A new method to measure aquaporin-facilitated membrane diffusion of hydrogen peroxide and cations in plant suspension cells.

Plant aquaporins (AQPs) facilitate the membrane diffusion of water and small solutes, including hydrogen peroxide (H2 O2 ) and, possibly, cations, essential signalling molecules in many physiological processes. While the determination of the channel activity generally depends on heterologous expression of AQPs in Xenopus oocytes or yeast cells, we established a genetic tool to determine whether they facilitate the diffusion of H2 O2 through the plasma membrane in living plant cells. We designed genetic constructs to co-express the fluorescent H2 O2 sensor HyPer and AQPs, with expression controlled by a heat shock-inducible promoter in Nicotiana tabacum BY-2 suspension cells. After induction of ZmPIP2;5 AQP expression, a HyPer signal was recorded when the cells were incubated with H2 O2 , suggesting that ZmPIP2;5 facilitates H2 O2 transmembrane diffusion; in contrast, the ZmPIP2;5W85A mutated protein was inactive as a water or H2 O2 channel. ZmPIP2;1, ZmPIP2;4 and AtPIP2;1 also facilitated H2 O2 diffusion. Incubation with abscisic acid and the elicitor flg22 peptide induced the intracellular H2 O2 accumulation in BY-2 cells expressing ZmPIP2;5. We also monitored cation channel activity of ZmPIP2;5 using a novel fluorescent photo-switchable Li+ sensor in BY-2 cells. BY-2 suspension cells engineered for inducible expression of AQPs as well as HyPer expression and the use of Li+ sensors constitute a powerful toolkit for evaluating the transport activity and the molecular determinants of PIPs in living plant cells.

Signaling Mechanisms and Pharmacological Modulators Governing Diverse Aquaporin Functions in Human Health and Disease.

The aquaporins (AQPs) are a family of small integral membrane proteins that facilitate the bidirectional transport of water across biological membranes in response to osmotic pressure gradients as well as enable the transmembrane diffusion of small neutral solutes (such as urea, glycerol, and hydrogen peroxide) and ions. AQPs are expressed throughout the human body. Here, we review their key roles in fluid homeostasis, glandular secretions, signal transduction and sensation, barrier function, immunity and inflammation, cell migration, and angiogenesis. Evidence from a wide variety of studies now supports a view of the functions of AQPs being much more complex than simply mediating the passive flow of water across biological membranes. The discovery and development of small-molecule AQP inhibitors for research use and therapeutic development will lead to new insights into the basic biology of and novel treatments for the wide range of AQP-associated disorders.

Novel Ion Channel Targets and Drug Delivery Tools for Controlling Glioblastoma Cell Invasiveness.

Comprising more than half of all brain tumors, glioblastoma multiforme (GBM) is a leading cause of brain cancer-related deaths worldwide. A major clinical challenge is presented by the capacity of glioma cells to rapidly infiltrate healthy brain parenchyma, allowing the cancer to escape control by localized surgical resections and radiotherapies, and promoting recurrence in other brain regions. We propose that therapies which target cellular motility pathways could be used to slow tumor dispersal, providing a longer time window for administration of frontline treatments needed to directly eradicate the primary tumors. An array of signal transduction pathways are known to be involved in controlling cellular motility. Aquaporins (AQPs) and voltage-gated ion channels are prime candidates as pharmacological targets to restrain cell migration in glioblastoma. Published work has demonstrated AQPs 1, 4 and 9, as well as voltage-gated potassium, sodium and calcium channels, chloride channels, and acid-sensing ion channels are expressed in GBM and can influence processes of cell volume change, extracellular matrix degradation, cytoskeletal reorganization, lamellipodial and filopodial extension, and turnover of cell-cell adhesions and focal assembly sites. The current gap in knowledge is the identification of optimal combinations of targets, inhibitory agents, and drug delivery systems that will allow effective intervention with minimal side effects in the complex environment of the brain, without disrupting finely tuned activities of neuro-glial networks. Based on published literature, we propose that co-treatments using AQP inhibitors in addition to other therapies could increase effectiveness, overcoming some limitations inherent in current strategies that are focused on single mechanisms. An emerging interest in nanobodies as drug delivery systems could be instrumental for achieving the selective delivery of combinations of agents aimed at multiple key targets, which could enhance success in vivo.

