell membrane potential (Vmem) displayed a distinctive time course after glycine application; initially, glycine evoked a rapid depolarization mediated by Na+-coupled glycine uptake via SNAT, followed by a further gradual depolarization, which was fully suppressed by DCPIB. Interestingly, glycine significantly increased migration of BV-2 cells, which was suppressed by MeAIB, suggesting that SNAT is involved in the migration process of microglial cells. CONCLUSION: We conclude that glycine acts as a chemoattractant for microglial cells presumably by a cell volume-dependent mechanism involving SNAT-mediated cell swelling.

Miniaturization of the Clonogenic Assay Using Confluence Measurement.

The clonogenic assay is a widely used method to study the ability of cells to 'infinitely' produce progeny and is, therefore, used as a tool in tumor biology to measure tumor-initiating capacity and stem cell status. However, the standard protocol of using 6-well plates has several disadvantages. By miniaturizing the assay to a 96-well microplate format, as well as by utilizing the confluence detection function of a multimode reader, we here describe a new and modified protocol that allows comprehensive experimental setups and a non-endpoint, label-free semi-automatic analysis. Comparison of bright field images with confluence images demonstrated robust and reproducible detection of clones by the confluence detection function. Moreover, time-resolved non-endpoint confluence measurement of the same well showed that semi-automatic analysis was suitable for determining the mean size and colony number. By treating cells with an inhibitor of clonogenic growth (PTC-209), we show that our modified protocol is suitable for comprehensive (broad concentration range, addition of technical replicates) concentration- and time-resolved analysis of the effect of substances or treatments on clonogenic growth. In summary, this protocol represents a time- and cost-effective alternative to the commonly used 6-well protocol (with endpoint staining) and also provides additional information about the kinetics of clonogenic growth.

Pharmacological Inhibition of Class IIA HDACs by LMK-235 in Pancreatic Neuroendocrine Tumor Cells.

Histone deacetylases (HDACs) play a key role in epigenetic mechanisms in health and disease and their dysfunction is implied in several cancer entities. Analysis of expression patterns in pancreatic neuroendocrine tumors (pNETs) indicated HDAC5 to be a potential target for future therapies. As a first step towards a possible treatment, the aim of this study was to evaluate the in vitro cellular and molecular effects of HDAC5 inhibition in pNET cells. Two pNET cell lines, BON-1 and QGP-1, were incubated with different concentrations of the selective class IIA HDAC inhibitor, LMK-235. Effects on cell viability were determined using the resazurin-assay, the caspase-assay, and Annexin-V staining. Western Blot and immunofluorescence microscopy were performed to assess the effects on HDAC5 functionality. LMK-235 lowered overall cell viability by inducing apoptosis in a dose- and time-dependent manner. Furthermore, acetylation of histone-H3 increased with higher LMK-235 concentrations, indicating functional inhibition of HDAC4/5. Immunocytochemical analysis showed that proliferative activity (phosphohistone H3 and Ki-67) decreased at highest concentrations of LMK-235 while chromogranin and somatostatin receptor 2 (SSTR2) expression increased in a dose-dependent manner. HDAC5 expression was found to be largely unaffected by LMK-235. These findings indicate LMK-235 to be a potential therapeutic approach for the development of an effective and selective pNET treatment.

In Vitro Cell Death Discrimination and Screening Method by Simple and Cost-Effective Viability Analysis.

BACKGROUND/AIMS: For in vitro cytotoxicity testing, discrimination of apoptosis and necrosis represents valuable information. Viability analysis performed at two different time points post treatment could serve such a purpose because the dynamics of metabolic activity of apoptotic and necrotic cells is different, i.e. a more rapid decline of cellular metabolism during necrosis whereas cellular metabolism is maintained during the entire execution phase of apoptosis. This study describes a straightforward approach to distinguish apoptosis and necrosis. METHODS: A431 human epidermoid carcinoma cells were treated with different concentrations/doses of actinomycin D (Act-D), 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), Ro 31-8220, H2O2 and photodynamic treatment (PDT). The resazurin viability signal was recorded at 2 and 24 hrs post treatment. Apoptosis and necrosis were verified by measuring caspase 3/7 and membrane integrity. RESULTS: Calculation of the difference curve between the 2 and 24 hrs resazurin signals yields the following information: a positive difference signal indicates apoptosis (i.e. high metabolic activity at early time points and low signal at 24 hrs post treatment) while an early reduction of the viability signal indicates necrosis. For all treatments, this dose-dependent sequence of cellular responses could be confirmed by independent assays. CONCLUSION: Simple and cost-effective viability analysis provides reliable information about the dose ranges of a cytotoxic agent where apoptosis or necrosis occurs. This may serve as a starting point for further in-depth characterisation of cytotoxic treatments.

