Matrine is a novel inhibitor of the TMEM16A chloride channel with antilung adenocarcinoma effects

Calcium-activated chloride channels (CaCCs) are ion channels with key roles in physiological processes. They are abnormally expressed in various cancers, including esophageal squamous cell cancer, head and neck squamous cell carcinoma, colorectal cancer, and gastrointestinal stromal tumors. The CaCC component TMEM16A/ANO1 was recently shown to be overexpressed in lung adenocarcinoma cells and may serve as a tumorigenic protein. In this study, we determined that matrine is a potent TMEM16A inhibitor that exerts anti-lung adenocarcinoma effects. Patch clamp experiments showed that matrine inhibited TMEM16A current in a concentration-dependent manner with an IC ₅₀ of 27.94 ± 4.78 μM. Molecular simulation and site-directed mutation experiments demonstrated that the matrine-sensitive sites of the TMEM16A channel involve the amino acids Y355, F411, and F415. Results of cell viability and wound healing assays showed that matrine significantly inhibited the proliferation and migration of LA795 cells, which exhibit high TMEM16A expression. In contrast, matrine has only weak inhibitory effect on CCD-19Lu and HeLa cells lacking TMEM16A expression. Matrine-induced effects on the proliferation and migration of LA795 cells were abrogated upon shRNA-mediated TMEM16A knockdown in LA795 cells. Finally, in vivo experiments demonstrated that matrine dramatically inhibited the growth of lung adenocarcinoma xenograft tumors in mice but did not affect mouse body weight. Collectively, these data indicate that matrine is an effective and safe TMEM16A inhibitor and that TMEM16A is the target of matrine anti-lung adenocarcinoma activity. These findings provide new insight for the development of novel treatments for lung adenocarcinoma.     

Recent advances in TMEM16A: Structure,function, and disease

TMEM16A (also known as anoctamin 1, ANO1) is the molecular basis of the calcium-activated chloride channels, with ten transmembrane segments. Recently, atomic structures of the transmembrane domains of mouse TMEM16A (mTMEM16A) were determined by single-particle electron cryomicroscopy. This gives us a solid ground to discuss the electrophysiological properties and functions of TMEM16A. TMEM16A is reported to be dually regulated by Ca²⁺ and voltage. In addition, the dysfunction of TMEM16A has been found to be involved in many diseases including cystic fibrosis, various cancers, hypertension, and gastrointestinal motility disorders. TMEM16A is overexpressed in many cancers, including gastrointestinal stromal tumors, gastric cancer, head and neck squamous cell carcinoma (HNSCC), colon cancer, pancreatic ductal adenocarcinoma, and esophageal cancer. Furthermore, overexpression of TMEM16A is related to the occurrence, proliferation, and migration of tumor cells. To date, several studies have shown that many natural compounds and synthetic compounds have regulatory effects on TMEM16A. These small molecule compounds might be novel drugs for the treatment of diseases caused by TMEM16A dysfunction in the future. In addition, recent studies have shown that TMEM16A plays different roles in different diseases through different signal transduction pathways. This review discusses the topology, electrophysiological properties, modulators and functions of TMEM16A in mediates nociception, gastrointestinal dysfunction, hypertension, and cancer and focuses on multiple regulatory mechanisms regarding TMEM16A.     

Systems Approaches to Unravel Molecular Function: High-content siRNA Screen Identifies TMEM16A Traffic Regulators as Potential Drug Targets for Cystic Fibrosis

An attractive approach to treat people with Cystic Fibrosis (CF), a life-shortening disease caused by mutant CFTR, is to compensate for the absence of this chloride/bicarbonate channel by activating alternative (non-CFTR) chloride channels. One obvious target for such “mutation-agnostic” therapeutic approach is TMEM16A (anoctamin-1/ANO1), a calcium-activated chloride channel (CaCC) which is also expressed in the airways of people with CF, albeit at low levels. To find novel TMEM16A regulators of both traffic and function, with the main goal of identifying candidate CF drug targets, we performed a fluorescence cell-based high-throughput siRNA microscopy screen for TMEM16A trafficking using a double-tagged construct expressed in human airway cells. About 700 genes were screened (2 siRNAs per gene) of which 262 were identified as candidate TMEM16A modulators (179 siRNAs enhanced and 83 decreased TMEM16A traffic), being G-protein coupled receptors (GPCRs) enriched on the primary hit list. Among the 179 TMEM16A traffic enhancer siRNAs subjected to secondary screening 20 were functionally validated. Further hit validation revealed that siRNAs targeting two GPCRs – ADRA2C and CXCR3 – increased TMEM16A-mediated chloride secretion in human airway cells, while their overexpression strongly diminished calcium-activated chloride currents in the same cell model. The knockdown, and likely also the inhibition, of these two TMEM16A modulators is therefore an attractive potential therapeutic strategy to increase chloride secretion in CF.     

