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Anoctamin1 (ANO1), a calcium-activated chloride channel, is frequently overexpressed in several cancers, including human prostate cancer and oral squamous cell carcinomas. ANO1 plays a critical role in tumor growth and maintenance of these cancers. In this study, we have isolated two new compounds (1 and 2) and four known compounds (3–6) from Mallotus apelta. These compounds were evaluated for their inhibitory effects on ANO1 channel activity and their cytotoxic effects on PC-3 prostate cancer cells. Interestingly, compounds 1 and 2 significantly reduced both ANO1 channel activity and cell viability. Electrophysiological study revealed that compound 2 (Ani-D2) is a potent and selective ANO1 inhibitor, with an IC50 value of 2.64 μM. Ani-D2 had minimal effect on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity and intracellular calcium signaling. Notably, Ani-D2 significantly reduced ANO1 protein expression levels and cell viability in an ANO1-dependent manner in PC-3 and oral squamous cell carcinoma CAL-27 cells. In addition, Ani-D2 strongly reduced cell migration and induced activation of caspase-3 and cleavage of PARP in PC-3 and CAL-27 cells. This study revealed that a novel ANO1 inhibitor, Ani-D2, has therapeutic potential for the treatment of several cancers that overexpress ANO1, such as prostate cancer and oral squamous cell carcinoma.

Neural stem cells (NSCs) are primary progenitor cells in the early developmental stage in the brain that initiate a diverse lineage of differentiated neurons and glia. Radial glial cells (RGCs), a type of neural stem cell in the ventricular zone, are essential for nurturing and delivering new immature neurons to the appropriate cortical target layers. Here we report that Anoctamin 1 (ANO1)/TMEM16A, a Ca2+-activated chloride channel, mediates the Ca2+-dependent process extension of RGCs. ANO1 is highly expressed and functionally active in RGCs of the mouse embryonic ventricular zone. Knockdown of ANO1 suppresses RGC process extension and protrusions, whereas ANO1 overexpression stimulates process extension. Among various trophic factors, brain-derived neurotrophic factor (BDNF) activates ANO1, which is required for BDNF-induced process extension in RGCs. More importantly, Ano1-deficient mice exhibited disrupted cortical layers and reduced cortical thickness. We thus conclude that the regulation of RGC process extension by ANO1 contributes to the normal formation of mouse embryonic brain.

DOG1 is a calcium-activated chloride channel that has gained attention as a promising drug target due to its involvement in several processes essential for tumor development and progression. DOG1 is overexpressed in >95% of gastrointestinal stromal tumors (GIST). The aim was to determine DOG1 inhibition antitumoral effects on GIST. Human GIST (GIST-T1 and GIST882) cell lines were used to study the effect of DOG1 inhibitors on chloride currents, viability, colony formation, and cell cycle. CaCC -A01 decreased chloride currents. CaCC -A01 and T16 -A01 reduced GIST cell viability and CaCC -A01 affected cell cycle distribution leading to G cell-cycle arrest. CaCC -A01 also increased the sub-G phase population, indicative of apoptosis, in GIST882. CaCC -A01 strongly reduced the colony forming ability of the cells, whereas T16 -A01 did not. DOG1 inhibition has antitumoral effects in GIST cells in vitro, and could potentially serve as a target for GIST therapy.

: Transmembrane member 16A (TMEM16A) is a component of calcium-activated chloride channels that regulate vascular smooth muscle cell (SMC) proliferation and remodeling. Autophagy, a highly conserved cellular catabolic process in eukaryotes, exerts important physiological functions in vascular SMCs. In the current study, we investigated the relationship between TMEM16A and autophagy during vascular remodeling. : We generated a transgenic mouse that overexpresses TMEM16A specifically in vascular SMCs to verify the role of TMEM16A in vascular remodeling. Techniques employed included immunofluorescence, electron microscopy, co-immunoprecipitation, and Western blotting. : Autophagy was activated in aortas from angiotensin II (AngII)-induced hypertensive mice with decreased TMEM16A expression. The numbers of light chain 3B (LC3B)-positive puncta in aortas correlated with the medial cross-sectional aorta areas and TMEM16A expression during hypertension. SMC-specific TMEM16A overexpression markedly inhibited AngII-induced autophagy in mouse aortas. Moreover, in mouse aortic SMCs (MASMCs), AngII-induced autophagosome formation and autophagic flux were blocked by TMEM16A upregulation and were promoted by TMEM16A knockdown. The effect of TMEM16A on autophagy was independent of the mTOR pathway, but was associated with reduced kinase activity of the vacuolar protein sorting 34 (VPS34) enzyme. Overexpression of VPS34 attenuated the effect of TMEM16A overexpression on MASMC proliferation, while the effect of TMEM16A downregulation was abrogated by a VPS34 inhibitor. Further, co-immunoprecipitation assays revealed that TMEM16A interacts with p62. TMEM16A overexpression inhibited AngII-induced p62-Bcl-2 binding and enhanced Bcl-2-Beclin-1 interactions, leading to suppression of Beclin-1/VPS34 complex formation. However, TMEM16A downregulation showed the opposite effects. : TMEM16A regulates the four-way interaction between p62, Bcl-2, Beclin-1, and VPS34, and coordinately prevents vascular autophagy and remodeling.

