RESUMEN
The opening of the Torpedo CLC-0 chloride (Cl-) channel is known to be regulated by two gating mechanisms: fast gating and slow (common) gating. The structural basis underlying the fast-gating mechanism is better understood than that of the slow-gating mechanism, which is still largely a mystery. Our previous study on the intracellular proton (H+i)-induced inhibition of the CLC-0 anionic current led to the conclusion that the inhibition results from the slow-gate closure (also called inactivation). The conclusion was made based on substantial evidence such as a large temperature dependence of the H+i inhibition similar to that of the channel inactivation, a resistance to the H+i inhibition in the inactivation-suppressed C212S mutant, and a similar voltage dependence between the current recovery from the H+i inhibition and the recovery from the channel inactivation. In this work, we further examine the mechanism of the H+i inhibition of wild-type CLC-0 and several mutants. We observe that an anion efflux through the pore of CLC-0 accelerates the recovery from the H+i-induced inhibition, a process corresponding to the slow-gate opening. Furthermore, various inactivation-suppressed mutants exhibit different current recovery kinetics, suggesting the existence of multiple inactivated states (namely, slow-gate closed states). We speculate that protonation of the pore of CLC-0 increases the binding affinity of permeant anions in the pore, thereby generating a pore blockage of ion flow as the first step of inactivation. Subsequent complex protein conformational changes further transition the CLC-0 channel to deeper inactivated states.
Asunto(s)
Canales de Cloruro , Activación del Canal Iónico , Protones , Canales de Cloruro/metabolismo , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/química , Canales de Cloruro/genética , Activación del Canal Iónico/efectos de los fármacos , Animales , Mutación , CinéticaRESUMEN
TMEM16 proteins, also known as anoctamins, are a family of ten membrane proteins with various tissue expression and subcellular localization. TMEM16A (anoctamin 1) is a plasma membrane protein that acts as a calcium-activated chloride channel. It is expressed in many types of epithelial cells, smooth muscle cells and some neurons. In airway epithelial cells, TMEM16A expression is particularly enhanced by inflammatory stimuli that also promote goblet cell metaplasia and mucus hypersecretion. Therefore, pharmacological modulation of TMEM16A could be beneficial to improve mucociliary clearance in chronic obstructive respiratory diseases. However, the correct approach to modulate TMEM16A activity (activation or inhibition) is still debated. Pharmacological inhibitors of TMEM16A could also be useful as anti-hypertensive agents given the TMEM16A role in smooth muscle contraction. In contrast to TMEM16A, TMEM16F (anoctamin 6) behaves as a calcium-activated phospholipid scramblase, responsible for the externalization of phosphatidylserine on cell surface. Inhibitors of TMEM16F could be useful as anti-coagulants and anti-viral agents. The role of other anoctamins as therapeutic targets is still unclear since their physiological role is still to be defined.
Asunto(s)
Anoctamina-1 , Humanos , Animales , Anoctamina-1/metabolismo , Anoctamina-1/antagonistas & inhibidores , Anoctaminas/metabolismo , Canales de Cloruro/metabolismo , Canales de Cloruro/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Transferencia de Fosfolípidos/antagonistas & inhibidoresRESUMEN
BACKGROUND/AIMS: Extracellular acidic conditions impair cellular activities; however, some cancer cells drive cellular signaling to adapt to the acidic environment. It remains unclear how ovarian cancer cells sense changes in extracellular pH. This study was aimed at characterizing acid-inducible currents in an ovarian cancer cell line and evaluating the involvement of these currents in cell viability. METHODS: The biophysical and pharmacological properties of membrane currents in OV2944, a mouse ovarian cancer cell line, were studied using the whole-cell configuration of the patch-clamp technique. Viability of this cell type in acidic medium was evaluated using the MTT assay. RESULTS: OV2944 had significant acid-sensitive outwardly rectifying (ASOR) Cl- currents at a pH50 of 5.3. The ASOR current was blocked by pregnenolone sulfate (PS), a steroid ion channel modulator that blocks the ASOR channel as one of its targets. The viability of the cells was reduced after exposure to an acidic medium (pH 5.3) but was slightly restored upon PS administration. CONCLUSION: These results offer first evidence for the presence of ASOR Cl- channel in ovarian cancer cells and indicate its involvement in cell viability under acidic environment.
