Anticancer Effect of Hemin through ANO1 Inhibition in Human Prostate Cancer Cells.

Anoctamin1 (ANO1), a calcium-activated chloride channel, is overexpressed in a variety of cancer cells, including prostate cancer, and is involved in cancer cell proliferation, migration, and invasion. Inhibition of ANO1 in these cancer cells exhibits anticancer effects. In this study, we conducted a screening to identify novel ANO1 inhibitors with anticancer effects using PC-3 human prostate carcinoma cells. Screening of 2978 approved and investigational drugs revealed that hemin is a novel ANO1 inhibitor with an IC50 value of 0.45 µM. Notably, hemin had no significant effect on intracellular calcium signaling and cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP (cAMP)-regulated chloride channel, and it showed a weak inhibitory effect on ANO2 at 3 µM, a concentration that completely inhibits ANO1. Interestingly, hemin also significantly decreased ANO1 protein levels and strongly inhibited the cell proliferation and migration of PC-3 cells in an ANO1-dependent manner. Furthermore, it strongly induced caspase-3 activation, PARP degradation, and apoptosis in PC-3 cells. These findings suggest that hemin possesses anticancer properties via ANO1 inhibition and could be considered for development as a novel treatment for prostate cancer.

Discovery of a novel natural compound, vitekwangin B, with ANO1 protein reduction properties and anticancer potential.

Background: Prostate cancer and non-small cell lung cancer (NSCLC) present significant challenges in the development of effective therapeutic strategies. Hormone therapies for prostate cancer target androgen receptors and prostate-specific antigen markers. However, treatment options for prostatic small-cell neuroendocrine carcinoma are limited. NSCLC, on the other hand, is primarily treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors but exhibits resistance. This study explored a novel therapeutic approach by investigating the potential anticancer properties of vitekwangin B, a natural compound derived from Vitex trifolia. Methods: Vitekwangin B was chromatographically isolated from the fruits of V. trifolia. ANO1 protein levels in prostate cancer and NSCLC cells were verified and evaluated again after vitekwangin B treatment. Results: Vitekwangin B did not inhibit anoctamin1 (ANO1) channel function but significantly reduced ANO1 protein levels. These results demonstrate that vitekwangin B effectively inhibited cancer cell viability and induced apoptosis in prostate cancer and NSCLC cells. Moreover, it exhibited minimal toxicity to liver cells and did not affect hERG channel activity, making it a promising candidate for further development as an anticancer drug. Conclusion: Vitekwangin B may offer a new direction for cancer therapy by targeting ANO1 protein, potentially improving treatment outcomes in patients with prostate cancer and NSCLC. Further research is needed to explore its full potential and overcome existing drug resistance challenges.

The role of anoctamin 1 in liver disease.

Liver diseases include all types of viral hepatitis, alcoholic liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), cirrhosis, liver failure (LF) and hepatocellular carcinoma (HCC). Liver disease is now one of the leading causes of disease and death worldwide, which compels us to better understand the mechanisms involved in the development of liver diseases. Anoctamin 1 (ANO1), a calcium-activated chloride channel (CaCC), plays an important role in epithelial cell secretion, proliferation and migration. ANO1 plays a key role in transcriptional regulation as well as in many signalling pathways. It is involved in the genesis, development, progression and/or metastasis of several tumours and other diseases including liver diseases. This paper reviews the role and molecular mechanisms of ANO1 in the development of various liver diseases, aiming to provide a reference for further research on the role of ANO1 in liver diseases and to contribute to the improvement of therapeutic strategies for liver diseases by regulating ANO1.

Airway basal cell­derived exosomes suppress epithelial­mesenchymal transition of lung cells by inhibiting the expression of ANO1.

