Chloride Intracellular Channel 1 Protein Research Articles

Abstract LB_B11: Transmembrane chloride intracellular channel 1 (tmCLIC1) protein as akey regulator of colorectal cancers' aggressiveness

Abstract Colorectal cancer is the third leading cause of death in developed countries. Although the huge progresses reached in therapeutically approach, metastases remain the main issue treating colorectal cancer. Ion channels have a prominent role in the acquirement of aggressive phenotype, but most of them are involved in normal physiological processes. Among all the new potential biomarkers, Chloride Intracellular Channel 1(CLIC1) is a promising candidate. As a matter of fact, in physiological homeostasis, CLIC1 is expressed by all cell types as a cytosolic monomer. CLIC1 is a metamorphic protein that coexist in both cytosolic and transmembrane form (tmCLIC1), the latter involved in several pathological states, including cancer. CLIC1 mRNA overexpression was identified in colorectal cancer cells and is related to a poor prognosis. In this scenario, the main aim of the present work is to understand the role of tmCLIC1 in tumorigenesis, invasion, and migration potential of colorectal cancer. We first investigated the level of expression of CLIC1 protein and the activity of its transmembrane form as an ion channel in several colorectal cancer cells at different stage of development. Our results have demonstrated that tmCLIC1 protein expression and activity is proportional to the level of aggressiveness of cells, and here its action is fundamental on cellular proliferation, migration, and invasion. The role of tmCLIC1 activity in colorectal cancer was confirmed also in in vivo models. Experiments performed in zebrafish and mice models have demonstrated that the silencing of CLIC1 results in the impairment of primary tumor development and a strong reduction of distal metastases formation. Moreover, many different metastatic genes are downregulated when CLIC1 protein is absent; in particular, genes related to oxidative stress conditions, typical of cancer cells. As a matter of fact, the peculiar activity of CLIC1 and its binding with glutathione makes this protein essential for redox balance of colorectal cancer cells. Altogether, all the results obtained might be considered a starting point to unveil a mechanism of action that could represent a new milestone in anticancer research. Citation Format: Francesca Cianci, Ivan Verduci, Carlotta Tacconi, Guido Rey, Luca Roz, Alessandro Fantin, Michele Mazzanti. Transmembrane chloride intracellular channel 1 (tmCLIC1) protein as akey regulator of colorectal cancers' aggressiveness [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B11.

Abstract LB_B15: The antiproliferative activity of metformin in glioblastoma stem cells is mediated by the direct binding and functional inhibition of the membrane configuration of CLIC1 protein

Abstract Glioblastoma (GBM) is the deadliest brain tumor and represents a major clinical challenge. Even with the current standard of care and cutting-edge strategies, patients’ life expectancy remains invariably poor. Tumor heterogeneity and its extensive invasiveness make GBM difficult to be radically resected by neurosurgery and recurrence is common. There is scientific consensus that tumor relapse originates from progenitor/stem-like cells known as glioblastoma stem cells (GSCs). GSCs are endowed with distinct tumorigenic potential, with properties of self-renewal and multi-lineage differentiation that contribute to tumor mass heterogeneity. Thus, GSCs represent an appealing target for new therapies. In recent years, metformin's anti-cancer properties have been highlighted. Metformin administration to patients with type 2 diabetes (T2D) has been positively linked to a decrease in the risk of developing several cancers and in cancer-related mortality, including glioblastoma. Additionally, numerous studies on glioblastoma demonstrate that metformin’s anti-cancer properties are directed against GSCs. Despite this evidence, metformin’s mechanism of action on cancer cells has not been clarified, although it has been proposed to target the oxidative phosphorylation (OXPHOS) pathway. GSCs are able to shuttle between glycolysis and OXPHOS energetic pathways, and this metabolic plasticity could be the major reason for metformin’s low effectiveness in clinical trials so far. Chloride Intracellular Channel 1 (CLIC1) is a metamorphic protein that is predominantly cytoplasmic under physiological conditions. In response to persistent stress, it translocates to the plasma membrane, inducing chloride conductance. When GSCs are treated with metformin and agents that impair the activity of the transmembrane form of CLIC1 (tmCLIC1), they show a parallel and non-additive inhibition of proliferation. Our previous studies comprehensively demonstrated that tmCLIC1 contributes to the progression of GBM both in vitro and in vivo. In addition, its specific localization and enrichment at the GSC plasma membrane position tmCLIC1 as a prime pharmacological target for GBM treatment. Our investigation found that in vitro, metformin’s antiproliferative effect is exerted at a millimolar concentration range. However, in vivo, with metformin administered through drinking water, mice exhibit an increase in survival with brain levels of metformin quantified in the nanomolar range. It is possible that the constant fresh drug circulation, continuously reaching the brain during prolonged administration in vivo (up to 100 days) creates a drug steady state with persistent saturation and inhibition of all the active tmCLIC1 expressed by GBM cells. The need for lower concentrations in vivo than in vitro is also important to translate these studies into the clinic; the already-available extended-release formulation may provide the same sustained activity on GBM cells as the drug in mouse drinking water. Citation Format: Michele M Mazzanti, Francesca M Cianci. The antiproliferative activity of metformin in glioblastoma stem cells is mediated by the direct binding and functional inhibition of the membrane configuration of CLIC1 protein [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B15.