Asunto(s)
Supervivencia Celular , Neoplasias Ováricas , Pregnenolona , Animales , Femenino , Ratones , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Línea Celular Tumoral , Pregnenolona/farmacología , Concentración de Iones de Hidrógeno , Supervivencia Celular/efectos de los fármacos , Canales de Cloruro/metabolismo , Canales de Cloruro/antagonistas & inhibidores , Técnicas de Placa-Clamp , Potenciales de la Membrana/efectos de los fármacosRESUMEN
Chemical or drug treatments are successfully used to treat parasitic nematode infections that impact human, animal and plant health. Many of these exert their effects through modifying neural function underpinning behaviours essential for parasite viability. Selectivity against the parasite may be achieved through distinct pharmacological properties of the parasite nervous system, as exemplified by the success of the ivermectin which target a glutamate-gated chloride channel found only in invertebrates. Despite the success of the ivermectins, emerging resistance and concerns around eco-toxicity are driving the search for new nematocidal chemicals or drugs. Here, we describe the potential of a 5-HT-gated chloride channel MOD-1, which is involved in vital parasite behaviours with constrained distribution in the invertebrate phyla. This ion channel has potential pharmacophores that could be targeted by new nematocidal chemicals and drugs. We have developed a microtiter based bioassay for MOD-1 pharmacology based on its ectopic expression in the Caenorhabditis elegans essential neuron M4. We have termed this technology 'PhaGeM4' for 'Pharmacogenetic targeting of M4 neuron'. Exposure of transgenic worms harbouring ectopically expressed MOD-1 to 5-HT results in developmental arrest. By additional expression of a fluorescence marker in body wall muscle to monitor growth we demonstrate that this assay is suitable for the identification of receptor agonists and antagonists. Indeed, the developmental progression is a robustly quantifiable bioassay that resolves MOD-1 activation by quipazine, 5-carboxyamidotryptamine and fluoxetine and highlight methiothepin as a potent antagonist. This assay has the intrinsic ability to highlight compounds with optimal bioavailability and furthermore to filter out off-target effects. It can be extended to the investigation of other classes of membrane receptors and modulators of neuronal excitation. This approach based on heterologous modulation of the essential M4 neuron function offers a route to discover new effective and selective anthelmintics potentially less confounded by disruptive environmental impact.
Asunto(s)
Caenorhabditis elegans , Canales de Cloruro , Neuronas , Animales , Antinematodos/farmacología , Caenorhabditis elegans/genética , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/genética , Ivermectina/farmacología , Neuronas/fisiología , Farmacogenética , Serotonina/metabolismoRESUMEN
BACKGROUND/AIM: Chloride intracellular channel protein 1 (CLIC1) is known as a promoter of cancer progression, metastasis, and angiogenesis. Thus, CLIC1 could be a future therapeutic target. This study aimed to evaluate the effect of anti-CLIC1 antibodies on tumour cells and vessels of human renal cell carcinoma (RCC) in rabbit cornea and chick embryo chorioallantoic membrane (CAM) models. MATERIALS AND METHODS: Human cc-RCC xenografts on rabbit cornea and CAM surface were performed. Anti-CLIC1 antibodies were applied for 5 consecutive days on both tumor models. We comparatively evaluated treated and untreated tumors by combining ultrasonography with microscopic techniques. RESULTS: RCC implants rapidly recruited blood vessels and had an exponential growth rate on both tumor models. Anti-CLIC1 antibodies suppressed tumor growth by inducing tumor cell necrosis. Tumor vessels regressed rapidly but not completely during anti-CLIC1 antibodies based therapy. CONCLUSION: Anti-CLIC1 antibodies induced tumor necrosis and tumor vasculature regression in human cc-RCC xenografts in both in vivo experimental models.