Disruption of the epithelial-mesenchymal transition (EMT) of activated lung cells is an important strategy to inhibit the progression of idiopathic pulmonary fibrosis (IPF). The present study investigated the role of exosomes derived from airway basal cells on EMT of lung cells and elucidate the underlying mechanism. Exosomes were characterized by nanoparticle tracking analysis, transmission electron microscopy imaging and markers detection. The role of exosome on the EMT of lung epithelial cells and lung fibroblasts induced by TGF-ß1 was detected. RNA sequencing screened dysregulated genes in exosome-treated group, followed by the bioinformatical analysis. One of the candidates, anoctamin-1 (ANO1), was selected for further gain-and-loss phenotype assays. A bleomycin-induced pulmonary fibrosis model was used to evaluate the treatment effect of exosomes. Exosomes were round-like and positively expressed CD63 and tumor susceptibility gene 101 protein. Treatment with exosomes inhibited the EMT of lung cells activated by TGF-ß1. 4158 dysregulated genes were identified in exosome-treated group under the threshold of |log2 fold-change| value >1 and they were involved in the metabolism of various molecules, as well as motility-related biological processes. A key gene, ANO1, was verified by reverse transcription-quantitative PCR, whose overexpression induced the EMT of lung cells. By contrast, ANO1 knockdown reversed the EMT induced by TGF-ß1. In vivo assay indicated that exosome treatment ameliorated pulmonary fibrosis and inhibited the upregulation of ANO1 induced by bleomycin. In conclusion, airway basal cell-derived exosomes suppressed the EMT of lung cells via the downregulation of ANO1. These exosomes represent a potential therapeutic option for patients with IPF.

Niclosamide, but not ivermectin, inhibits anoctamin 1 and 6 and attenuates inflammation of the respiratory tract.

Inflammatory airway diseases like cystic fibrosis, asthma and COVID-19 are characterized by high levels of pulmonary cytokines. Two well-established antiparasitic drugs, niclosamide and ivermectin, are intensively discussed for the treatment of viral inflammatory airway infections. Here, we examined these repurposed drugs with respect to their anti-inflammatory effects in airways in vivo and in vitro. Niclosamide reduced mucus content, eosinophilic infiltration and cell death in asthmatic mouse lungs in vivo and inhibited release of interleukins in the two differentiated airway epithelial cell lines CFBE and BCi-NS1.1 in vitro. Cytokine release was also inhibited by the knockdown of the Ca2+-activated Cl- channel anoctamin 1 (ANO1, TMEM16A) and the phospholipid scramblase anoctamin 6 (ANO6, TMEM16F), which have previously been shown to affect intracellular Ca2+ levels near the plasma membrane and to facilitate exocytosis. At concentrations around 200 nM, niclosamide inhibited inflammation, lowered intracellular Ca2+, acidified cytosolic pH and blocked activation of ANO1 and ANO6. It is suggested that niclosamide brings about its anti-inflammatory effects at least in part by inhibiting ANO1 and ANO6, and by lowering intracellular Ca2+ levels. In contrast to niclosamide, 1 µM ivermectin did not exert any of the effects described for niclosamide. The present data suggest niclosamide as an effective anti-inflammatory treatment in CF, asthma, and COVID-19, in addition to its previously reported antiviral effects. It has an advantageous concentration-response relationship and is known to be well tolerated.

Anoctamin-1 is induced by TGF-ß and contributes to lung myofibroblast differentiation.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-ß (TGF-ß) is one of the most established drivers of fibrotic processes. TGF-ß promotes the transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-ß robustly upregulates the expression of the calcium-activated chloride channel anoctamin-1 (ANO1) in human lung fibroblasts (HLFs) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle α-actin (SMA)-positive myofibroblasts. TGF-ß-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1, and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate profibrotic TGF-ß signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-ß treatment of HLFs results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-ß-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that 1) ANO1 is a TGF-ß-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-ß; and 2) ANO1 mediates TGF-ß-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein but independent of WNK1 kinase activity.NEW & NOTEWORTHY This study describes a novel mechanism of differentiation of human lung fibroblasts (HLFs) to myofibroblasts: the key process in the pathogenesis of pulmonary fibrosis. Transforming growth factor-ß (TGF-ß) drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive with-no-lysine (K) kinase (WNK1) to activate profibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.

Researches of calcium-activated chloride channel ANO1 intervening amyotrophic lateral sclerosis progression by activating EGFR and CaMKII signaling.