Abstract 2407: Transmembrane chloride intracellular channel 1 (tmCLIC1) protein is a key regulator of colorectal cancers' metastasis

Abstract Colorectal cancer (CRC) is the third most common tumor worldwide and the fourth cause of cancer-related death. The presence of distal metastasis at the time of the first diagnosis represents a worst prognosis factor for patients’ long-term survival. Therefore, finding new targets to arrest the metastatic process is a main challenge for successful CRC therapy. Our laboratory focused its attention on the Chloride Intracellular Channel 1 (CLIC1), a metamorphic protein able to switch from a cytoplasmic to a transmembrane form associated to ion channel activity. Previous experiments demonstrate that tmCLIC1 functional activity is negligible in normal colon cells, while increases proportionally to CRC cells’ aggressiveness. Moreover, we also reveal that tmCLIC1 supports CRC cells abnormal proliferation rate. Here, we show that tmCLIC1 pharmacological inhibition or its stable downregulation led to a significative reduction of CRC cells migrative and invasive potential, in vitro. To further confirm our hypothesis, we analysed downregulation of metalloproteinase-7 (MMP-7) protein and p38 mitogen-activated protein kinase (MAPK) signalling, both involved in migration and metastatic processes. Moreover, we demonstrate in murine model that CLIC1 knock-down impairs tumor growth and limits cancer cells dissemination. We tried to investigate the alteration of metastatic pathways after CLIC1 silencing. We obtained that many different genes are downregulated when CLIC1 protein is absent; in particular, genes regulated by Reactive Species of Oxygen (ROS) levels. In this scenario, we tried to define the mechanism of action in which CLIC1 is involved. Altogether, all the results obtained in the present work could be considered a starting point to design a new therapeutic strategy to counteract against metastatic colorectal cancer. Citation Format: Francesca Cianci, Ivan Verduci, Valentina Carlini, Carlotta Tacconi, Matteo Ranucci, Alessandro Fantin, Tullio Florio, Michele Mazzanti. Transmembrane chloride intracellular channel 1 (tmCLIC1) protein is a key regulator of colorectal cancers' metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2407.

Transmembrane Chloride Intracellular Channel 1 (tmCLIC1) as a Potential Biomarker for Personalized Medicine.

Identification of potential pathological biomarkers has proved to be essential for understanding complex and fatal diseases, such as cancer and neurodegenerative diseases. Ion channels are involved in the maintenance of cellular homeostasis. Moreover, loss of function and aberrant expression of ion channels and transporters have been linked to various cancers, and to neurodegeneration. The Chloride Intracellular Channel 1 (CLIC1), CLIC1 is a metamorphic protein belonging to a partially unexplored protein superfamily, the CLICs. In homeostatic conditions, CLIC1 protein is expressed in cells as a cytosolic monomer. In pathological states, CLIC1 is specifically expressed as transmembrane chloride channel. In the following review, we trace the involvement of CLIC1 protein functions in physiological and in pathological conditions and assess its functionally active isoform as a potential target for future therapeutic strategies.