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos Inmunológicos/farmacología , Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/tratamiento farmacológico , Canales de Cloruro/antagonistas & inhibidores , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/tratamiento farmacológico , Neovascularización Patológica , Animales , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Canales de Cloruro/metabolismo , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Masculino , Terapia Molecular Dirigida , Necrosis , Conejos , Transducción de Señal , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND AND PURPOSE: Nematode glutamate-gated chloride channels (GluCls) are targets of ivermectin (IVM) and moxidectin (MOX), structurally dissimilar macrocyclic lactone (ML) anthelmintics. IVM and MOX possess different pharmacokinetics and efficacy profiles but are thought to have the same binding site, through which they allosterically activate GluCls, apart from the GLC-2 receptor, which is antagonized by IVM. Our goal was to determine GLC-2 sensitivity to MOX, investigate residues involved in antagonism of GLC-2, and to identify differences in receptor-level pharmacology between IVM and MOX. EXPERIMENTAL APPROACH: Two-electrode voltage clamp electrophysiology was used to study the pharmacology of Caenorhabditis elegans GLC-2 receptors heterologously expressed in Xenopus laevis oocytes. In silico homology modeling identified Cel-GLC-2 residues Met291 and Gln292 at the IVM binding site that differ from other GluCls; we mutated these residues to those found in ML-sensitive GluCls, and those of filarial nematode GLC-2. KEY RESULTS: We discovered that MOX inhibits wild-type C. elegans GLC-2 receptors roughly 10-fold more potently than IVM, and with greater maximal inhibition of glutamate activation (MOX = 86.9 ± 2.5%; IVM = 57.8 ± 5.9%). IVM was converted into an agonist in the Met291Gln mutant, but MOX remained an antagonist. Glutamate responses were abrogated in a Met291Leu Gln292Thr double mutant (mimicking filarial nematode GLC-2), but MOX and IVM were converted into positive allosteric modulators of glutamate at this construct. CONCLUSIONS AND IMPLICATIONS: Our data provides new insights into differences in receptor-level pharmacology between IVM and MOX and identify residues responsible for ML antagonism of GLC-2.
Asunto(s)
Antihelmínticos/farmacología , Canales de Cloruro/antagonistas & inhibidores , Ivermectina/farmacología , Macrólidos/farmacología , Animales , Sitios de Unión , Caenorhabditis elegans , Femenino , Oocitos , Técnicas de Placa-Clamp , Xenopus laevisRESUMEN
Transmembrane protein 206 (TMEM206), a proton-activated chloride channel, has been implicated in various biochemical processes, including bone metabolism, and has emerged as a novel cancer-related protein in multiple tumor types. However, its role in primary malignant bone tumors, particularly in osteosarcoma (OS), remains unclear. This study is aimed at exploring the effects of TMEM206 gene silencing on the proliferation, migration, invasion, and metastasis of human OS cells in vitro and in vivo using an shRNA-knockdown strategy. We found that TMEM206 is frequently overexpressed and that high levels of TMEM206 correlated with clinical stage and pulmonary metastasis in patients with OS. We provided evidence that TMEM206-silenced OS cancer cells exhibit decreased proliferation, migration, and invasion in vitro. Mechanistically, we identified ß-catenin, a key member of Wnt/ß-catenin signaling, as a downstream effector of TMEM206. TMEM206 silencing inhibits the Wnt/ß-catenin signaling pathway in expression rescue experiments, confirming that TMEM206 silencing attenuates OS cell tumorigenic behavior, at least in part, via the ß-catenin mediated downregulation of Wnt/ß-catenin signaling. More importantly, TMEM206 knockdown-related phenotype changes were replicated in a xenograft nude mouse model where pulmonary metastases of OS cells were suppressed. Together, our results demonstrate that silencing TMEM206 negatively modulates the Wnt/ß-catenin signaling pathway via ß-catenin to suppress proliferation, migration, invasion, and metastasis in OS carcinogenesis, suggesting TMEM206 as a potential oncogenic biomarker and a potential target for OS treatment.