BACKGROUND: ANO1 is closely correlated with the activation of EGFR and CaMKII, while EGFR and CaMKII show low activation in amyotrophic lateral sclerosis (ALS) models. Therefore, we designed experiments to verify that ANO1 may play a protective role on motor neurons in ALS by activating EGFR and CaMKII. METHODS: The expression changes of ANO1, EGFR, CaMKII, pEGFR, and pCaMKII, cell survival status, and apoptosis were studied by western blot, real-time quantitative PCR, immunofluorescence, immunohistochemistry, CCK-8, and flow cytometry. The role of ANO1 in the ALS model by activating EGFR and CaMKII was studied by applying corresponding activators, inhibitors, gene silencing, and overexpression. RESULTS: In hSOD1G93A transgenic animals and cell lines, low expression of ANO1 and low activation of EGFR and CaMKII were identified. ANO1 expression decreased gradually with the progression of ALS. Overexpression of ANO1 in the hSOD1G93A cell line and primary neurons of hSOD1G93A transgenic mice increased cell viability and decreased cell apoptosis. After the application of ANO1 inhibitor CaCC-inhA01 in hSOD1G93A cell line and primary neurons of hSOD1G93A transgenic mice, EGFR activator EGF and CaMKII activator Carbachol, increased cell viability and reduced cell apoptosis. After ANO1 was overexpressed in the hSOD1G93A cell line and primary neurons of hSOD1G93A transgenic mice, EGFR inhibitor AEE788 and CaMKII inhibitor KN93 decreased cell viability and increased cell apoptosis. CONCLUSIONS: Our results suggest that ANO1 plays an important role in the survival of ALS motor neurons. ANO1 can increase cell activity and reduce apoptosis by activating EGFR and CaMKII signals.

ANO1-downregulation induced by schisandrathera D: a novel therapeutic target for the treatment of prostate and oral cancers.

Anoctamin 1 (ANO1), a drug target for various cancers, including prostate and oral cancers, is an intracellular calcium-activated chloride ion channel that plays various physiopathological roles, especially in the induction of cancer growth and metastasis. In this study, we tested a novel compound isolated from Schisandra sphenanthera, known as schisandrathera D, for its inhibitory effect on ANO1. Schisandrathera D dose-dependently suppressed the ANO1 activation-mediated decrease in fluorescence of yellow fluorescent protein; however, it did not affect the adenosine triphosphate-induced increase in the intracellular calcium concentration or forskolin-induced cystic fibrosis transmembrane conductance regulator activity. Specifically, schisandrathera D gradually decreased the levels of ANO1 protein and significantly reduced the cell viability in ANO1-expressing cells when compared to those in ANO1-knockout cells. These effects could be attributed to the fact that schisandrathera D displayed better binding capacity to ANO1 protein than the previously known ANO1 inhibitor, Ani9. Finally, schisandrathera D increased the levels of caspase-3 and cleaved poly (ADP-ribose) polymerase 1, thereby indicating that its anticancer effect is mediated through apoptosis. Thus, this study highlights that schisandrathera D, which reduces ANO1 protein levels, has apoptosis-mediated anticancer effects in prostate and oral cancers, and thus, can be further developed into an anticancer agent.

Interstitial cells of Cajal: clinical relevance in pediatric gastrointestinal motility disorders.

Interstitial cells of Cajal (ICCs) are pacemaker cells of gastrointestinal motility that generate and transmit electrical slow waves to smooth muscle cells in the gut wall, thus inducing phasic contractions and coordinated peristalsis. Traditionally, tyrosine-protein kinase Kit (c-kit), also known as CD117 or mast/stem cell growth factor receptor, has been used as the primary marker of ICCs in pathology specimens. More recently, the Ca2+-activated chloride channel, anoctamin-1, has been introduced as a more specific marker of ICCs. Over the years, various gastrointestinal motility disorders have been described in infants and young children in which symptoms of functional bowel obstruction arise from ICC-related neuromuscular dysfunction of the colon and rectum. The current article provides a comprehensive overview of the embryonic origin, distribution, and functions of ICCs, while also illustrating the absence or deficiency of ICCs in pediatric patients with Hirschsprung disease intestinal neuronal dysplasia, isolated hypoganglionosis, internal anal sphincter achalasia, and congenital smooth muscle cell disorders such as megacystis microcolon intestinal hypoperistalsis syndrome.