The H+/K+ ATPase Inhibitor SCH-28080 Inhibits Insulin Secretion and Induces Cell Death in INS-1E Rat Insulinoma Cells.

BACKGROUND/AIMS: Glucose-stimulated insulin secretion (GSIS) of pancreatic ß-cells involves glucose uptake and metabolism, closure of KATP channels and depolarization of the cell membrane potential (Vmem), activation of voltage-activated Ca2+ currents (ICav) and influx of Ca2+, which eventually triggers hormone exocytosis. Beside this classical pathway, KATP-independent mechanisms such as changes in intracellular pH (pHi) or cell volume, which also affect ß-cell viability, can elicit or modify insulin release. In ß-cells the regulation of pHi is mainly accomplished by Na+/H+ exchangers (NHEs). To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the non-gastric H+/K+ ATPase in rat insulinoma cells and assessed effects of the H+/K+ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis. METHODS: In INS-1E cell cultures, H+/K+ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pHi) recovery after acute acidic load was measured by NH4Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. Vmem, K+ and Ca2+ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability) and cytotoxicity assays. The mean cell volume (MCV), cell granularity (side-scatter; SSC), phosphatidylserine (PS) exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (ΔΨm) were measured by flow cytometry. RESULTS: We found that the α-subunit of the non-gastric H+/K+ ATPase (HKα2) is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH4Cl prepulsing experiments no K+-dependent pHi recovery was observed under Na+-free extracellular conditions. Nonetheless within 1 h, 20 µM SCH-28080 inhibited GSIS by ∼50%, while basal release was unaffected. The L-type ICav blocker nifedipine caused a full inhibition of GSIS at 10 and 20 µM. At 20 µM, SCH-28080 inhibited ICav comparable to 20 µM nifedipine and in addition augmented IKATP recorded at -60 mV and hyperpolarized Vmem by ∼15 mV. Cell viability 2 and 24 h post treatment with SCH-28080 was dose-dependently inhibited with IC50 values of 22.9 µM and 15.3 µM, respectively. At 20 µM the percentages of Annexin-V+, caspase+ and propidium iodide+ cells were significantly increased after 24 and 48 h. Concurrently, the MCV was significantly decreased (apoptotic volume decrease, AVD) and the SSC signal was increased. At concentrations >40-50 µM, SCH-28080 became progressively cytotoxic causing a steep increase in necrotic cells already 2 h post treatment and a breakdown of ΔΨm within 4 h under 50 and 100 µM while 10 and 20 µM had no effect on ΔΨm within 24 h. CONCLUSION: We demonstrate expression of HKα2 in rat INS-1E cells. However, the pump is apparently non-functional under the given conditions. Nonetheless the H+/K+ ATPase blocker SCH-28080 inhibits insulin secretion and induces cell death. Importantly, we show that SCH-28080 inhibits ICav - and activates KATP channels identifying them as novel "off-targets" of the inhibitor, causing hyperpolarization of Vmem and inhibition of insulin secretion.

Quercetin Stimulates Insulin Secretion and Reduces the Viability of Rat INS-1 Beta-Cells.