Asunto(s)
Neoplasias Óseas/prevención & control , Canales de Cloruro/antagonistas & inhibidores , Silenciador del Gen , Osteosarcoma/prevención & control , Vía de Señalización Wnt , Animales , Apoptosis , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Proliferación Celular , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pentameric ligand-gated ion channels (pLGICs) activated by the inhibitory neurotransmitter γ-aminobutyric acid (GABA) are expressed widely in both vertebrate and invertebrate species. One of the best characterised insect GABA-gated chloride channels is RDL, an abbreviation of 'resistance to dieldrin', that was originally identified by genetic screening in Drosophila melanogaster. Here we have cloned the analogous gene from the bumblebee Bombus terrestris audax (BtRDL) and examined its pharmacological properties by functional expression in Xenopus oocytes. Somewhat unexpectedly, the sensitivity of BtRDL to GABA, as measured by its apparent affinity (EC50), was influenced by heterologous expression conditions. This phenomenon was observed in response to alterations in the amount of cRNA injected; the length of time that oocytes were incubated before functional analysis; and by the presence or absence of a 3' untranslated region. In contrast, similar changes in expression conditions were not associated with changes in apparent affinity with RDL cloned from D. melanogaster (DmRDL). Changes in apparent affinity with BtRDL were also observed following co-expression of a chaperone protein (NACHO). Similar changes in apparent affinity were observed with an allosteric agonist (propofol) and a non-competitive antagonist (picrotoxinin), indicating that expression-depended changes are not restricted to the orthosteric agonist binding site. Interestingly, instances of expression-dependent changes in apparent affinity have been reported previously for vertebrate glycine receptors, which are also members of the pLGIC super-family. Our observations with BtRDL are consistent with previous data obtained with vertebrate glycine receptors and indicates that agonist and antagonist apparent affinity can be influenced by the level of functional expression in a variety of pLGICs.
Asunto(s)
Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/metabolismo , Drosophila melanogaster/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Regiones no Traducidas 3'/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Abejas/metabolismo , Agonistas de los Canales de Cloruro/farmacología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efectos de los fármacos , Femenino , Picrotoxina/análogos & derivados , Picrotoxina/farmacología , Propofol/farmacología , Receptores de Glicina/metabolismo , Sesterterpenos , Xenopus laevis/metabolismoRESUMEN
EGFR tyrosine kinase inhibitors (TKIs) are mainly used to treat non-small cell lung cancer; however, adverse effects such as severe diarrhea represent a major obstacle towards the continuation of EGFR-TKIs therapy. Chloride channels, which control the fluid flow in the intestinal lumen, are proposed as an important target to remediate EGFR-TKIs-induced diarrhea, but the mechanism remains unclear. The aim of this study was to clarify the mechanism underlying EGFR-TKIs-induced diarrhea with a particular focus on the role of intestinal chloride channels. Here, we show that osimertinib-treated rats exhibit diarrhea and an increase in fecal water content without showing any severe histopathological changes. This diarrhea was attenuated by intraperitoneal treatment with the calcium-activated chloride channel (CaCC) inhibitor CaCCinh-A01. These findings were confirmed in afatinib-treated rats with diarrhea. Moreover, treatment with the Japanese traditional herbal medicine, hangeshashinto (HST), decreased fecal water content and improved fecal appearance in rats treated with EGFR-TKIs. HST inhibited the ionomycin-induced CaCC activation in HEK293 cells in patch-clamp current experiments and its active ingredients were identified. In conclusion, secretory diarrhea induced by treatment with EGFR-TKIs might be partially mediated by the activation of CaCC. Therefore, blocking the CaCC could be a potential new treatment for EGFR-TKI-induced diarrhea.
Asunto(s)
Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/metabolismo , Diarrea/inducido químicamente , Receptores ErbB/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/toxicidad , Acrilamidas/toxicidad , Afatinib/toxicidad , Compuestos de Anilina/toxicidad , Animales , Diarrea/patología , Heces/química , Células HEK293 , Humanos , Masculino , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Tiofenos/farmacología , Agua/químicaRESUMEN
INTRODUCTION: Placental oxidative stress features in pregnancy pathologies but in clinical trials antioxidant supplementation has not improved outcomes. N-acetylcysteine (NAC) stimulates glutathione production and is proposed as a therapeutic agent in pregnancy. However, key elements of N-acetylcysteine biology, including its cellular uptake mechanism, remains unclear. This study explores how the cystine/glutamate transporter xCT may mediate N-acetylcysteine uptake and how N-acetylcysteine alters placental redox status. METHODS: The involvement of xCT in NAC uptake by the human placenta was studied in perfused placenta and Xenopus oocytes. The effect of short-term N-acetylcysteine exposure on the placental villous proteome was determined using LC-MS. The effect of N-acetylcysteine on Maxi-chloride channel activity was investigated in perfused placenta, villous fragments and cell culture. RESULTS: Maternoplacental N-acetylcysteine administration stimulated intracellular glutamate efflux suggesting a role of the exchange transporter xCT, which was localised to the microvillous membrane of the placental syncytiotrophoblast. Placental exposure to a bolus of N-acetylcysteine inhibited subsequent activation of the redox sensitive Maxi-chloride channel independently of glutathione synthesis. Stable isotope quantitative proteomics of placental villi treated with N-acetylcysteine demonstrated changes in pathways associated with oxidative stress, apoptosis and the acute phase response. DISCUSSION: This study suggests that xCT mediates N-acetylcysteine uptake into the placenta and that N-acetylcysteine treatment of placental tissue alters the placental proteome while regulating the redox sensitive Maxi-chloride channel. Interestingly N-acetylcysteine had antioxidant effects independent of the glutathione pathway. Effective placental antioxidant therapy in pregnancy may require maintaining the balance between normalising redox status without inhibiting physiological redox signalling.