CLIC1 Protein Accumulates in Circulating Monocyte Membrane during Neurodegeneration

Pathologies that lead to neurodegeneration in the central nervous system (CNS) represent a major contemporary medical challenge. Neurodegenerative processes, like those that occur in Alzheimer’s disease (AD) are progressive, and at the moment, they are unstoppable. Not only is an adequate therapy missing but diagnosis is also extremely complicated. The most reliable method is the measurement of beta amyloid and tau peptides concentration in the cerebrospinal fluid (CSF). However, collecting liquid samples from the CNS is an invasive procedure, thus it is not suitable for a large-scale prevention program. Ideally, blood testing is the most manageable and appropriate diagnostic procedure for a massive population screening. Recently, a few candidates, including proteins or microRNAs present in plasma/serum have been identified. The aim of the present work is to propose the chloride intracellular channel 1 (CLIC1) protein as a potential marker of neurodegenerative processes. CLIC1 protein accumulates in peripheral blood mononuclear cells (PBMCs), and increases drastically when the CNS is in a chronic inflammatory state. In AD patients, both immunolocalization and mRNA quantification are able to show the behavior of CLIC1 during a persistent inflammatory state of the CNS. In particular, confocal microscopy analysis and electrophysiological measurements highlight the significant presence of transmembrane CLIC1 (tmCLIC1) in PBMCs from AD patients. Recent investigations suggest that tmCLIC1 has a very specific role. This provides an opportunity to use blood tests and conventional technologies to discriminate between healthy individuals and patients with ongoing neurodegenerative processes.

Establish cisplatin-resistant gastric cancer cell line MGC-803/cisplatin and reveal its mechanism

Objective To establish a cisplatin-resistant gastric cancer cell line MGC-803/cisplatin (DDP) and to explore its drug resistance mechanism. Methods The cisplatin was used to induce the drug resistance of MGC-803 cells. The half maximal inhibitory concentration (IC50) of the drug-resistant strains was detected by the cell counting kit-8 (CCK-8) method. The expression of chloride intracellular channel 1 (CLIC1) was detected by Western blotting analyses. The wild-type CLIC1 plasmid and the short hairpin RNA (shRNA) plasmid targeting CLIC1 were constructed. After Lipo3000 was transfected into gastric cancer cells, the IC50 of gastric cancer cells to cisplatin was detected, and then the intracellular Cl-concentration was detected by MQAE fluorescent probe. Results The cisplatin-resistant cell line MGC-803/DDP was successfully induced with an IC50 of (7.02±0.13) mg/L, while the IC50 of MGC803 was (1.29±0.09) mg/L, (t=36.090, P<0.05). The CLIC1 protein of MGC803/DDP was up-regulated by (2.27±0.15) times relative to MGC803 (t=7.841, P<0.05). After silencing the CLIC1 gene of MGC803/DDP, the IC50 of the CON, CN, and KD groups were (6.96±0.09), (6.93±0.15) and (3.02±0.20) mg/L, respectively, which was significantly decreased in the KD group (F=0.209, P<0.05). After overexpression of the CLIC1 gene in MGC803 cells, the IC50 of the CON, CN, and OE groups were (1.35±0.07), (1.25±0.07) and (4.77±0.12) mg/L, respectively, and the OE group was significantly elevated (F=0.508, P<0.05). The concentration of chloride ion was detected by MQAE. The relative fluorescence intensity of MGC-803, MGC-803/OE, MGC-803/DDP and MGC-803/DDP-KD were (1.02±0.03), (0.61±0.02), (0.67±0.01) and (1.39±0.02), respectively. The concentration of Cl- in MGC-803/OE cells was higher than that in MGC-803 (t=10.800, P<0.05), while the concentration of Cl- in MGC-803/DDP was higher than that in MGC-803 and MGC-803/DDP-KD, and the concentration of Cl- in KD group was the lowest (F=229, P<0.05). Conclusion The CLIC1 gene can induce the resistance of gastric cancer cell line MGC-803 to cisplatin in vitro. The mechanism may be related to the up-regulation of CLIC1 expression, which leads to an increase of intracellular chloride ion concentration. Key words: Gastric cancer; Chloride intracellular channel 1; Cisplatin; Drug resistance

Mutual Influence of ROS, pH, and CLIC1 Membrane Protein in the Regulation of G1-S Phase Progression in Human Glioblastoma Stem Cells.