5-Hydroxymethyl-Furfural and Structurally Related Compounds Block the Ion Conductance in Human Aquaporin-1 Channels and Slow Cancer Cell Migration and Invasion.

Aquaporin-1 (AQP1) dual water and ion channels enhance migration and invasion when upregulated in leading edges of certain classes of cancer cells. Work here identifies structurally related furan compounds as novel inhibitors of AQP1 ion channels. 5-Hydroxymethyl-2-furfural (5HMF), a component of natural medicinal honeys, and three structurally related compounds, 5-nitro-2-furoic acid (5NFA), 5-acetoxymethyl-2-furaldehyde (5AMF), and methyl-5-nitro-2-furoate (M5NF), were analyzed for effects on water and ion channel activities of human AQP1 channels expressed in Xenopus oocytes. Two-electrode voltage clamp showed dose-dependent block of the AQP1 ion current by 5HMF (IC50 0.43 mM), 5NFA (IC50 1.2 mM), and 5AMF (IC50 ∼3 mM) but no inhibition by M5NF. In silico docking predicted the active ligands interacted with glycine 165, located in loop D gating domains surrounding the intracellular vestibule of the tetrameric central pore. Water fluxes through separate intrasubunit pores were unaltered by the furan compounds (at concentrations up to 5 mM). Effects on cell migration, invasion, and cytoskeletal organization in vitro were tested in high-AQP1-expressing cancer lines, colon cancer (HT29) and AQP1-expressing breast cancer (MDA), and low-AQP1-expressing SW480. 5HMF, 5NFA, and 5AMF selectively impaired cell motility in the AQP1-enriched cell lines. In contrast, M5NF immobilized all the cancer lines by disrupting actin cytoskeleton. No reduction in cell viability was observed at doses that were effective in blocking motility. These results define furans as a new class of AQP1 ion channel inhibitors for basic research and potential lead compounds for development of therapeutic agents targeting aquaporin channel activity. SIGNIFICANCE STATEMENT: 5-Hydroxymethyl-2-furfural (5HMF), a component of natural medicinal honeys, blocks the ion conductance but not the water flux through human Aquaporin-1 (AQP1) channels and impairs AQP1-dependent cell migration and invasiveness in cancer cell lines. Analyses of 5HMT and structural analogs demonstrate a structure-activity relationship for furan compounds, supported by in silico docking modeling. This work identifies new low-cost pharmacological antagonists for AQP1 available to researchers internationally. Furans merit consideration as a new class of therapeutic agents for controlling cancer metastasis.

Development of a Photoswitchable Lithium-Sensitive Probe to Analyze Nonselective Cation Channel Activity in Migrating Cancer Cells.

This is the first work to use a newly designed Li+-selective photoswitchable probe Sabrina Heng Lithium (SHL) in living colon cancer cells to noninvasively monitor cation channel activity in real time by the appearance of lithium hot spots detected by confocal microscopy. Punctate Li+ hot spots are clustered in the lamellipodial leading edges of HT29 human colon cancer cells and are colocalized with aquaporin-1 (AQP1) channels. AQP1 is a dual water and cyclic-nucleotide-gated cation channel located in lamellipodia and is essential for rapid cell migration in a subset of aggressive cancers. Both the Li+ hot spots and cell migration are blocked in HT29 cells by the AQP1 ion channel antagonist AqB011. In contrast, Li+ hot spots are not evident in a poorly migrating colon cancer cell line, SW620, which lacks comparable membrane expression of AQP1. Knockdown of AQP1 by RNA interference in HT29 cells significantly impairs Li+ hot spot activity. The SHL probe loaded in living cells shows signature chemical properties of ionic selectivity and reversibility. Dynamic properties of the Li+ hot spots, turning on and off, are confirmed by time-lapse imaging. SHL is a powerful tool for evaluating cation channel function in living cells in real time, with particular promise for studies of motile cells or interlinked networks not easily analyzed by electrophysiological methods. The ability to reset SHL by photoswitching allows monitoring of dynamic signals over time. Future applications of the Li+ probe could include high-throughput optical screening for discovering new classes of channels, or finding new pharmacological modulators for nonselective cation channels.