Asunto(s)
Acetilcisteína/farmacología , Sistema de Transporte de Aminoácidos y+/genética , Canales de Cloruro/antagonistas & inhibidores , Placenta , Acetilcisteína/metabolismo , Sistema de Transporte de Aminoácidos y+/metabolismo , Animales , Canales de Cloruro/metabolismo , Vellosidades Coriónicas/efectos de los fármacos , Vellosidades Coriónicas/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Ácido Glutámico/efectos de los fármacos , Ácido Glutámico/metabolismo , Células HEK293 , Humanos , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Placenta/efectos de los fármacos , Placenta/metabolismo , Embarazo , Proteoma/efectos de los fármacos , Proteoma/metabolismo , Xenopus laevisRESUMEN
Oral squamous cell carcinoma (OSCC) is a highly aggressive carcinoma with a high incidence of recurrence and distant metastasis. However, the mechanism of epithelial to mesenchymal transition (EMT) during tumor progression and metastasis in OSCC has not yet been fully elucidated. It is well known that the Cl- channel controls cell volume and activates several signaling pathways for cell differentiation. The aim of the present study was to investigate the role of the Cl- channel on EMT in the OSC 20 cell line, which is an OSCC line. OSC-20 cells were cultured with low serum medium containing a Cl- channel blocker NPPB. Morphological changes, gene expression, immunoreactivity, cell volume, and signaling pathway of the NPPB-treated OSC-20 cells were evaluated. The NPPB-treated OSC-20 cells showed typical morphology of mesenchymal cells. The expression levels of the epithelial marker E-cadherin in the NPPB-treated OSC-20 cells were lower than those of the untreated and TGF-ß1-treated OSC-20 cells. On the other hand, mesenchymal markers such as vimentin, ZEB1, and Snail, in the NPPB-treated OSC-20 cells were higher than those in the untreated and TGF-ß1-treated OSC-20 cells. Furthermore, a large number of vimentin-positive cells also appeared in the NPPB-treated OSC-20 cells. Additionally, the cell volume of these cells was significantly increased compared to that of the untreated and TGF-ß1-treated cells. Interestingly, NPPB did not activate the TGF-ß/smad signaling pathway, but activated the Wnt/ß-catenin signaling pathway. These results suggest that Cl- channel dysfunction promoted EMT via activation of the Wnt/ß-catenin signaling pathway in OSCC.
Asunto(s)
Carcinoma de Células Escamosas/patología , Canales de Cloruro/metabolismo , Transición Epitelial-Mesenquimal , Neoplasias de la Boca/patología , Antracenos/farmacología , Antígenos CD/metabolismo , Cadherinas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Tamaño de la Célula/efectos de los fármacos , Canales de Cloruro/antagonistas & inhibidores , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/fisiología , Humanos , Neoplasias de la Boca/metabolismo , Nitrobenzoatos/farmacología , Tripsina/farmacología , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Cataracts have a high incidence and prevalence rate worldwide, and they are the leading cause of blindness. Lens epithelial cell (LEC) apoptosis is often analysed in cataract research since it is the pathological basis of cataracts, except for congenital cataract. Chloride channels are present in ocular tissues, such as in trabecular cells, LECs and other cells. They serve an important role in apoptosis and participate in endoplasmic reticulum (ER) stress and oxidative stress. However, their role in the apoptosis of LECs has not been discussed. The present study examined the effects of the chloride channel blocker 5nitro2(3phenylpropylamino) benzoic acid (NPPB) in human LECs (HLECs) to elucidate the role of NPPB in HLECs and investigate the role and mechanism of chloride channels in cataract formation. HLECs were exposed to NPPB. Cell survival rate was evaluated using Cell Counting Kit8 assays. Oxidative stress was detected as reactive oxygen species (ROS) in cells by using a ROS assay kit. Apoptosis was examined by assessing mitochondrial membrane potential and using a JC1 assay kit, and western blot analysis was performed to measure the expression levels of mitochondrialdependent apoptosis pathwayassociated proteins. ER stress was evaluated by determining the intracellular calcium ion fluorescence intensity, and western blot analysis was performed to measure ER stressassociated protein expression. The results revealed that NPPB treatment decreased the viability of HLECs and increased apoptosis. Additionally, NPPB increased intracellular ROS levels, as well as the number of JC1 monomers and the protein expression levels of Bcell lymphoma2 (Bcl2)associated X and cleaved caspase3, and decreased Bcl2 protein expression. NPPB increased intracellular calcium ions, the protein expression levels of activating transcription factor 6, JNK, C/EBP homologous protein and caspase12, and the phosphorylation of protein kinase Rlike endoplasmic reticulum kinase. Nacetylcysteine and 4phenylbutyric acid inhibited NPPBinduced oxidative stress, ER stress and apoptosis. Therefore, NPPB treatment decreased cell viability and promoted apoptosis of HLECs via the promotion of oxidative and ER stress.