Glioblastoma (GB) is the most lethal, aggressive, and diffuse brain tumor. The main challenge for successful treatment is targeting the cancer stem cell (CSC) subpopulation responsible for tumor origin, progression, and recurrence. Chloride Intracellular Channel 1 (CLIC1), highly expressed in CSCs, is constitutively present in the plasma membrane where it is associated with chloride ion permeability. In vitro, CLIC1 inhibition leads to a significant arrest of GB CSCs in G1 phase of the cell cycle. Furthermore, CLIC1 knockdown impairs tumor growth in vivo Here, we demonstrate that CLIC1 membrane localization and function is specific for GB CSCs. Mesenchymal stem cells (MSC) do not show CLIC1-associated chloride permeability, and inhibition of CLIC1 protein function has no influence on MSC cell-cycle progression. Investigation of the basic functions of GB CSCs reveals a constitutive state of oxidative stress and cytoplasmic alkalinization compared with MSCs. Both intracellular oxidation and cytoplasmic pH changes have been reported to affect CLIC1 membrane functional expression. We now report that in CSCs these three elements are temporally linked during CSC G1-S transition. Impeding CLIC1-mediated chloride current prevents both intracellular ROS accumulation and pH changes. CLIC1 membrane functional impairment results in GB CSCs resetting from an allostatic tumorigenic condition to a homeostatic steady state. In contrast, inhibiting NADPH oxidase and NHE1 proton pump results in cell death of both GB CSCs and MSCs. Our results show that CLIC1 membrane protein is crucial and specific for GB CSC proliferation, and is a promising pharmacologic target for successful brain tumor therapies. Mol Cancer Ther; 17(11); 2451-61. ©2018 AACR.

Abstract 4299: Mutual influence of ROS, pH and CLIC1 membrane protein in the regulation of G1/S phase progression in human glioblastoma stem cells

Abstract Glioblastoma (GB) is the most lethal, aggressive and diffuse brain tumor. The main challenge of successful treatment is targeting the cancer stem cell (CSC) sub-population responsible for tumor origin, progression and recurrence. Chloride Intracellular Channel 1 (CLIC1), highly expressed in CSCs, is constitutively present in the plasma membrane where it is associated with chloride ion permeability. In vitro, CLIC1 inhibition leads to a significant arrest of GB CSCs in G1 phase of the cell cycle. Furthermore, CLIC1 knockdown impairs tumor growth in vivo. Here we demonstrate that CLIC1 membrane localization and function is specific for GB CSCs. Mesenchymal stem cells (MSCs) do not show CLIC1-associated chloride permeability and inhibition of CLIC1 protein function has no influence on MSC cell cycle progression. Investigation of the basic functions of GB CSCs reveals a constitutive state of oxidative stress and cytoplasmic alkalinization compared with MSCs. We now report that these three elements are temporally linked during CSC G1/S transition. Impeding CLIC1-mediated chloride current prevents both intracellular ROS accumulation and pH changes. CLIC1 membrane functional impairment results in GB CSCs resetting from an allostatic tumorigenic condition to a homeostatic steady state. In contrast, inhibiting NADPH oxidase and the NHE1 proton pump results in cell death of both GB CSCs and MSCs. Our results show that CLIC1 membrane protein is crucial and specific for GB CSC proliferation, and is a promising pharmacological target for successful brain tumor therapies. This work was supported by grant n.16713, IG 2015 to MM Italian Association for Cancer Research (AIRC). Citation Format: Ivan Verduci, Marta Peretti, Federica Maddalena Raciti, Valentina Carlini, Alessandra Patarozzi, Sara Barozzi, Massimiliano Garrè, Sarah Sertic, Alex Costa, Antonio Daga, Federica Barbieri, Tullio Florio, Michele Mazzanti. Mutual influence of ROS, pH and CLIC1 membrane protein in the regulation of G1/S phase progression in human glioblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4299.

Identification of Potent Chloride Intracellular Channel Protein 1 Inhibitors from Traditional Chinese Medicine through Structure-Based Virtual Screening and Molecular Dynamics Analysis.