Bumetanide-Derived Aquaporin 1 Inhibitors, AqB013 and AqB050 Inhibit Tube Formation of Endothelial Cells through Induction of Apoptosis and Impaired Migration In Vitro.

AqB013 and AqB050 compounds inhibit aquaporin 1 (AQP1), a dual water and ion channel implicated in tumour angiogenesis. We tested AqB013 and AqB050 either as monotherapy or in combination on tube formation of murine endothelial cells (2H-11 and 3B-11) and human umbilical vascular endothelial cells (HUVECs). The mechanism underlying their anti-tubulogenic effect was explored by examining cell viability, induction of apoptosis and migration using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, Annexin V/propidium iodide apoptosis assay and scratch wound assay. Tube formation of all the cell lines was inhibited by AqB013, AqB050 and the combination of the two compounds. The inhibition of 2H-11 and 3B-11 was frequently accompanied by impaired migration, whereas that of HUVEC treated with AqB050 and the combination was associated with reduced cell viability due to apoptosis. AqB013 and AqB050 exhibited an anti-tubulogenic effect through inhibition of AQP1-mediated cell migration and induction of apoptosis. Together with previously reported anti-tumour cell effect of AqB013 and AqB050, our findings support further evaluation of these compounds as potential cancer therapeutics.

Bacopasides I and II Act in Synergy to Inhibit the Growth, Migration and Invasion of Breast Cancer Cell Lines.

Bacopaside (bac) I and II are triterpene saponins purified from the medicinal herb Bacopa monnieri. Previously, we showed that bac II reduced endothelial cell migration and tube formation and induced apoptosis in colorectal cancer cell lines. The aim of the current study was to examine the effects of treatment with combined doses of bac I and bac II using four cell lines representative of the breast cancer subtypes: triple negative (MDA-MB-231), estrogen receptor positive (T47D and MCF7) and human epidermal growth factor receptor 2 (HER2) positive (BT-474). Drug treatment outcome measures included cell viability, proliferation, cell cycle, apoptosis, migration, and invasion assays. Relationships were analysed by one- and two-way analysis of variance with Bonferroni post-hoc analysis. Combined doses of bac I and bac II, each below their half maximal inhibitory concentration (IC50), were synergistic and reduced the viability and proliferation of the four breast cancer cell lines. Cell loss occurred at the highest dose combinations and was associated with G2/M arrest and apoptosis. Migration in the scratch wound assay was significantly reduced at apoptosis-inducing combinations, but also at non-cytotoxic combinations, for MDA-MB-231 and T47D (p < 0.0001) and BT-474 (p = 0.0003). Non-cytotoxic combinations also significantly reduced spheroid invasion of MDA-MB-231 cells by up to 97% (p < 0.0001). Combining bac I and II below their IC50 reduced the viability, proliferation, and migration and invasiveness of breast cancer cell lines, suggesting synergy between bac I and II.

Fundamental structural and functional properties of Aquaporin ion channels found across the kingdoms of life.

Aquaporin (AQP) channels in the major intrinsic protein (MIP) family are known to facilitate transmembrane water fluxes in prokaryotes and eukaryotes. Some classes of AQPs also conduct ions, glycerol, urea, CO2 , nitric oxide, and other small solutes. Ion channel activity has been demonstrated for mammalian AQPs 0, 1, 6, Drosophila Big Brain (BIB), soybean nodulin 26, and rockcress AtPIP2;1. More classes are likely to be discovered. Newly identified blockers are providing essential tools for establishing physiological roles of some of the AQP dual water and ion channels. For example, the arylsulfonamide AqB011 which selectively blocks the central ion pore of mammalian AQP1 has been shown to impair migration of HT29 colon cancer cells. Traditional herbal medicines are sources of selective AQP1 inhibitors that also slow cancer cell migration. The finding that plant AtPIP2;1 expressed in root epidermal cells mediates an ion conductance regulated by calcium and protons provided insight into molecular mechanisms of environmental stress responses. Expression of lens MIP (AQP0) is essential for maintaining the structure, integrity and transparency of the lens, and Drosophila BIB contributes to neurogenic signalling pathways to control the developmental fate of fly neuroblast cells; however, the ion channel roles remain to be defined for MIP and BIB. A broader portfolio of pharmacological agents is needed to investigate diverse AQP ion channel functions in situ. Understanding the dual water and ion channel roles of AQPs could inform the development of novel agents for rational interventions in diverse challenges from agriculture to human health.