Solithromycin inhibits IL-13-induced goblet cell hyperplasia and MUC5AC, CLCA1, and ANO1 in human bronchial epithelial cells.

Solithromycin is a novel fluoroketolide antibiotic belonging to the class of macrolide antibiotics. Activation of the interleukin (IL)-13 receptor leads to STAT6 activation and subsequent induction of SAM pointed domain containing ETS transcription factor (SPDEF), chloride channel accessory 1 (CLCA1), and anoctamin-1 (ANO1), all of which are associated with the induction of MUC5AC. We examined the effects of solithromycin on mucin production led by IL-13 signaling. Normal human bronchial epithelial cells were grown at the air-liquid interface with IL-13 with/without solithromycin for 14 days. Histochemical analysis was performed using hematoxylin and eosin staining and MUC5AC immunostaining. MUC5AC, SPDEF, CLCA1, and ANO1 mRNA expressions were examined using real-time polymerase chain reaction. Western blot analysis was performed to assess CLCA1 and ANO1 proteins, and phosphorylation of STAT6 and ERK. Solithromycin attenuated IL-13 induction of goblet cell hyperplasia and MUC5AC, CLCA1 and ANO1 mRNA and protein expression induced by IL-13, but had no effect on the phosphorylation of STAT6 and ERK. Our results indicate that solithromycin could attenuate goblet cell hyperplasia and MUC5AC induced by IL-13 through inhibition of CLCA1 and ANO1 mRNA and protein expression. However, much more information is required to clarify the molecular mechanisms underlying the inhibition of CLCA1 and ANO1 by solithromycin.

Inhibition of ANO1 by Cis- and Trans-Resveratrol and Their Anticancer Activity in Human Prostate Cancer PC-3 Cells.

Anoctamin1 (ANO1), a calcium-activated chloride channel, is involved in the proliferation, migration, and invasion of various cancer cells including head and neck squamous cell carcinoma, lung cancer, and prostate cancer. Inhibition of ANO1 activity or downregulation of ANO1 expression in these cancer cells is known to exhibit anticancer effects. Resveratrol, a natural polyphenol abundant in wines, grapes, berries, soybeans, and peanuts, shows a wide variety of biological effects including anti-inflammatory, antioxidant, and anticancer activities. In this study, we investigated the effects of two stereoisomers of resveratrol on ANO1 activity and found that cis- and trans-resveratrol inhibited ANO1 activity with different potencies. Cis- and trans-resveratrol inhibited ANO1 channel activity with IC50 values of 10.6 and 102 µM, respectively, and had no significant effect on intracellular calcium signaling at 10 and 100 µM, respectively. In addition, cis-resveratrol downregulated mRNA and protein expression levels of ANO1 more potently than trans-resveratrol in PC-3 prostate cancer cells. Cis- and trans-resveratrol significantly reduced cell proliferation and cell migration in an ANO1-dependent manner, and both resveratrol isomers strongly increased caspase-3 activity, PARP cleavage, and apoptotic sub-G1 phase ratio in PC-3 cells. These results revealed that cis-resveratrol is a potent inhibitor of ANO1 and exhibits ANO1-dependent anticancer activity against human metastatic prostate cancer PC-3 cells.

Anoctamin 1 and c-Kit immunohistochemical study of interstitial cells of Cajal in the muscularis externa of human gastrointestinal tract.

BACKGROUND: Interstitial cells of Cajal (ICC) are widely distributed in human gastrointestinal (GI) tract, especially in the layer of muscularis externa between neurons and smooth muscles. They play a very important role of coordination of GI tract motility. The aims of this research were to study the morphology and distribution of ICC in the muscularis externa of the GI tract, using immunohistochemistry staining methods, to determine the distribution of immune reactivity of anoctamin 1 (Ano1) compared with c-Kit, and to determine if Ano1 is a reliable marker for ICC in human GI tract. MATERIALS AND METHODS: Specimens from the wall of stomach, small intestine, and colon were taken from human cadavers and processed for histological and immunohistochemical study using c-Kit and Ano1 primary antibodies. RESULTS: Interstitial cells of Cajal appeared as bipolar cells, not forming network, in both the circular and longitudinal muscle layers, while in the myenteric area they appeared as multipolar interconnected cells. They were unevenly distributed in and between the muscle layers of the muscularis externa of human GI tract. They were more numerous in the stomach followed by the colon then the small intestine, and more numerous in the myenteric area followed by the circular muscle layer then the longitudinal muscle layer, in the three organs. Our results also showed that Ano1 is a more reliable marker for human ICC than c-Kit. CONCLUSIONS: Interstitial cells of Cajal differed in morphology and were unevenly distributed between muscle layers of muscularis externa and between different parts of human GI tract.