BACKGROUND/AIMS: Previously we described insulinotropic effects of Leonurus sibiricus L. plant extracts used for diabetes mellitus treatment in Traditional Mongolian Medicine. The flavonoid quercetin and its glycoside rutin, which exert anti-diabetic properties in vivo by interfering with insulin signaling in peripheral target tissues, are constituents of these extracts. This study was performed to better understand short- and long-term effects of quercetin and rutin on beta-cells. METHODS: Cell viability, apoptosis, phospho-protein abundance and insulin release were determined using resazurin, annexin-V binding assays, Western blot and ELISA, respectively. Membrane potentials (Vmem), whole-cell Ca2+ (ICa)- and ATP-sensitive K+ (IKATP) currents were measured by patch clamp. Intracellular Ca2+ (Cai) levels were measured by time-lapse imaging using the ratiometric Ca2+ indicator Fura-2. RESULTS: Rutin, quercetin and the phosphoinositide-3-kinase (PI3K) inhibitor LY294002 caused a dose-dependent reduction in cell viability with IC50 values of ∼75 µM, ∼25 µM and ∼3.5 µM, respectively. Quercetin (50 µM) significantly increased the percentage of Annexin-V+ cells within 48 hrs. The mean cell volume (MCV) of quercetin-treated cells was significantly lower. Within 2 hrs, quercetin significantly decreased basal- and insulin-stimulated Akt(T308) phosphorylation and increased Erk1/2 phosphorylation, without affecting P-Akt(S473) abundance. Basal- and glucose-stimulated insulin release were significantly stimulated by quercetin. Quercetin significantly depolarized Vmem by ∼25 mV which was prevented by the KATP-channel opener diazoxide, but not by the L-type ICa inhibitor nifedipine. Quercetin significantly stimulated ICa and caused a 50% inhibition of IKATP. The effects on Vmem, ICa and IKATP rapidly reached peak values and then gradually diminished to control values within ∼1 minute. With a similar time-response quercetin induced an elevation in Cai which was completely abolished in the absence of Ca2+ in the bath solution. Rutin (50 µM) did not significantly alter the percentage of Annexin-V+ cells, MCV, Akt or Erk1/2 phosphorylation, insulin secretion, or the electrophysiological behavior of INS-1 cells. CONCLUSION: We conclude that quercetin acutely stimulates insulin release, presumably by transient KATP channel inhibition and ICa stimulation. Long term application of quercetin inhibits cell proliferation and induces apoptosis, most likely by inhibition of PI3K/Akt signaling.

The green tea catechin epigallocatechin gallate induces cell cycle arrest and shows potential synergism with cisplatin in biliary tract cancer cells.

BACKGROUND: The green tea catechin epigallocatechin gallate (EGCG) was shown to effectively inhibit tumor growth in various types of cancer including biliary tract cancer (BTC). For most BTC patients only palliative therapy is possible, leading to a median survival of about one year. Chemoresistance is a major problem that contributes to the high mortality rates of BTC. The aim of this study was to investigate the cytotoxic effect of EGCG alone or in combination with cisplatin on eight BTC cell lines and to investigate the cellular anti-cancer mechanisms of EGCG. METHODS: The effect of EGCG treatment alone or in combination with the standard chemotherapeutic cisplatin on cell viability was analyzed in eight BTC cell lines. Additionally, we analyzed the effects of EGCG on caspase activity, cell cycle distribution and gene expression in the BTC cell line TFK-1. RESULTS: EGCG significantly reduced cell viability in all eight BTC cell lines (p < 0.05 or p < 0.01, respectively, for most cell lines and EGCG concentrations > 5 µM). Combined EGCG and cisplatin treatment showed a synergistic cytotoxic effect in five cell lines and an antagonistic effect in two cell lines. Furthermore, EGCG reduced the mRNA levels of various cell cycle-related genes, while increasing the expression of the cell cycle inhibitor p21 and the apoptosis-related death receptor 5 (p < 0.05). This observation was accompanied by an increase in caspase activity and cells in the sub-G1 phase of the cell cycle, indicating induction of apoptosis. EGCG also induced a down-regulation of expression of stem cell-related genes and genes that are associated with an aggressive clinical character of the tumor, such as cd133 and abcg2. CONCLUSIONS: EGCG shows various anti-cancer effects in BTC cell lines and might therefore be a potential anticancer drug for future studies in BTC. Additionally, EGCG displays a synergistic cytotoxic effect with cisplatin in most tested BTC cell lines. Graphical abstract Summary illustration.

The putative role of the non-gastric H⁺/K⁺-ATPase ATP12A (ATP1AL1) as anti-apoptotic ion transporter: effect of the H⁺/K⁺ ATPase inhibitor SCH28080 on butyrate-stimulated myelomonocytic HL-60 cells.