The Aquaporin 1 Inhibitor Bacopaside II Reduces Endothelial Cell Migration and Tubulogenesis and Induces Apoptosis.

Expression of aquaporin-1 (AQP1) in endothelial cells is critical for their migration and angiogenesis in cancer. We tested the AQP1 inhibitor, bacopaside II, derived from medicinal plant Bacopa monnieri, on endothelial cell migration and tube-formation in vitro using mouse endothelial cell lines (2H11 and 3B11) and human umbilical vein endothelial cells (HUVEC). The effect of bacopaside II on viability, apoptosis, migration and tubulogenesis was assessed by a proliferation assay, annexin-V/propidium iodide flow cytometry, the scratch wound assay and endothelial tube-formation, respectively. Cell viability was reduced significantly for 2H11 at 15 µM (p = 0.037), 3B11 at 12.5 µM (p = 0.017) and HUVEC at 10 µM (p < 0.0001). At 15 µM, the reduced viability was accompanied by an increase in apoptosis of 38%, 50% and 32% for 2H11, 3B11 and HUVEC, respectively. Bacopaside II at ≥10 µM significantly reduced migration of 2H11 (p = 0.0002) and 3B11 (p = 0.034). HUVECs were most sensitive with a significant reduction at ≥7.5 µM (p = 0.037). Tube-formation was reduced with a 15 µM dose for all cell lines and 10 µM for 3B11 (p < 0.0001). These results suggest that bacopaside II is a potential anti-angiogenic agent.

Role of Aquaporin 1 Signalling in Cancer Development and Progression.

Cancer is a major health burden worldwide. Despite the advances in our understanding of its pathogenesis and continued improvement in cancer management and outcomes, there remains a strong clinical demand for more accurate and reliable biomarkers of metastatic progression and novel therapeutic targets to abrogate angiogenesis and tumour progression. Aquaporin 1 (AQP1) is a small hydrophobic integral transmembrane protein with a predominant role in trans-cellular water transport. Recently, over-expression of AQP1 has been associated with many types of cancer as a distinctive clinical prognostic factor. This has prompted researchers to evaluate the link between AQP1 and cancer biological functions. Available literature implicates the role of AQP1 in tumour cell migration, invasion and angiogenesis. This article reviews the current understanding of AQP1-facilitated tumour development and progression with a focus on regulatory mechanisms and downstream signalling pathways.

Divalent Cations Regulate the Ion Conductance Properties of Diverse Classes of Aquaporins.

Aquaporins (AQPs) are known to facilitate water and solute fluxes across barrier membranes. An increasing number of AQPs are being found to serve as ion channels. Ion and water permeability of selected plant and animal AQPs (plant Arabidopsis thaliana AtPIP2;1, AtPIP2;2, AtPIP2;7, human Homo sapiens HsAQP1, rat Rattus norvegicus RnAQP4, RnAQP5, and fly Drosophilamelanogaster DmBIB) were expressed in Xenopus oocytes and examined in chelator-buffered salines to evaluate the effects of divalent cations (Ca2+, Mg2+, Ba2+ and Cd2+) on ionic conductances. AtPIP2;1, AtPIP2;2, HsAQP1 and DmBIB expressing oocytes had ionic conductances, and showed differential sensitivity to block by external Ca2+. The order of potency of inhibition by Ca2+ was AtPIP2;2 > AtPIP2;1 > DmBIB > HsAQP1. Blockage of the AQP cation channels by Ba2+ and Cd2+ caused voltage-sensitive outward rectification. The channels with the highest sensitivity to Ca2+ (AtPIP2;1 and AtPIP2;2) showed a distinctive relief of the Ca2+ block by co-application of excess Ba2+, suggesting that divalent ions act at the same site. Recognizing the regulatory role of divalent cations may enable the discovery of other classes of AQP ion channels, and facilitate the development of tools for modulating AQP ion channels. Modulators of AQPs have potential value for diverse applications including improving salinity tolerance in plants, controlling vector-borne diseases, and intervening in serious clinical conditions involving AQPs, such as cancer metastasis, cardiovascular or renal dysfunction.