Chloride intracellular channel 1 (CLIC1) is involved in the development of most aggressive human tumors, including gastric, colon, lung, liver, and glioblastoma cancers. It has become an attractive new therapeutic target for several types of cancer. In this work, we aim to identify natural products as potent CLIC1 inhibitors from Traditional Chinese Medicine (TCM) database using structure-based virtual screening and molecular dynamics (MD) simulation. First, structure-based docking was employed to screen the refined TCM database and the top 500 TCM compounds were obtained and reranked by X-Score. Then, 30 potent hits were achieved from the top 500 TCM compounds using cluster and ligand-protein interaction analysis. Finally, MD simulation was employed to validate the stability of interactions between each hit and CLIC1 protein from docking simulation, and Molecular Mechanics/Generalized Born Surface Area (MM-GBSA) analysis was used to refine the virtual hits. Six TCM compounds with top MM-GBSA scores and ideal-binding models were confirmed as the final hits. Our study provides information about the interaction between TCM compounds and CLIC1 protein, which may be helpful for further experimental investigations. In addition, the top 6 natural products structural scaffolds could serve as building blocks in designing drug-like molecules for CLIC1 inhibition.

Chloride intracellular channel 1 regulates migration and invasion in gastric cancer by triggering the ROS-mediated p38 MAPK signaling pathway.

Mol Med Rep 12:[Related article:] 8041–8047, 2015; DOI: 10.3892/mmr.2015.4459 Following the publication of this article, an interested reader drew to our attention anomalies associated with the presentation of Fig. 4A. In the original presentation of the Figure, different representations of the same photomicrograph were inadvertently used for the panels labelled 'N', 'H-R', 'H-R + NAC' and 'H-R + SB203580'. This error arose as a consequence of an incorrect ordering of our original image numbers. A corrected version of Fig. 4A is provided below, featuring the appropriate data for each of the five panels listed above. This Figure was intended to show how, compared with the normoxic control group, the invasiveness of the SGC-7901 gastric cancer cells was markedly increased under hypoxia-reoxygenation (H-R) conditions, although pretreatment with N-acetyl cysteine (NAC; 30 mmol/l) or indanyloxyacetic acid 94 (IAA-94; 40 μmol/l), inhibited the invasiveness resulting from H-R exposure. Similarly, treatment with the inhibitor of p38 mitogen-activated protein kinase, SB203580 (10 μmol/l), also inhibited the cell invasiveness induced under the H-R conditions. We sincerely apologize for this mistake, and thank the reader of our article who drew this matter to our attention. Furthermore, we regret any inconvenience this mistake has caused.

Chloride intracellular channel 1 regulates migration and invasion in gastric cancer by triggering the ROS-mediated p38 MAPK signaling pathway.

Chloride intracellular channel 1 (CLIC1) has been demonstrated to be overexpressed in gastric cancer, and elevated CLIC1 expression levels are markedly associated with the processes of tumor cell migration and invasion. However, the regulatory mechanism and signaling pathway underlying these processes have remained to be elucidated. The present study examined the impact of N-acetyl cysteine (NAC), indanyloxyacetic acid (IAA)-94 and SB203580, inhibitors of reactive oxygen species (ROS), as well as CLIC1 and p38 mitogen-activated protein kinase (MAPK) on the migration and invasion of SGC-7901 gastric cancer cells in a hypoxia-reoxygenation (H-R) microenvironment. The results demonstrated that intracellular ROS and CLIC1 levels were increased under H-R conditions, and that functional inhibition of CLIC1 significantly decreased the H-R-elevated ROS generation and p-p38 MAPK levels in SGC-7901 cells, as well as inhibited the migration and invasion of SGC-7901 cells. In addition, the expression levels of MMP-2 and MMP-9 were inhibited by NAC, IAA-94 and SB203580. These results indicated that CLIC1 regulates gastric cancer-cell migration and invasion via the ROS-mediated p38 MAPK signaling pathway.

Extracellular vesicle-mediated transfer of CLIC1 protein is a novel mechanism for the regulation of glioblastoma growth.

Little progresses have been made in the treatment of glioblastoma (GBM), the most aggressive and lethal among brain tumors. Recently we have demonstrated that Chloride Intracellular Channel-1 (CLIC1) is overexpressed in GBM compared to normal tissues, with highest expression in patients with poor prognosis. Moreover, CLIC1-silencing in cancer stem cells (CSCs) isolated from human GBM patients negatively influences proliferative capacity and self-renewal properties in vitro and impairs the in vivo tumorigenic potential. Here we show that CLIC1 exists also as a circulating protein, secreted via extracellular vesicles (EVs) released by either cell lines or GBM-derived CSCs. Extracellular vesicles (EVs), comprising exosomes and microvesicles based on their composition and biophysical properties, have been shown to sustain tumor growth in a variety of model systems, including GBM. Interestingly, treatment of GBM cells with CLIC1-containing EVs stimulates cell growth both in vitro and in vivo in a CLIC1-dose dependent manner. EVs derived from CLIC1-overexpressing GBM cells are strong inducers of proliferation in vitro and tumor engraftment in vivo. These stimulations are significantly attenuated by treatment of GBM cells with EVs derived from CLIC1-silenced cells. However, CLIC1 modulation appears to have no direct role in EV structure, biogenesis and secretion. These findings reveal that, apart from the function of CLIC1 cellular reservoir, CLIC1 contained in EVs is a novel regulator of GBM growth.