BACKGROUND/AIMS: The ATP12A gene codes for a non-gastric H(+)/K(+) ATPase, which is expressed in a wide variety of tissues. The aim of this study was to test for the molecular and functional expression of the non-gastric H(+)/K(+) ATPase ATP12A/ATP1AL1 in unstimulated and butyrate-stimulated (1 and 10 mM) human myelomonocytic HL-60 cells, to unravel its potential role as putative apoptosis-counteracting ion transporter as well as to test for the effect of the H(+)/K(+) ATPase inhibitor SCH28080 in apoptosis. METHODS: Real-time reverse-transcription PCR (qRT-PCR) was used for amplification and cloning of ATP12A transcripts and to assess transcriptional regulation. BCECF microfluorimetry was used to assess changes of intracellular pH (pHi) after acute intracellular acid load (NH4Cl prepulsing). Mean cell volumes (MCV) and MCV-recovery after osmotic cell shrinkage (Regulatory Volume Increase, RVI) were assessed by Coulter counting. Flow-cytometry was used to measure MCV (Coulter principle), to assess apoptosis (phosphatidylserine exposure to the outer leaflet of the cell membrane, caspase activity, 7AAD staining) and differentiation (CD86 expression). RESULTS: We found by RT-PCR, intracellular pH measurements, MCV measurements and flow cytometry that ATP12A is expressed in human myelomonocytic HL-60 cells. Treatment of HL-60 cells with 1 mM butyrate leads to monocyte-directed differentiation whereas higher concentrations (10 mM) induce apoptosis as assessed by flow-cytometric determination of CD86 expression, caspase activity, phosphatidylserine exposure on the outer leaflet of the cell membrane and MCV measurements. Transcriptional up-regulation of ATP12A and CD86 is evident in 1 mM butyrate-treated HL-60 cells. The H(+)/K(+) ATPase inhibitor SCH28080 (100 µM) diminishes K(+)-dependent pHi recovery after intracellular acid load and blocks RVI after osmotic cell shrinkage. After seeding, HL-60 cells increase their MCV within the first 24 h in culture, and subsequently decrease it over the course of the next 48 h. This effect can be observed in the overall- and non-apoptotic fraction of both untreated and 1 mM butyrate-treated HL-60 cells, but not in 1 mM butyrate-stimulated phosphatidylserine-positive cells. These cells do not shrink from 24 h to 72 h and have finally a higher MCV than untreated cells unless they are exposed to SCH28080. 10 mM butyrate induces apoptosis within 24 h. CONCLUSION: In summary we show that in HL-60 cells ATP12A is a functionally active H(+)/K(+) ATPase that may counteract events during early apoptosis like intracellular acidosis, loss of intracellular K(+) ions and apoptotic volume decrease. Its expression and/or susceptibility to the H(+)/K(+) ATPase inhibitor SCH28080 becomes most evident in cells exposing phosphatidylserine on the outer leaflet of the cell membrane and therefore during early apoptosis.

Glycine modulates membrane potential, cell volume, and phagocytosis in murine microglia.

Phagocytes form engulfment pseudopodia at the contact area with their target particle by a process resembling cell volume (CV) regulatory mechanisms. We evaluated whether the osmoregulatory active neutral amino acid glycine, which contributes to CV regulation via activation of sodium-dependent neutral amino acid transporters (SNATs) improves phagocytosis in isotonic and hypertonic conditions in the murine microglial cell line BV-2 and primary microglial cells (pMG). In BV-2 cells and pMG, RT-PCR analysis revealed expression of SNATs (Slc38a1, Slc38a2), but not of GlyRs (Glra1-4). In BV-2 cells, glycine (5 mM) led to a rapid Na(+)-dependent depolarization of membrane potential (V mem). Furthermore, glycine increased CV by about 9%. Visualizing of phagocytosis of polystyrene microspheres by scanning electron microscopy revealed that glycine (1 mM) increased the number of BV-2 cells containing at least one microsphere by about 13%. Glycine-dependent increase in phagocytosis was suppressed by the SNAT inhibitor α-(methylamino)isobutyric acid (MeAIB), by replacing extracellular Na(+) with choline, and under hypertonic conditions, but not by the GlyR antagonist strychnine or the GlyR agonist taurine. Interestingly, hypertonicity-induced suppression of phagocytosis was rescued by glycine. These findings demonstrate that glycine increases phagocytosis in iso- and hypertonic conditions by activation of SNATs.