Asunto(s)
Apoptosis/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Cristalino/efectos de los fármacos , Nitrobenzoatos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Caspasa 3/metabolismo , Catarata/tratamiento farmacológico , Catarata/patología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Canales de Cloruro/antagonistas & inhibidores , Retículo Endoplásmico/metabolismo , Humanos , Cristalino/citología , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
OBJECTIVES: Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line. METHODS: Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays. RESULTS: VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane. CONCLUSIONS: We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.
Asunto(s)
Anoctamina-1/metabolismo , Proliferación Celular , Canales de Cloruro/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Neoplasias de la Lengua/metabolismo , Anoctamina-1/antagonistas & inhibidores , Anoctamina-1/genética , Antineoplásicos/farmacología , Apoptosis , Bestrofinas/genética , Bestrofinas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/genética , Ciclopentanos/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Indanos/farmacología , Activación del Canal Iónico , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Unión Proteica , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/secundario , Neoplasias de la Lengua/tratamiento farmacológico , Neoplasias de la Lengua/genética , Neoplasias de la Lengua/patologíaRESUMEN
Chloride intracellular channel 1 (CLIC1) is a sensor of oxidative stress in endothelial cells (EC). However, the mechanism by which CLIC1 mediate the regulation of endothelial dysfunction has not been established. In this study, overexpressed CLIC1 impaired the ability of the vascular cells to resist oxidative damage and promoted cellular senescence. Besides, suppressed CLIC1 protected against cellular senescence and dysfunction in Human Umbilical Vein Endothelial Cells (HUVECs) through the Nrf2/HO-1 pathway. We also found that ROS-activated CLIC1-induced oxidative stress in HUVECs. Nrf2 nuclear translocation was inhibited by CLIC1 overexpression, but was enhanced by IAA94 (CLICs inhibitor) treatment or knockdown of CLIC1. The Nrf2/HO-1 pathway plays a critical role in the anti-oxidative effect of suppressing CLIC1. And inhibition of CLIC1 decreases oxidative stress injury by downregulating the levels of ROS, MDA, and the expression of EC effectors (ICAM1 and VCAM1) protein expression and promotes the activity of superoxide dismutase (SOD). The AMPK-mediated signaling pathway activates Nrf2 through Nrf2 phosphorylation and nuclear translocation, which is also regulated by CLIC1. Moreover, the activation of CLIC1 contributes to H2O2-induced mitochondrial dysfunction and activation of mitochondrial fission. Therefore, elucidation of the mechanisms by which CLIC1 is involved in these pivotal pathways may uncover its therapeutic potential in alleviating ECs oxidative stress and age-related cardiovascular disease development.