Calcium-activated chloride channels (CaCCs) mediate numerous physiological functions and are best known for the transport of electrolytes and water in epithelia. In the intestine, CaCC currents are considered necessary for the secretion of fluid to protect the intestinal epithelium. Although genetic ablation of ANO1/TMEM16A, a gene encoding a CaCC, reduces the carbachol-induced secretion of intestinal fluid, its mechanism of action is still unknown. Here, we confirm that ANO1 is essential for the secretion of intestinal fluid. Carbachol-induced transepithelial currents were reduced in the proximal colon of Ano1- deficient mice. Surprisingly, cholera toxin-induced and cAMP-induced fluid secretion, believed to be mediated by CFTR, were also significantly reduced in the intestine of Ano1 -deficient mice. ANO1 is largely expressed in the apical membranes of intestines, as predicted for CaCCs. The Ano1 -deficient colons became edematous under basal conditions and had a greater susceptibility to dextran sodium sulfate-induced colitis. However, Ano1 depletion failed to affect tumor development in a model of colorectal cancer. We thus conclude that ANO1 is necessary for cAMP- and carbachol-induced Cl − secretion in the intestine, which is essential for the protection of the intestinal epithelium from colitis. Colitis: Intestinal membrane protein implicated in defective fluid secretion An ion channel, a membrane protein allowing ion transport, that controls the flow of chloride is needed for proper secretion of protective fluids in the intestine. Uhtaek Oh from the Korea Institute of Science & Technology in Seoul, South Korea, and colleagues showed that cells lining the intestinal surface express a calcium-activated chloride channel called anoctamin-1 (ANO1) that regulates fluid secretion in the gut. Compared to control animals, ANO1-deficient mice released less fluid into their intestines following exposure to a diarrhea-inducing toxin or to a chloride transport–stimulating signaling molecule. This fluid secretion was previously thought to be mediated via a different ion channel. The ANO1-deficient mice accumulated fluid within colonic tissues, which increased their susceptibility to colitis. The findings point to ANO1 activation as a potential therapeutic strategy for treating colitis.

Recent studies have indicated that the intracellular concentration of chloride ions (Cl−) regulates gene expression in several types of cells and that Cl− modulators positively or negatively regulate the PI3K/AKT/mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription (STAT)3 signaling pathways. We previously reported that the Ca2+‐activated Cl− channel anoctamine (ANO)1 regulated human epidermal growth factor receptor 2 (HER2) transcription in breast cancer YMB‐1 cells. However, the mechanisms underlying ANO1‐regulated HER2 gene expression have not yet been elucidated. In the present study, we showed the involvement of intracellular organelle ClC‐3 Cl−/H+ transporter in HER2 transcription in breast cancer MDA‐MB‐453 cells. The siRNA‐mediated inhibition of ClC‐3, but not ANO1, markedly repressed HER2 transcription in MDA‐MB‐453 cells. Subsequently, treatments with the AKT inhibitor AZD 5363 and mTOR inhibitor everolimus significantly enhanced HER2 transcription in MDA‐MB‐453 cells, whereas that with the STAT3 inhibitor 5,15‐diphenylporphyrin (5,15‐DPP) inhibited it. AKT and mTOR inhibitors also significantly enhanced HER2 transcription in YMB‐1 cells. The siRNA‐mediated inhibition of ClC‐3 and ANO1 resulted in increased AKT phosphorylation and decreased STAT3 phosphorylation in MDA‐MB‐453 and YMB‐1 cells, respectively. The intracellular Cl− channel protein CLIC1 was expressed in both cells; however, its siRNA‐mediated inhibition did not elicit the transcriptional repression of HER2. Collectively, our results demonstrate that intracellular Cl− regulation by ANO1/ClC‐3 participates in HER2 transcription, mediating the PI3K/AKT/mTOR and/or STAT3 signaling pathway(s) in HER2‐positive breast cancer cells, and support the potential of ANO1/ClC‐3 blockers as therapeutic options for patients with resistance to anti‐HER2 therapies. Inhibition of intracellular organelle ClC‐3 Cl−/H+ transporter repressed HER2 transcription in breast cancer cells. Intracellular Cl‐ regulation participated in HER2 transcription, mediating the PI3K/AKT/mTOR and/or STAT3 signaling pathway(s) in HER2‐positive breast cancer cells. ANO1/ClC‐3 blockers may be potential therapeutic options for patients with resistance to anti‐HER2 therapies.