Ca2+ channel blockers reverse iron overload by a new mechanism via divalent metal transporter-1.

Hereditary hemochromatosis and transfusional iron overload are frequent clinical conditions associated with progressive iron accumulation in parenchymal tissues, leading to eventual organ failure. We have discovered a new mechanism to reverse iron overload-pharmacological modulation of the divalent metal transporter-1 (DMT-1). DMT-1 mediates intracellular iron transport during the transferrin cycle and apical iron absorption in the duodenum. Its additional functions in iron handling in the kidney and liver are less well understood. We show that the L-type calcium channel blocker nifedipine increases DMT-1-mediated cellular iron transport 10- to 100-fold at concentrations between 1 and 100 microM. Mechanistically, nifedipine causes this effect by prolonging the iron-transporting activity of DMT-1. We show that nifedipine mobilizes iron from the liver of mice with primary and secondary iron overload and enhances urinary iron excretion. Modulation of DMT-1 function by L-type calcium channel blockers emerges as a new pharmacological therapy for the treatment of iron overload disorders.

Chloride channel blockers suppress formation of engulfment pseudopodia in microglial cells.

BACKGROUND/AIMS: Phagocytosis depends on the formation of engulfment pseudopodia surrounding the target. We tested in microglia, monocyte-derived cells in the brain, whether a swelling-activated Cl(-)-current (I(Cl,swell)), required for global cell volume (CV) regulation, also contributes to local expansion and retraction of engulfment pseudopodia. METHODS: We used scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to visualize and quantify the uptake of polystyrene microbeads (MBs) by microglial cells. Flow cytometry was used for cell volume measurments and I(Cl,swell) was measured by whole-cell patch clamp. RESULTS: We found that exposure of microglial BV-2 cells to MBs in Cl(-)-free extracellular solution attenuated MB uptake and that the Cl(-)-channel blockers DIOA, flufenamic acid, NPPB and DCPIB suppressed the uptake of MBs in BV-2 cells and in primary microglial cells. Microglial cells exposed to MBs in the presence of Cl(-) channel blockers failed to extend engulfment pseudopodia. We observed that cells containing at least three MBs revealed an about twofold increase in current density of I(Cl, swell) compared to cells without MB. Osmotic challenges to stimulate global CV regulation before exposure to MBs modulated phagocytosis. Pre-conditioning of cells in hypo- or hypertonic medium for 12-16 hours caused a decrease in MB uptake. CONCLUSION: These findings indicate that I(Cl,swell) contributes to formation of engulfment pseudopodia and participates in engulfment and particle uptake in microglial cells.

Evidence for the formation of symmetric and asymmetric DLPC-DAPC lipid bilayer domains.

BACKGROUND/AIMS: We investigated if mixtures of the phosphatidylcholine (PC) lipids 1,2-dilauroyl-sn-glycero-3-phosphocholine (C12:0 PC; DLPC) and 1,2-diarachidoyl-sn-glycero-3-phosphocholine (C20:0 PC; DAPC), which differ by eight methylene groups in acyl chain length, lead to the spontaneous formation of distinct lipid rafts and asymmetric bilayers. METHODS: The experiments were performed using Atomic Force Microscopy (AFM). RESULTS: We show that DLPC and DAPC mixed at a molar ratio of 1:1 lead to the formation of single, double and triple bilayers with peaks at 6.14 ± 0.11, 13.27 ± 0.17 and 20.54 ± 0.46 nm, respectively (n=750). Within these formations discrete height steps of 0.92 nm can be resolved (n=422). CONCLUSION: The most frequently observed height steps value of 0.92 nm matches best with the calculated mean lipid hydrophobic thickness difference for asymmetric C12:0 PC and C20:0 PC lipid bilayers of 0.88 nm. This indicates the ability of DLPC and DAPC to form asymmetric lipid bilayers.

Evaluation of Tazemetostat as a Therapeutically Relevant Substance in Biliary Tract Cancer.