Chloride intracellular channel 1 regulates colon cancer cell migration and invasion through ROS/ERK pathway

To investigate the mechanisms of chloride intracellular channel 1 (CLIC1) in the metastasis of colon cancer under hypoxia-reoxygenation (H-R) conditions. Fluorescent probes were used to detect reactive oxygen species (ROS) in LOVO cells. Wound healing assay and transwell assay were performed to examine the migration and invasion of LOVO cells. Expression of CLIC1 mRNA and protein, p-ERK, MMP-2 and MMP-9 proteins was analyzed by reverse transcription-polymerase chain reaction and Western blot. H-R treatment increased the intracellular ROS level in LOVO cells. The mRNA and protein expression of CLIC1 was elevated under H-R conditions. Functional inhibition of CLIC1 markedly decreased the H-R-enhanced ROS generation, cell migration, invasion and phosphorylation of ERK in treated LOVO cells. Additionally, the expression of MMP-2 and MMP-9 could be regulated by CLIC1-mediated ROS/ERK pathway. Our results suggest that CLIC1 protein is involved in the metastasis of colon cancer LOVO cells via regulating the ROS/ERK pathway in the H-R process.

Chloride intracellular channel 1 is overexpression in hepatic tumor and correlates with a poor prognosis

Chloride intracellular channel 1 (CLIC1) is expressed in many human tissues and has been reported to be involved in the regulation of cell cycle, cell proliferation, and differentiation. Its roles in human hepatic tumor, however, remain unclear. The aim of this study was to investigate the clinicopathological significance and expression pattern of CLIC1 in human primary hepatic tumors. We examined the expression pattern of CLIC1 mRNA and protein in hepatic tumors using real-time quantitative RT-PCR and Western blot, respectively. CLIC1 protein and mRNA levels were significantly higher in cancerous tissues compared with corresponding normal tissue. In 85 hepatic tumor tissues, CLIC1 was significantly higher in 69 cases (81.2%), as determined by immunohistochemical staining. Increased CLIC1 expression was correlated with tumor size (p = 0.021), distant metastasis (p = 0.025), pathological TNM (pTNM) stage (p = 0.023), and poor survival (25.11 ± 2.27 vs 45.29 ± 4.28 months, p = 0.001). Our data show that increased CLIC1 protein expression is associated with clinicopathological factors and a poor prognosis of hepatic tumors, and suggest that CLIC1 might represent a valuable prognostic marker for human hepatic tumors.

Chloride intracellular channel 1 is an important factor in the lymphatic metastasis of hepatocarcinoma

We have previously demonstrated that chloride intracellular channel 1 (CLIC1) is involved in the lymphatic metastasis of tumors. In this study, a self-designed shRNA sequence of mouse CLIC1 gene was synthesized and inserted into a pGPU6/GFP/Neo plasmid, then stably transfected into mouse hepatic carcinoma cell line Hca-F cells to down-regulate the expression of CLIC1 gene. The levels of expression of CLIC1 mRNA and protein were detected by real-time quantitative polymerase chain reaction (qRT-PCR) and western blot (WB) analysis, respectively. The down-regulation of CLIC1 enhanced proliferative activity, increased the ratio of G2/M and decreased percentage of apoptosis. In addition, the capability of migration and invasion decreased significantly. The results indicate that CLIC1 is a critical factor in the development of lymphatic metastasis.