Bumetanide Derivatives AqB007 and AqB011 Selectively Block the Aquaporin-1 Ion Channel Conductance and Slow Cancer Cell Migration.

Aquaporins (AQPs) in the major intrinsic family of proteins mediate fluxes of water and other small solutes across cell membranes. AQP1 is a water channel, and under permissive conditions, a nonselective cation channel gated by cGMP. In addition to mediating fluid transport, AQP1 expression facilitates rapid cell migration in cell types including colon cancers and glioblastoma. Work here defines new pharmacological derivatives of bumetanide that selectively inhibit the ion channel, but not the water channel, activity of AQP1. Human AQP1 was analyzed in the Xenopus laevis oocyte expression system by two-electrode voltage clamp and optical osmotic swelling assays. The aquaporin ligand bumetanide derivative AqB011 was the most potent blocker of the AQP1 ion conductance (IC50 of 14 µM), with no effect on water channel activity (at up to 200 µM). The order of potency for inhibition of the ionic conductance was AqB011 > AqB007 >> AqB006 ≥ AqB001. Migration of human colon cancer (HT29) cells was assessed with a wound-closure assay in the presence of a mitotic inhibitor. AqB011 and AqB007 significantly reduced migration rates of AQP1-positive HT29 cells without affecting viability. The order of potency for AQP1 ion channel block matched the order for inhibition of cell migration, as well as in silico modeling of the predicted order of energetically favored binding. Docking models suggest that AqB011 and AqB007 interact with the intracellular loop D domain, a region involved in AQP channel gating. Inhibition of AQP1 ionic conductance could be a useful adjunct therapeutic approach for reducing metastasis in cancers that upregulate AQP1 expression.

Differential Inhibition of Water and Ion Channel Activities of Mammalian Aquaporin-1 by Two Structurally Related Bacopaside Compounds Derived from the Medicinal Plant Bacopa monnieri.

Aquaporin-1 (AQP1) is a major intrinsic protein that facilitates flux of water and other small solutes across cell membranes. In addition to its function as a water channel in maintaining fluid homeostasis, AQP1 also acts as a nonselective cation channel gated by cGMP, a property shown previously to facilitate rapid cell migration in a AQP1-expressing colon cancer cell line. Here we report two new modulators of AQP1 channels, bacopaside I and bacopaside II, isolated from the medicinal plant Bacopa monnieri Screening was conducted in the Xenopus oocyte expression system, using quantitative swelling and two-electrode voltage clamp techniques. Results showed bacopaside I blocked both the water (IC50 117 µM) and ion channel activities of AQP1 but did not alter AQP4 activity, whereas bacopaside II selectively blocked the AQP1 water channel (IC50 18 µM) without impairing the ionic conductance. These results fit with predictions from in silico molecular modeling. Both bacopasides were tested in migration assays using HT29 and SW480 colon cancer cell lines, with high and low levels of AQP1 expression, respectively. Bacopaside I (IC50 48 µM) and bacopaside II (IC50 14 µM) impaired migration of HT29 cells but had minimal effect on SW480 cell migration. Our results are the first to identify differential AQP1 modulators isolated from a medicinal plant. Bacopasides could serve as novel lead compounds for pharmaceutic development of selective aquaporin modulators.

AqF026 is a pharmacologic agonist of the water channel aquaporin-1.