Biliary tract cancer (BTC) is a gastrointestinal malignancy associated with a poor survival rate. Current therapies encompass palliative and chemotherapeutic treatment as well as radiation therapy, which results in a median survival of only one year due to standard therapeutic ineffectiveness or resistance. Tazemetostat is an FDA-approved inhibitor of enhancer of Zeste homolog 2 (EZH2), a methyltransferase involved in BTC tumorigenesis via trimethylation of histone 3 at lysine 27 (H3K27me3), an epigenetic mark associated with silencing of tumor suppressor genes. Up to now, there are no data available regarding tazemetostat as a possible treatment option against BTC. Therefore, the aim of our study is a first-time investigation of tazemetostat as a potential anti-BTC substance in vitro. In this study, we demonstrate that tazemetostat affects cell viability and the clonogenic growth of BTC cells in a cell line-dependent manner. Furthermore, we found a strong epigenetic effect at low concentrations of tazemetostat, which was independent of the cytotoxic effect. We also observed in one BTC cell line that tazemetostat increases the mRNA levels and protein expression of the tumor suppressor gene Fructose-1,6-bisphosphatase 1 (FBP1). Interestingly, the observed cytotoxic and epigenetic effects were independent of the mutation status of EZH2. To conclude, our study shows that tazemetostat is a potential anti-tumorigenic substance in BTC with a strong epigenetic effect.

Ouabain at nanomolar concentrations is cytotoxic for biliary tract cancer cells.

Biliary tract cancer is a deadly disease with limited therapeutic options. Ouabain is a well-known inhibitor of the pumping function of Na+/K+-ATPase, though there is evidence that low concentrations of ouabain lead to a reduction of cell viability of cancer cells independent of its inhibition of the pumping function of the Na+/K+-ATPase. Regarding the impact of ouabain on biliary tract cancer, no data is currently available. Therefore, we aimed for a first-time investigation of ouabain as a potential anti-neoplastic biliary tract cancer agent using comprehensive human biliary tract cancer in vitro models. We found that ouabain has a strong cell line-dependent cytotoxic effect with IC50 levels in the (low) nanomolar-range and that this effect was not associated with the mRNA expression levels of the Na+/K+-ATPase α, ß and fxyd-subunits. Regarding the mode of cytotoxicity, we observed induction of apoptosis in biliary tract cancer cells upon treatment with ouabain. Interestingly, cytotoxic effects of ouabain at sub-saturating (< µM) levels were independent of cellular membrane depolarization and changes in intracellular sodium levels. Furthermore, using a 3D cell culture model, we found that ouabain disturbs spheroid growth and reduces the viability of biliary tract cancer cells within the tumor spheroids. In summary, our data suggest that ouabain possesses anti-biliary tract cancer potential at low µM-concentration in 2D and 3D in vitro biliary tract cancer models and encourage further detailed investigation.

Effect of the AMP-kinase modulators AICAR, metformin and compound C on insulin secretion of INS-1E rat insulinoma cells under standard cell culture conditions.

BACKGROUND/AIMS: The function of ß-cells is regulated by nutrient uptake and metabolism. The cells' metabolic state can be expressed as concentration ratios of AMP, ADP and ATP. Relative changes in these ratios regulate insulin release. An increase in the intracellular ATP concentration causes closure of K(ATP) channels and cell membrane depolarization, which triggers stimulus-secretion coupling (SSC). In addition to K(ATP) channels, the AMP-dependent protein kinase (AMPK), a major cellular fuel sensor in a variety of cells and tissues, also affects insulin secretion and ß-cell survival. In a previous study we found that the widely used AMPK inhibitor compound C retards proliferation and induces apoptosis in the rat ß-cell line INS-1E. We therefore tested the effects of AMPK activators (AICAR and metformin), and compound C on AMPK phosphorylation, insulin secretion, K(ATP) channel currents, cell membrane potential, intracellular calcium concentration, apoptosis and cell cycle distribution of INS-1E cells under standard cell culture conditions (11 mM glucose). METHODS: Western blotting, ELISA, patch-clamp, calcium imaging and flow cytometry. RESULTS: We found that basal AMPK phosphorylation is enhanced by AICAR (1 mM) and metformin (1 mM) but remained unaffected by compound C (10 µM). Both AICAR and compound C stimulated basal insulin secretion whereas metformin had no effect. Pre-incubation with AICAR (1 mM) caused an inhibition of K(ATP) currents but did not significantly alter the average cell membrane potential (Vm) or the threshold potential of electrical activity. Acute administration of AICAR (300 µM) led to a depolarization of Vm, which was not due to an inhibition of the basal- or glucose-induced chloride conductance, and was not accompanied by elevations of intracellular calcium (Ca(i)). AICAR had no additive blocking effect on K(ATP) currents when applied together with tolbutamide. Compound C applied over 24 hours induced an increase in the percentage of cells positive for caspase activity, whereas AICAR (1 mM) applied for 48 hours was without effect. Medium glucose concentration <3 mM caused cell cycle arrest, caspase activation and an increase of cell granularity. CONCLUSION: We conclude that under standard cell culture conditions the AMPK modulators AICAR and compound C, but not metformin, stimulate insulin secretion by AMPK-independent mechanisms.