Single Channel and Ensemble Measurements of CLIC1 in Lipid Bilayers

AbstractChloride intracellular channel 1 (CLIC1) is an ion channel protein which has been hypothesized to play a fundamental role in neurodegenerative diseases like Alzheimer's disease (AD) [1]. To date, single-channel characteristics of this protein have been obtained by reconstituting it in lipid bilayers and whole cell patch clamp measurements of overexpressing cells. CLIC1 protein can exist in both soluble and integral membrane form[2] and localizes on the plasma membrane and intracellular organelles. Obtaining ensemble measurements of CLIC1 by whole cell patch clamp is very difficult, due to its subcellular localization and its solubility in cytoplasm [3]. We report single channel and ensemble measurements of reconstituted CLIC1 in artificial lipid bilayers formed using sessile droplets, a measurement platform amenable to parallel and automated ion channel studies.[4] We also measured dose dependent inhibition of CLIC1 multi-channel currents by a known blocker, IAA94. This work may be applicable to measurement and screening of other intracellular ion channels as well.[1] Stephania Averaimo, Rosemary H. Milton, Michael R. Duchen, Michele Mazzanti. ‘Chloride intracellular channel 1 (CLIC1): sensor and effector during oxidative stress’. FEBS Letters 584 (2010) 2076-2084.[2] Singh, H. ‘Two decades of dimorphic chloride intracellular channels’. FEBS Letters 584 (2010) 2112-2121.[3] Michele Mazzanti et al. ‘Involvement of the Intracellular Ion Channel CLIC1 in Microglia-Mediated -Amyloid-Induced Neurotoxicity’. The Journal of Neuroscience (2004) 5322-5330.[4] J L Poulos, S A Portonovo, H Bang and J J Schmidt. ‘Automatable Lipid Bilayer Formation and ion Channel Measurement Using Sessile Droplets’. Journal of Physics: Condensed Matter (2010) 22 (45), 454105.

Function of chloride intracellular channel 1 in gastric cancer cells

To investigate the effect of chloride intracellular channel 1 (CLIC1) on the cell proliferation, apoptosis, migration and invasion of gastric cancer cells. CLIC1 expression was evaluated in human gastric cancer cell lines SGC-7901 and MGC-803 by real time polymerase chain reaction (RT-PCR). Four segments of small interference RNA (siRNA) targeting CLIC1 mRNA and a no-sense control segment were designed by bioinformatics technology. CLIC1 siRNA was selected using Lipofectamine 2000 and transfected transiently into human gastric cancer SGC-7901 and MGC-803 cells. The transfected efficiency was observed under fluorescence microscope. After transfection, mRNA expression of CLIC1 was detected with RT-PCR and Western blotting was used to detect the protein expression. Proliferation was examined by methyl thiazolyl tetrazolium and apoptosis was detected with flow cytometry. Polycarbonate membrane transwell chamber and Matrigel were used for the detection of the changes of invasion and migration of the two cell lines. In gastric cancer cell lines SGC-7901 and MGC-803, CLIC1 was obviously expressed and CLIC1 siRNA could effectively suppress the expression of CLIC1 protein and mRNA. Proliferation of cells transfected with CLIC1 siRNA3 was enhanced notably, and the highest proliferation rate was 23.3% (P = 0.002) in SGC-7901 and 35.55% (P = 0.001) in MGC-803 cells at 48 h. The G2/M phase proportion increased, while G0/G1 and S phase proportions decreased. The apoptotic rate of the CLIC1 siRNA3 group obviously decreased in both SGC-7901 cells (62.24%, P = 0.000) and MGC-803 cells (52.67%, P = 0.004). Down-regulation of CLIC1 led to the inhibition of invasion and migration by 54.31% (P = 0.000) and 33.62% (P = 0.001) in SGC-7901 and 40.74% (P = 0.000) and 29.26% (P = 0.002) in MGC-803. However, there was no significant difference between the mock group cells and the negative control group cells. High CLIC1 expression can efficiently inhibit proliferation and enhance apoptosis, migration and invasion of gastric cancer cells in vitro. CLIC1 might be a promising target for the treatment of gastric cancer.