Aquaporin-1 (AQP1) facilitates the osmotic transport of water across the capillary endothelium, among other cell types, and thereby has a substantial role in ultrafiltration during peritoneal dialysis. At present, pharmacologic agents that enhance AQP1-mediated water transport, which would be expected to increase the efficiency of peritoneal dialysis, are not available. Here, we describe AqF026, an aquaporin agonist that is a chemical derivative of the arylsulfonamide compound furosemide. In the Xenopus laevis oocyte system, extracellular AqF026 potentiated the channel activity of human AQP1 by >20% but had no effect on channel activity of AQP4. We found that the intracellular binding site for AQP1 involves loop D, a region associated with channel gating. In a mouse model of peritoneal dialysis, AqF026 enhanced the osmotic transport of water across the peritoneal membrane but did not affect the osmotic gradient, the transport of small solutes, or the localization and expression of AQP1 on the plasma membrane. Furthermore, AqF026 did not potentiate water transport in Aqp1-null mice, suggesting that indirect mechanisms involving other channels or transporters were unlikely. Last, in a mouse gastric antrum preparation, AqF026 did not affect the Na-K-Cl cotransporter NKCC1. In summary, AqF026 directly and specifically potentiates AQP1-mediated water transport, suggesting that it deserves additional investigation for applications such as peritoneal dialysis or clinical situations associated with defective water handling.

Protective roles of peroxiporins AQP0 and AQP11 in human astrocyte and neuronal cell lines in response to oxidative and inflammatory stressors.

In addition to aquaporin (AQP) classes AQP1, AQP4 and AQP9 known to be expressed in mammalian brain, our recent transcriptomic analyses identified AQP0 and AQP11 in human cortex and hippocampus at levels correlated with age and Alzheimer's disease (AD) status; however, protein localization remained unknown. Roles of AQP0 and AQP11 in transporting hydrogen peroxide (H2O2) in lens and kidney prompted our hypothesis that up-regulation in brain might similarly be protective. Established cell lines for astroglia (1321N1) and neurons (SHSY5Y, differentiated with retinoic acid) were used to monitor changes in transcript levels for human AQPs (AQP0 to AQP12) in response to inflammation (simulated with 10-100 ng/ml lipopolysaccharide [LPS], 24 h), and hypoxia (5 min N2, followed by 0 to 24 h normoxia). AQP transcripts up-regulated in both 1321N1 and SHSY5Y included AQP0, AQP1 and AQP11. Immunocytochemistry in 1321N1 cells confirmed protein expression for AQP0 and AQP11 in plasma membrane and endoplasmic reticulum; AQP11 increased 10-fold after LPS and AQP0 increased 0.3-fold. In SHSY5Y cells, AQP0 expression increased 0.2-fold after 24 h LPS; AQP11 showed no appreciable change. Proposed peroxiporin roles were tested using melondialdehyde (MDA) assays to quantify lipid peroxidation levels after brief H2O2. Boosting peroxiporin expression by LPS pretreatment lowered subsequent H2O2-induced MDA responses (∼50%) compared with controls; conversely small interfering RNA knockdown of AQP0 in 1321N1 increased lipid peroxidation (∼17%) after H2O2, with a similar trend for AQP11 siRNA. Interventions that increase native brain peroxiporin activity are promising as new approaches to mitigate damage caused by aging and neurodegeneration.

Aquaporins and Ion Channels as Dual Targets in the Design of Novel Glioblastoma Therapeutics to Limit Invasiveness.

Current therapies for Glioblastoma multiforme (GBM) focus on eradicating primary tumors using radiotherapy, chemotherapy and surgical resection, but have limited success in controlling the invasive spread of glioma cells into a healthy brain, the major factor driving short survival times for patients post-diagnosis. Transcriptomic analyses of GBM biopsies reveal clusters of membrane signaling proteins that in combination serve as robust prognostic indicators, including aquaporins and ion channels, which are upregulated in GBM and implicated in enhanced glioblastoma motility. Accumulating evidence supports our proposal that the concurrent pharmacological targeting of selected subclasses of aquaporins and ion channels could impede glioblastoma invasiveness by impairing key cellular motility pathways. Optimal sets of channels to be selected as targets for combined therapies could be tailored to the GBM cancer subtype, taking advantage of differences in patterns of expression between channels that are characteristic of GBM subtypes, as well as distinguishing them from non-cancerous brain cells such as neurons and glia. Focusing agents on a unique channel fingerprint in GBM would further allow combined agents to be administered at near threshold doses, potentially reducing off-target toxicity. Adjunct therapies which confine GBM tumors to their primary sites during clinical treatments would offer profound advantages for treatment efficacy.