Expression of the non-gastric H+/K+ ATPase ATP12A in normal and pathological human prostate tissue.

Altered cellular proton handling and cell volume regulation are hallmarks of tumorigenesis. To investigate a possible involvement of the non-gastric H(+)/K(+) ATPase ATP12A (ATP1AL1) in prostate cancer, we performed immunohistochemistry in formalin-fixed, paraffin-embedded histological sections from benign and malignant human prostate lesions. Normal prostate tissue displayed a membrane-bound ATP12A staining with focal accumulated pattern, whereas in the benign prostate hyperplasia (BPH) and cancerous prostate tissue (tumor grade I-III) the protein appears to be displaced in the luminal cells of the glandular epithelium. Hence, the expression pattern of ATP12A is markedly altered in BPH and prostate cancer. To test for altered gene expression of ATP12A we performed quantitative reverse transcriptase PCR (QRT-PCR) in normal (tumor-free) prostate tissue, BPH and tumor stages I-III using a prostate cancer cDNA array. However, no significantly different expression levels could be detected in the various disease states compared to normal tissue, which contrasts the findings from immunohistochemistry and points to the possibility of altered post-translational processing and/or sorting of the protein. We further show that ATP12A mRNA is expressed at different levels in PC-3 and LNCaP prostate cancer cells, with a significant ~26-fold higher expression in the latter cell type. Protein expression in these tumor cell lines was verified by Western blot.

Low pH Attenuates Apoptosis by Suppressing the Volume-Sensitive Outwardly Rectifying (VSOR) Chloride Current in Chondrocytes.

In a variety of physiological and pathophysiological conditions, cells are exposed to acidic environments. Severe synovial fluid acidification also occurs in a progressive state of osteoarthritis (OA) affecting articular chondrocytes. In prior studies extracellular acidification has been shown to protect cells from apoptosis but the underlying mechanisms remain elusive. In the present study, we demonstrate that the inhibition of Cl- currents plays a significant role in the antiapoptotic effect of acidification in human articular chondrocytes. Drug-induced apoptosis was analyzed after exposure to staurosporine by caspase 3/7 activity and by annexin-V/7-actinomycin D (7-AAD) staining, followed by flow cytometry. Cell viability was assessed by resazurin, CellTiter-Glo and CellTiter-Fluor assays. Cl- currents and the mean cell volume were determined using the whole cell patch clamp technique and the Coulter method, respectively. The results reveal that in C28/I2 cells extracellular acidification decreases caspase 3/7 activity, enhances cell viability following staurosporine treatment and gradually deactivates the volume-sensitive outwardly rectifying (VSOR) Cl- current. Furthermore, the regulatory volume decrease (RVD) as well as the apoptotic volume decrease (ADV), which represents an early event during apoptosis, were absent under acidic conditions after hypotonicity-induced cell swelling and staurosporine-induced apoptosis, respectively. Like acidosis, the VSOR Cl- current inhibitor DIDS rescued chondrocytes from apoptotic cell death and suppressed AVD after induction of apoptosis with staurosporine. Similar to acidosis and DIDS, the VSOR channel blockers NPPB, niflumic acid (NFA) and DCPIB attenuated the staurosporine-induced AVD. NPPB and NFA also suppressed staurosporine-induced caspase 3/7 activation, while DCPIB and Tamoxifen showed cytotoxic effects per se. From these data, we conclude that the deactivation of VSOR Cl- currents impairs cell volume regulation under acidic conditions, which is likely to play an important role in the survivability of human articular chondrocytes.