CLIC1 Functional Expression in the Plasma Membrane Correlates with Human Glioblastoma Aggressiveness

Glioblastomas are brain tumors composed by two cell types: cancer stem cells (CSCs), which are a small population able to self-renew and generate progeny, and bulk cells, consisting of a larger population committed to a precise fate. Glioblastomas are aggressive tumors because of CSC brain infiltration efficiency and their resistance to chemotherapies. Therefore, CSCs represent the most tumorigenic component of glioblastomas. Several forms of glioblastomas exhibit a level of Chloride Intracellular Channel 1 (CLIC1) expression higher than in normal brains. CLIC1 mainly localizes in the cytoplasm and in the nucleoplasm and, in stress conditions, it is able to translocate into plasma and nuclear membranes where it acts as a Cl- permeability. Four human glioblastoma biopsies were cultured in a CSCs selecting medium. By knocking down CLIC1 expression using siRNA lentiviral infection (siCLIC1), we found that CLIC1-deficient cells migrated about 50% less efficiently than cells treated with siRNA for luciferase (siLUC, control) in Boyden chamber assays. To determine whether this phenotype results from the lack of CLIC1 plasma membrane expression, CLIC1-mediated currents were estimated by using a specific inhibitor (IAA94, 100 μM) in voltage clamp experiments from perforated patches. We consistently recorded an IAA94-sensitive current in siLUC cells that was absent in siCLIC1 cells. Moreover, in non-treated CSCs we estimated the relative abundance of CLIC1-mediated current compared to the other Cl- currents in the cell. By calculating the ratio of IAA94-sensitive current over DIDS-sensitive current we found a positive correlation between tumor aggressiveness and the relative abundance of CLIC1-mediated currents in the four human glioblastomas analyzed. Our experiments highlight the involvement of CLIC1 protein in the spreading of the human glioblastoma CSCs, suggesting this protein as a possible pharmacological target to contrast the invasiveness of the brain tumor.

Cell Secretome Analysis Using Hollow Fiber Culture System Leads to the Discovery of CLIC1 Protein as a Novel Plasma Marker for Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in Southeast Asia. Unfortunately, most NPC victims have had metastasis when first diagnosed due to its deep location and vague symptoms. To date, discovery of sensitive and specific biomarkers for improving detection of NPC remains a challenge. Our previous study established a strategy for cell secretome analysis using a hollow fiber culture (HFC) system combined with liquid chromatography mass spectrometry. Herein, the above platform was used to collect NPC secretome for the discovery of relevant clinical biomarkers. Among 66 identified proteins, chloride intracellular channel 1 (CLIC1) was sieved out for intended use as a potential NPC biomarker candidate. Approximately 75% of NPC tissue specimens showed positive CLIC1 staining by IHC. The plasma levels of CLIC1 in NPC patients (N = 70), as presented by sandwich ELISA, were significantly higher than those in the healthy controls (N = 74) (mean +/- SD, 16.38 +/- 26.53 vs 2.39 +/- 5.32 microg/mL; p = 0.00005). Using a cutoff point of 2.58 microg/mL, CLIC1 successfully discriminated NPC from the benign healthy control group with a sensitivity of 63% and a specificity of 77%. The area under the receiver operating characteristic curve was determined to be 0.74 (95% CI, 0.652-0.818). The statistical analysis of CLIC1 level in plasma shows that CLIC1 could be applied as a marker for early detection of NPC. This is the first report for the detection of CLIC1 as a plasma marker. Our results indicate that the analytical platform could provide a feasible strategy to profile tumor cell secretome for identifying cancer biomarkers, and CLIC1 may be a novel plasma tumor marker for NPC.

Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity.

It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimer's disease, for example, is characterized by an accumulation of beta-amyloid protein (Abeta) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although Abeta and its fragment 25-35 exert a direct toxic effect on neurons, they also activate microglia. Microglial activation is accompanied by morphological changes, cell proliferation, and release of various cytokines and growth factors. A number of scientific reports suggest that the increased proliferation of microglial cells is dependent on ionic membrane currents and in particular on chloride conductances. An unusual chloride ion channel known to be associated with macrophage activation is the chloride intracellular channel-1 (CLIC1). Here we show that Abeta stimulation of neonatal rat microglia specifically leads to the increase in CLIC1 protein and to the functional expression of CLIC1 chloride conductance, both barely detectable on the plasma membrane of quiescent cells. CLIC1 protein expression in microglia increases after 24 hr of incubation with Abeta, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-alpha (TNF-alpha). We demonstrate that reducing CLIC1 chloride conductance by a specific blocker [IAA-94 (R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] prevents neuronal apoptosis in neurons cocultured with Abeta-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-alpha release induced by Abeta stimulation. These results provide a direct link between Abeta-induced microglial activation and CLIC1 functional expression.