Unexpected Classes of Aquaporin Channels Detected by Transcriptomic Analysis in Human Brain Are Associated with Both Patient Age and Alzheimer's Disease Status.

The altered expression of known brain Aquaporin (AQP) channels 1, 4 and 9 has been correlated with neuropathological AD progression, but possible roles of other AQP classes in neurological disease remain understudied. The levels of transcripts of all thirteen human AQP subtypes were compared in healthy and Alzheimer's disease (AD) brains by statistical analyses of microarray RNAseq expression data from the Allen Brain Atlas database. Previously unreported, AQPs 0, 6 and 10, are present in human brains at the transcript level. Three AD-affected brain regions, hippocampus (HIP), parietal cortex (PCx) and temporal cortex (TCx), were assessed in three subgroups: young controls (n = 6, aged 24-57); aged controls (n = 26, aged 78-99); and an AD cohort (n = 12, aged 79-99). A significant positive correlation (p < 10-10) was seen for AQP transcript levels as a function of the subject's age in years. Differential expressions correlated with brain region, age, and AD diagnosis, particularly between the HIP and cortical regions. Interestingly, three classes of AQPs (0, 6 and 8) upregulated in AD compared to young controls are permeable to H2O2. Of these, AQPs 0 and 8 were increased in TCx and AQP6 in HIP, suggesting a role of AQPs in AD-related oxidative stress. The outcomes here are the first to demonstrate that the expression profile of AQP channels in the human brain is more diverse than previously thought, and transcript levels are influenced by both age and AD status. Associations between reactive oxygen stress and neurodegenerative disease risk highlight AQPs 0, 6, 8 and 10 as potential therapeutic targets.

Pharmacological Inhibition of Membrane Signaling Mechanisms Reduces the Invasiveness of U87-MG and U251-MG Glioblastoma Cells In Vitro.

Impairing the motility of glioblastoma multiforme (GBM) cells is a compelling goal for new approaches to manage this highly invasive and rapidly lethal human brain cancer. Work here characterized an array of pharmacological inhibitors of membrane ion and water channels, alone and in combination, as tools for restraining glioblastoma spread in human GBM cell lines U87-MG and U251-MG. Aquaporins, AMPA glutamate receptors, and ion channel classes (shown to be upregulated in human GBM at the transcript level and linked to mechanisms of motility in other cell types) were selected as pharmacological targets for analyses. Effective compounds reduced the transwell invasiveness of U87-MG and U251-MG glioblastoma cells by 20-80% as compared with controls, without cytotoxicity. The compounds and doses used were: AqB013 (14 µM); nifedipine (25 µM); amiloride (10 µM); apamin (10 µM); 4-aminopyridine (250 µM); and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; 30 µM). Invasiveness was quantified in vitro across transwell filter chambers layered with extracellular matrix. Co-application of each of the ion channel agents with the water channel inhibitor AqB013 augmented the inhibition of invasion (20 to 50% greater than either agent alone). The motility impairment achieved by co-application of pharmacological agents differed between the GBM proneural-like subtype U87-MG and classical-like subtype U251-MG, showing patterns consistent with relative levels of target channel expression (Human Protein Atlas database). In addition, two compounds, xanthurenic acid and caelestine C (from the Davis Open Access Natural Product-based Library, Griffith University QLD), were discovered to block invasion at micromolar doses in both GBM lines (IC50 values from 0.03 to 1 µM), without cytotoxicity, as measured by full mitochondrial activity under conditions matching those in transwell assays and by normal growth in spheroid assays. Mechanisms of action of these agents based on published work are likely to involve modulation of glutamatergic receptor signaling. Treating glioblastoma by the concurrent inhibition of multiple channel targets could be a powerful approach for slowing invasive cell spread without cytotoxic side effects, potentially enhancing the effectiveness of clinical interventions focused on eradicating primary tumors.