Anoctamin 1 Inhibition Suppresses Cystogenesis by Enhancing Ciliogenesis and the Ciliary Dosage of Polycystins.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy characterized by abnormal tubular epithelial proliferation and fluid secretion. Anoctamin 1 (ANO1) is a calcium-dependent chloride channel. However, how ANO1 contributes to ADPKD is largely unexplored. METHODS: Kidney tissues from ADPKD patients, Pkd1RC/RC mice model, WT9-7 human PKD1+⁣/- cells, and 3D culture models in vitro were used. Localization of ANO1 and cilium length were investigated by confocal immunofluorescence. RESULTS: We found that ANO1 was consistently upregulated in human and mouse PKD kidneys. Intriguingly, ANO1 located in a vesicle-like pattern at the ciliary base but not on the ciliary surface. ANO1 deficiency enhanced ciliogenesis and the ciliary dosage of polycystin-2 in human PKD cells, and reduced cyst formation in 3D culture models. Moreover, inhibition of ANO1 abolished the activation of STAT3 and ERK pathways in PKD cells. CONCLUSIONS: Our data indicate ANO1 is a negative regulator for both cilia length and cilia trafficking of polycystin-2 and provide mechanistic insights regarding the therapeutic potential of ANO1 pathway in ADPKD treatment.

Anticancer effect of verteporfin on non-small cell lung cancer via downregulation of ANO1.

Anoctamin 1 (ANO1) is a calcium-activated chloride channel found in various cell types and is overexpressed in non-small cell lung cancer (NSCLC), a major cause of cancer-related mortality. With the rising interest in development of druggable compounds for NSCLC, there has been a corresponding rise in interest in ANO1, a novel drug target for NSCLC. However, as ANO1 inhibitors that have been discovered simultaneously exhibit both the functions of an inhibition of ANO1 channel as well as a reduction of ANO1 protein levels, it is unclear which of the two functions directly causes the anticancer effect. In this study, verteporfin, a chemical compound that reduces ANO1 protein levels was identified through high-throughput screening. Verteporfin did not inhibit ANO1-induced chloride secretion but reduced ANO1 protein levels in a dose-dependent manner with an IC50 value of ~300 nM. Moreover, verteporfin inhibited neither P2Y receptor-induced intracellular Ca2+ mobilization nor cystic fibrosis transmembrane conductance regulator (CFTR) channel activity, and molecular docking studies revealed that verteporfin bound to specific sites of ANO1 protein. Confirming that verteporfin reduces ANO1 protein levels, we then investigated the molecular mechanisms involved in its effect on NSCLC cells. Interestingly, verteporfin decreased ANO1 protein levels, the EGFR-STAT3 pathway as well as ANO1 mRNA expression. Verteporfin reduced the viability of ANO1-expressing cells (PC9, and gefitinib-resistant PC9) and induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage. However, it did not affect hERG channel activity. These results show that the anticancer mechanism of verteporfin is caused via the down-regulation of ANO1.

The pharmacology of the TMEM16A channel: therapeutic opportunities.

The TMEM16A Ca2+-gated Cl- channel is involved in a variety of vital physiological functions and may be targeted pharmacologically for therapeutic benefit in diseases such as hypertension, stroke, and cystic fibrosis (CF). The determination of the TMEM16A structure and high-throughput screening efforts, alongside ex vivo and in vivo animal studies and clinical investigations, are hastening our understanding of the physiology and pharmacology of this channel. Here, we offer a critical analysis of recent developments in TMEM16A pharmacology and reflect on the therapeutic opportunities provided by this target.

In-vitro and in-vivo anti-allergic effects of magnolol on allergic rhinitis via inhibition of ORAI1 and ANO1 channels.