Asunto(s)
Senescencia Celular , Canales de Cloruro/metabolismo , Transducción de Señal , Acetilcisteína/farmacología , Senescencia Celular/efectos de los fármacos , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/genética , Hemo-Oxigenasa 1/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Peróxido de Hidrógeno/farmacología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Dinámicas Mitocondriales/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
CLC-2 is a voltage-gated chloride channel that is widely expressed in mammalian tissues. In the central nervous system, CLC-2 appears in neurons and glia. Studies to define how this channel contributes to normal and pathophysiological function in the central nervous system raise questions that remain unresolved, in part due to the absence of precise pharmacological tools for modulating CLC-2 activity. Herein, we describe the development and optimization of AK-42, a specific small-molecule inhibitor of CLC-2 with nanomolar potency (IC50 = 17 ± 1 nM). AK-42 displays unprecedented selectivity (>1,000-fold) over CLC-1, the closest CLC-2 homolog, and exhibits no off-target engagement against a panel of 61 common channels, receptors, and transporters expressed in brain tissue. Computational docking, validated by mutagenesis and kinetic studies, indicates that AK-42 binds to an extracellular vestibule above the channel pore. In electrophysiological recordings of mouse CA1 hippocampal pyramidal neurons, AK-42 acutely and reversibly inhibits CLC-2 currents; no effect on current is observed on brain slices taken from CLC-2 knockout mice. These results establish AK-42 as a powerful tool for investigating CLC-2 neurophysiology.
Asunto(s)
Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Sitios de Unión , Células CHO , Canales de Cloruro CLC-2 , Línea Celular , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Cricetulus , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Hipocampo/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Simulación del Acoplamiento Molecular , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Chrysin, a natural flavonoid available in honey, propolis and medicinal plants, has been shown to be vasorelaxant in some vascular beds. Proper intake of an alimental vasodilator as a food additive may be a promising strategy for prevention and treatment of coronary spasmodic disorders. PURPOSE: TMEM16A-encoded anoctamin 1 (ANO1), a Ca2+ activated Cl- channel (CaCC), plays an important role in the modulation of vascular tone. We tested the possibility that inhibition of CaCCs contributes to chrysin-induced coronary arterial relaxation. METHODS: The vascular tone of the rat coronary artery (RCA) was recorded with a wire myograph. CaCC currents were assessed using whole-cell patch clamp in arterial smooth muscle cell (ASMC) freshly isolated from RCAs. An inhibitor study was performed to explore the mechanisms underlying the vasomotor and electrophysiological effects of chrysin. RESULTS: Pre-incubation with chrysin depressed the contractions elicited by thromboxane A2 analog U46619, vasopressin (VP), depolarization and extracellular Ca2+ elevation/depolarization without significant preference among these vasoconstrictors. Besides, chrysin inhibited both intracellular Ca2+ release-dependent and extracellular Ca2+ influx-dependent components of contractions induced by U46619 or VP. In RCAs pre-contracted with U46619, VP or KCl, chrysin elicited concentration-dependent relaxations, which were weakened by Cl- -deprivation. The electrophysiological study showed that chrysin reduced ANO1-antibody-sensitive CaCC currents and depressed CaCC increments induced by U46619. Inhibitor study showed that both the vasorelaxation and the CaCC current reduction induced by chrysin were attenuated by blocking CaCCs and inhibiting cAMP/PKA and NO/PKG pathways. CONCLUSION: The present findings indicate that inhibition of RCA ASMC CaCC currents, which may be consequential following intracellular Ca2+ availability reduction and activation of cAMP/PKA and NO/cGMP signaling pathways, contributes to chrysin-induced RCA relaxation.
Asunto(s)
Anoctamina-1/metabolismo , Canales de Cloruro/antagonistas & inhibidores , Flavonoides/farmacología , Vasodilatación/efectos de los fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Calcio/metabolismo , Canales de Cloruro/metabolismo , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Flavonoides/administración & dosificación , Masculino , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Vasoconstricción/efectos de los fármacosRESUMEN
PURPOSE: In the present study, we attempted to explore the role of chloride channel 3 (CLC-3) in colorectal cancer (CRC) and its related mechanism. METHODS: First, the expression level of CLC-3 in CRC tumor tissues and cell lines were measured by RT-qPCR, immunohistochemistry or western blot analysis. CLC-3 expression knockdown in CRC cells was achieved by siRNA transfection. The effect of CLC-3 silence on cell viability, cell cycle, invasion and migration of CRC was estimated by CCK8, flow cytometry based cell cycle assay, and transwell assay, respectively. In order to investigate whether Wnt/ß-catenin signaling was perturbed by CLC-3 knockdown, CLC-3 knockdown cells were treated with pathway activator LiCl, followed by the measurement of the expressions of pathway related genes, cell viability, cell cycle, metastasis ability. RESULTS: The expression of CLC-3 was gradually increased from normal adjacent tissues to CRC tumor tissues, and the increase in tumor tissues was related to TNM stages. CLC-3 was overexpressed in four CRC cell lines (HCT116, SW480, LoVo and SW620), compared with NCM460 cells. CLC-3 knockdown significantly reduced cell proliferation, invasion and migration ability, reflected by declined cell viability, arrested G0/G1 cell cycle, decreased invasion and migration ability. In contrast, the declined cell proliferation, invasion and migration of LoVo and SW620 cells induced by CLC-3 knockdown were reversed by the addition of Wnt/ß-catenin activator LiCl. CONCLUSION: CLC-3 contributed to the CRC development and metastasis through Wnt/ß-catenin signaling pathway. CLC-3 could be proposed as the candidate target for CRC treatment.