Reducing the Invasiveness of Low- and High-Grade Endometrial Cancers in Both Primary Human Cancer Biopsies and Cell Lines by the Inhibition of Aquaporin-1 Channels.

Aquaporin (AQP) channels in endometrial cancer (EC) cells are of interest as pharmacological targets to reduce tumor progression. A panel of compounds, including AQP1 ion channel inhibitors (AqB011 and 5-(phenoxymethyl) furan-2-carbaldehyde, PMFC), were used to test the hypothesis that inhibition of key AQPs can limit the invasiveness of low- and high-grade EC cells. We evaluated the effects on transwell migration in EC cell lines (Ishikawa, MFE-280) and primary EC cells established from surgical tissues (n = 8). Quantitative PCR uncovered classes of AQPs not previously reported in EC that are differentially regulated by hormonal signaling. With estradiol, Ishikawa showed increased AQPs 5, 11, 12, and decreased AQPs 0 and 4; MFE-280 showed increased AQPs 0, 1, 3, 4, 8, and decreased AQP11. Protein expression was confirmed by Western blot and immunocytochemistry. AQPs 1, 4, and 11 were colocalized with plasma membrane marker; AQP8 was intracellular in Ishikawa and not detectable in MFE-280. AQP1 ion channel inhibitors (AqB011; PMFC) reduced invasiveness of EC cell lines in transwell chamber and spheroid dispersal assays. In Ishikawa cells, transwell invasiveness was reduced ~41% by 80 µM AqB011 and ~55% by 0.5 mM 5-PMFC. In MFE-280, 5-PMFC inhibited invasion by ~77%. In contrast, proposed inhibitors of AQP water pores (acetazolamide, ginsenoside, KeenMind, TGN-020, IMD-0354) were not effective. Treatments of cultured primary EC cells with AqB011 or PMFC significantly reduced the invasiveness of both low- and high-grade primary EC cells in transwell chambers. We confirmed the tumors expressed moderate to high levels of AQP1 detected by immunohistochemistry, whereas expression levels of AQP4, AQP8, and AQP11 were substantially lower. The anti-invasive potency of AqB011 treatment for EC tumor tissues showed a positive linear correlation with AQP1 expression levels. In summary, AQP1 ion channels are important for motility in both low- and high-grade EC subtypes. Inhibition of AQP1 is a promising strategy to inhibit EC invasiveness and improve patient outcomes.

Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties.

Human aquaporin 1 (hAQP1) forms homotetrameric channels that facilitate fluxes of water and small solutes across cell membranes. In addition to water channel activity, hAQP1 displays non-selective monovalent cation-channel activity gated by intracellular cyclic GMP. Dual water and ion-channel activity of hAQP1, thought to regulate cell shape and volume, could offer a target for novel therapeutics relevant to controlling cancer cell invasiveness. This study probed properties of hAQP1 ion channels using proteoliposomes, which, unlike conventional cell-based systems such as Xenopus laevis oocytes, are relatively free of background ion channels. Histidine-tagged recombinant hAQP1 protein was synthesized and purified from the methylotrophic yeast, Pichia pastoris, and reconstituted into proteoliposomes for biophysical analyses. Osmotic water channel activity confirmed correct folding and channel assembly. Ion-channel activity of hAQP1-Myc-His6 was recorded by patch-clamp electrophysiology with excised patches. In symmetrical potassium, the hAQP1-Myc-His6 channels displayed coordinated gating, a single-channel conductance of approximately 75 pS, and multiple subconductance states. Applicability of this method for structure-function analyses was tested using hAQP1-Myc-His6 D48A/D185A channels modified by site-directed mutations of charged Asp residues estimated to be adjacent to the central ion-conducting pore of the tetramer. No differences in conductance were detected between mutant and wild-type constructs, suggesting the open-state conformation could differ substantially from expectations based on crystal structures. Nonetheless, the method pioneered here for AQP1 demonstrates feasibility for future work defining structure-function relationships, screening pharmacological inhibitors, and testing other classes in the broad family of aquaporins for previously undiscovered ion-conducting capabilities.