ETHNOPHARMACOLOGICAL RELEVANCE: Flos Magnoliae (the dried flower buds of Magnolia biondii Pamp, FM) is a known herbal traditional medicine used for the symptomatic relief of nasal congestion and rhinorrhea caused by rhinitis and sinusitis. Magnolol, a neolignan from the magnolia family, is a secondary metabolite known to have anti-allergic and anti-inflammatory effects. However, the underlying mechanisms and therapeutic effect of magnolol in the treatment of allergic rhinitis (AR) remain elusive. AIMS OF THE STUDY: Anoctamin 1 (ANO1), a calcium-activated anion channel, mediates mucus and electrolyte secretion in nasal airway epithelial cells, whereas calcium release-activated calcium channel protein 1 (ORAI1) participates in the activation of T-lymphocytes and mast cells. The aim of our study is to understand the mechanisms of action of magnolol against AR, i.e., whether it acts through the modulation of ANO1 and ORAI1 channels that are expressed in nasal epithelial cells and T-lymphocytes, respectively. MATERIALS AND METHODS: Whole-cell patch clamp was used to record the activity of ORAI1 and ANO1 ion channels in ORAI1 or ANO1 overexpressed HEK293T cells, while the Ussing chamber apparatus was used to measure electrolyte transport via the epithelium, in Calu-3 cells cultured in an air-liquid interface. Additionally, calcium imaging of Jurkat T-lymphocytes was used to assess changes in the intracellular calcium concentration. Magnolol toxicity was assessed using the CCK-8 assay, and its effect on T-lymphocyte proliferation was measured by labeling human primary T-lymphocytes with carboxyfluorescein succinimidyl ester. Finally, OVA-induced Balb/c mice were employed to evaluate the effect of magnolol on nasal symptoms, as well as cytokine and eosinophil infiltration in AR. RESULTS: Magnolol inhibits ORAI1 and ANO1 channels in a concentration-dependent manner. Magnolol (30 µM) inhibits anti-CD3 induced cellular proliferation and production of IL-2 via ORAI1 channels in T-lymphocytes. Further, ATP-induced electrolyte transport mediated by ANO1 channels is significantly inhibited by magnolol in IL-4 sensitized Calu-3 cells. Notably, 300 µM magnolol significantly attenuates cytokine and eosinophil infiltration, thus alleviating AR symptoms in mice OVA-induced AR. CONCLUSION: Magnolol may be a promising therapeutic agent for the treatment and prevention of AR.

Role of the IgG4-related cholangitis autoantigen annexin A11 in cholangiocyte protection.

BACKGROUND & AIMS: Annexin A11 was identified as autoantigen in IgG4-related cholangitis (IRC), a B-cell driven disease. Annexin A11 modulates calcium-dependent exocytosis, a crucial mechanism for insertion of proteins into their target membranes. Human cholangiocytes form an apical 'biliary bicarbonate umbrella' regarded as defense against harmful hydrophobic bile acid influx. The bicarbonate secretory machinery comprises the chloride/bicarbonate exchanger AE2 and the chloride channel ANO1. We aimed to investigate the expression and function of annexin A11 in human cholangiocytes and a potential role of IgG1/IgG4-mediated autoreactivity against annexin A11 in the pathogenesis of IRC. METHODS: Expression of annexin A11 in human liver was studied by immunohistochemistry and immunofluorescence. In human control and ANXA11 knockdown H69 cholangiocytes, intracellular pH, AE2 and ANO1 surface expression, and bile acid influx were examined using ratio microspectrofluorometry, cell surface biotinylation, and 22,23-3H-glycochenodeoxycholic acid permeation, respectively. The localization of annexin A11-mEmerald and ANO1-mCherry was investigated by live-cell microscopy in H69 cholangiocytes after incubation with IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies or disease control serum. RESULTS: Annexin A11 was strongly expressed in human cholangiocytes, but not hepatocytes. Knockdown of ANXA11 led to reduced plasma membrane expression of ANO1, but not AE2, alkalization of intracellular pH and uncontrolled bile acid influx. High intracellular calcium conditions led to annexin A11 membrane shift and colocalization with ANO1. Incubation with IRC patient serum inhibited annexin A11 membrane shift and reduced ANO1 surface expression. CONCLUSION: Cholangiocellular annexin A11 mediates apical membrane abundance of the chloride channel ANO1, thereby supporting biliary bicarbonate secretion. Insertion is inhibited by IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies. Anti-annexin A11 autoantibodies may contribute to the pathogenesis of IRC by weakening the 'biliary bicarbonate umbrella'. LAY SUMMARY: We previously identified annexin A11 as a specific autoantigen in immunoglobulin G4-related cholangitis (IRC), a B-cell driven disease affecting the bile ducts. Human cholangiocytes are protected against harmful hydrophobic bile acid influx by a defense mechanism referred to as the 'biliary bicarbonate umbrella'. We found that annexin A11 is required for the formation of a robust bicarbonate umbrella. Binding of patient-derived annexin A11 autoantibodies inhibits annexin A11 function, possibly contributing to bile duct damage by weakening the biliary bicarbonate umbrella in patients with IRC.