Maintenance of fecal continence requires a continuous or basal tone of the internal anal sphincter (IAS). Paradoxically, the basal tone results largely from high-frequency rhythmic contractions of the IAS smooth muscle. However, the cellular and molecular mechanisms that initiate these contractions remain elusive. Here we show that the IAS contains multiple pacemakers. These pacemakers spontaneously generate propagating calcium waves that drive rhythmic contractions and establish the basal tone. These waves are myogenic and act independently of nerve, paracrine or autocrine signals. Using cell-specific gene knockout mice, we further found that TMEM16A Cl − channels in smooth muscle cells (but not in the interstitial cells of Cajal) are indispensable for pacemaking, rhythmic contractions, and basal tone. Our results identify TMEM16A in smooth muscle cells as a critical pacemaker channel that enables the IAS to contract rhythmically and continuously. This study provides cellular and molecular insights into fecal continence. TMEM16A acts in smooth muscle cells as a critical pacemaker channel that enables the internal anal sphincter to contract rhythmically and continuously, a critical phenomenon to allow for fecal continence.

Anoctamin-1 Ca 2+ -activated Cl − channels (ANO1) are proposed to modulate contractility of urethra smooth muscle cells (USMC), but their cellular expression and contribution to agonist/neural evoked activity is unclear. ANO1 is implicated as a potential target to treat incontinence, thus this is an important issue to resolve. We sought to clarify roles of ANO1 in contractility of mouse USMC. We found expression of Ano1 transcripts in murine urethra, with no difference between male and females. Immunolabelling revealed ANO1 was expressed in USMC and not in specialized populations of interstitial cells (c-kit + interstitial of Cajal-like cells (ICC-LC) and PDGFRα + cells). However, a specific ANO1 channel inhibitor, Ani9, failed to affect urethral contractions elicited by phenylephrine, arginine vasopressin or electrical field stimulation of intrinsic nerves. CaCC inh A01 also failed to affect urethral contractions. In addition, Ani9 had no effect on Ca 2+ signals generated by USMC in situ. In contrast, Ani9 effectively reduced spontaneous contractions and Ca 2+ signals of mouse proximal colon. In addition, Ani9 inhibited ANO1 currents recorded in HEK 293 cells, at concentrations 30 times less than those used in organ bath experiments. Our data suggest that despite expression of ANO1 in USMC, these channels do not contribute to basal Ca 2+ signalling, or agonist and neurally-evoked contractions in murine urethra.

Gastrointestinal (GI) cancers are main causes of poor health, with most remaining difficult to treat effectively. Identifying new targets for treatment is crucial for improving the efficacy of tumour therapies and enhancing patient quality of life. Anoctamin-1 (ANO1), a crucial component of calcium-activated chloride channels (CaCCs), is expressed widely in various cell types, including epithelial cells, vascular smooth muscle cells, and tumour cells, and influences cell proliferation and migration. Nonetheless, the exact pathways through which ANO1 contributes to malignant transformation and immune responses remain elusive. This review comprehensively examines the regulatory functions and potential therapeutic applications of ANO1 in GI cancers. The goal of this work is to offer new perspectives for further study on the role of ANO1 in gastrointestinal cancers and to support improvements in therapeutic strategies for cancer diagnosis and treatment through the targeting of ANO1.

Mechanotransduction channels are widely expressed in both vertebrates and invertebrates, mediating various physiological processes such as touch, hearing and blood-pressure sensing. While previously known mechanotransduction channels in metazoans are primarily cation-selective, we identified Anoctamin-1 (ANOH-1), the C. elegans homolog of mammalian calcium-activated chloride channel ANO1/TMEM16A, as an essential component of a mechanosensory channel complex that contributes to the nose touch mechanosensation in C. elegans . Ectopic expression of either C. elegans or human Anoctamin-1 confers mechanosensitivity to touch-insensitive neurons, suggesting a cell-autonomous role of ANOH-1/ANO1 in mechanotransduction. Additionally, we demonstrated that the mechanosensory function of ANOH-1/ANO1 relies on CIB (calcium- and integrin- binding) proteins. Thus, our results reveal an evolutionarily conserved chloride channel involved in mechanosensory transduction in metazoans, highlighting the importance of anion channels in mechanosensory processes. Mechanotransduction channels play key roles in various sensory processes. Here, the authors identify the chloride channel ANOH-1/ANO1 as a core component of a mechanosensory channel complex, highlighting its evolutionary conservation.