Asunto(s)
Canales de Cloruro/fisiología , Neoplasias Colorrectales/metabolismo , Vía de Señalización Wnt , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Técnicas de Silenciamiento del Gen , Humanos , Metástasis de la Neoplasia , beta Catenina/metabolismoRESUMEN
The use of synthetic ion transporters for alteration of the concentration of ions across cell membranes has drawn attention from scientists over the last two decades. This ion transport property has been sensibly used to reduce the viability of cancer cells mainly due to the disruption of their ion homeostasis, leading to the perturbation of their abnormal pH gradient. The use of the proanionophore strategy has been recently adopted to increase cellular deliverability and reduce unwanted cytotoxicity towards normal cells. Meanwhile, various anionophores exhibiting non-toxic behavior in epithelial cells have shown a great propensity to be used as a putative treatment for channelopathies like cystic fibrosis. Anionophores with Cl- ion transport mediated antibacterial activities have also been successfully used against clinically relevant bacterial strains, many of which are tolerant towards commercial antibiotics. Recent developments in the chloride ion transport mediated biological activities of anionophores have been mainly discussed in this article. It also highlights other aspects, such as design criteria, targeted delivery options, and others, which can be useful for the further development of selective anionophores for specific biological applications.
Asunto(s)
Antibacterianos/farmacología , Productos Biológicos/farmacología , Canales de Cloruro/antagonistas & inhibidores , Antibacterianos/química , Productos Biológicos/química , Canales de Cloruro/metabolismo , Humanos , Estructura MolecularRESUMEN
Using the patch-clamp method in the whole cell configuration, it was shown that new conjugates of 2-aminothiophene-3-carboxylic acid with adamantane derivatives exhibit the ability to modulate CaCC activity in the single Purkinje neurons of rat cerebellum. It was noted that, depending on the nature of the substitution in the thiophene fragment, the nature of the effect on CaCC varies from inhibition to potentiation of CaCC currents. The described compounds are also blockers of the NMDA receptor ifenprodile site, which may have an additional neuroprotective contribution to the spectrum of biological activity of these compounds.
Asunto(s)
Adamantano/farmacología , Canales de Cloruro/antagonistas & inhibidores , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Tiofenos/farmacología , Animales , Células Cultivadas , Masculino , Neuronas/metabolismo , Neuronas/fisiología , Técnicas de Placa-Clamp/métodos , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
SCO-101 (Endovion) was discontinued 20 years ago as a new drug under development against sickle cell anaemia. Data from the phase 1 studies remained unpublished. New data indicate that SCO-101 might be efficacious as add-on therapy in cancer. Thus, we report the results from the four phase 1 trials performed between 2001 and 2002. Adult volunteers received SCO-101 or placebo in four independent trials. Adverse events were recorded, and SCO-101 was determined for pharmacokinetic analysis. Ninety-two volunteers completed the trials. The most remarkable adverse effect was a transient and dose-dependent increase in unconjugated bilirubin. Plasma SCO-101 elimination was approximately log linear, with apparent oral clearances of between 315 and 2103 mL/h for single doses, and between 121 and 2433 mL/h at steady state following oral administration. There was a marked decrease in clearance with increasing dose, and for repeated dose versus single dose. Tmax was greater, and Cmax and AUC∞ were lower in the fed state compared to the fasted state. Exposure was equivalent in males and females and for African Americans and Caucasians. In conclusion, SCO-101 appears to be a safe drug with a predictable PK profile. Its efficacy as add-on to standard anticancer drugs has yet to be defined.