TMEM16A/ANO1: Current Strategies and Novel Drug Approaches for Cystic Fibrosis.

Cystic fibrosis (CF) is the most common of rare hereditary diseases in Caucasians, and it is estimated to affect 75,000 patients globally. CF is a complex disease due to the multiplicity of mutations found in the CF transmembrane conductance regulator (CFTR) gene causing the CFTR protein to become dysfunctional. Correctors and potentiators have demonstrated good clinical outcomes for patients with specific gene mutations; however, there are still patients for whom those treatments are not suitable and require alternative CFTR-independent strategies. Although CFTR is the main chloride channel in the lungs, others could, e.g., anoctamin-1 (ANO1 or TMEM16A), compensate for the deficiency of CFTR. This review summarizes the current knowledge on calcium-activated chloride channel (CaCC) ANO1 and presents ANO1 as an exciting target in CF.

Cinobufagin Exerts Anticancer Activity in Oral Squamous Cell Carcinoma Cells through Downregulation of ANO1.

Anoctamin1 (ANO1), a calcium-activated chloride channel, is frequently overexpressed in several cancers, including oral squamous cell carcinoma (OSCC). OSCC is a highly aggressive cancer and the most common oral malignancy. ANO1 has been proposed as a potential candidate for targeted anticancer therapy. In this study, we performed a cell-based screening to identify novel regulators leading to the downregulation of ANO1, and discovered cinobufagin, which downregulated ANO1 expression in oral squamous cell carcinoma CAL-27 cells. ANO1 protein levels were significantly reduced by cinobufagin in a dose-dependent manner with an IC50 value of ~26 nM. Unlike previous ANO1 inhibitors, short-term (≤10 min) exposure to cinobufagin did not alter ANO1 chloride channel activity and ANO1-dependent intestinal smooth muscle contraction, whereas long-term (24 h) exposure to cinobufagin significantly reduced phosphorylation of STAT3 and mRNA expression of ANO1 in CAL-27 cells. Notably, cinobufagin inhibited cell proliferation of CAL-27 cells expressing high levels of ANO1 more potently than that of ANO1 knockout CAL-27 cells. In addition, cinobufagin significantly reduced cell migration and induced caspase-3 activation and PARP cleavage in CAL-27 cells. These results suggest that downregulation of ANO1 by cinobufagin is a potential mechanism for the anticancer effect of cinobufagin in OSCC.

The Ca2+-activated chloride channel ANO1/TMEM16A: An emerging therapeutic target for epithelium-originated diseases?

Anoctamin 1 (ANO1) or TMEM16A gene encodes a member of Ca2+ activated Cl- channels (CaCCs) that are critical for physiological functions, such as epithelial secretion, smooth muscle contraction and sensory signal transduction. The attraction and interest in ANO1/TMEM16A arise from a decade long investigations that abnormal expression or dysfunction of ANO1 is involved in many pathological phenotypes and diseases, including asthma, neuropathic pain, hypertension and cancer. However, the lack of specific modulators of ANO1 has impeded the efforts to validate ANO1 as a therapeutic target. This review focuses on the recent progress made in understanding of the pathophysiological functions of CaCC ANO1 and the current modulators used as pharmacological tools, hopefully illustrating a broad spectrum of ANO1 channelopathy and a path forward for this target validation.