3-Hydroxychromones as cyclin-dependent kinase inhibitors: synthesis and biological evaluation

A novel series of 3-hydroxychromones were prepared and found to be CDK inhibitors. Isothiazolidine 1,1-dioxide analogues showed potent CDK1 and CDK2 inhibitory activities and inhibited proliferation of EJ, HCT116, SW620, and MDAMB468 cancer cells.     

TMEM16A inhibitors reveal TMEM16A as a minor component of calcium-activated chloride channel conductance in airway and intestinal epithelial cells

TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC). We developed pharmacological tools to investigate the contribution of TMEM16A to CaCC conductance in human airway and intestinal epithelial cells. A screen of ～110,000 compounds revealed four novel chemical classes of small molecule TMEM16A inhibitors that fully blocked TMEM16A chloride current with an IC(50) < 10 μM, without interfering with calcium signaling. Following structure-activity analysis, the most potent inhibitor, an aminophenylthiazole (T16A(inh)-A01), had an IC(50) of ～1 μM. Two distinct types of inhibitors were identified. Some compounds, such as tannic acid and the arylaminothiophene CaCC(inh)-A01, fully inhibited CaCC current in human bronchial and intestinal cells. Other compounds, including T16A(inh)-A01 and digallic acid, inhibited total CaCC current in these cells poorly, but blocked mainly an initial, agonist-stimulated transient chloride current. TMEM16A RNAi knockdown also inhibited mainly the transient chloride current. In contrast to the airway and intestinal cells, all TMEM16A inhibitors fully blocked CaCC current in salivary gland cells. We conclude that TMEM16A carries nearly all CaCC current in salivary gland epithelium, but is a minor contributor to total CaCC current in airway and intestinal epithelia. The small molecule inhibitors identified here permit pharmacological dissection of TMEM16A/CaCC function and are potential development candidates for drug therapy of hypertension, pain, diarrhea, and excessive mucus production.     

The multifaceted role of TMEM16A in cancer

The calcium-activated chloride channel TMEM16A is intimately linked to cancers. Over decades, TMEM16A over-expression and contribution to prognosis have been widely studied for multiple cancers strengthening the idea that TMEM16A could be a valuable biomarker and a promising therapeutic target. Surprisingly, from the survey of the literature, it appears that TMEM16A has been involved in multiple cancer-related functions and a large number of molecular targets of TMEM16A have been proposed. Thus, TMEM16A appears to be an ion channel with a multifaceted role in cancers.In this review, we summarize the latest development regarding TMEM16A contribution to cancers. We will survey TMEM16A contribution in cancer prognosis, the origins of its over-expression in cancer cells, the multiple biological functions and molecular pathways regulated by TMEM16A. Then, we will consider the question regarding the molecular mechanism of TMEM16A in cancers and the possible basis for the multifaceted role of TMEM16A in cancers.     

Pharmacological characterization of TMEM16A currents

Recent studies have shown that transmembrane protein 16 A (TMEM16A) is a subunit of calcium-activated chloride channels (CACCs). Pharmacological agents have been used to probe the functional role of CACCs, however their effect on TMEM16A currents has not been systematically investigated. In the present study, we characterized the voltage and concentration-dependent effects of 2 traditional CACC inhibitors (niflumic acid and anthracene-9-carboxcylic acid) and 2 novel CACC / TMEM16A inhibitors (CACC_inhA01 and T16A_inhA01) on TMEM16A currents. The whole cell patch clamp technique was used to record TMEM16A currents from HEK 293 cells that stably expressed human TMEM16A. Niflumic acid, A-9-C, CACC_inhA01 and T16A_inhA01 inhibited TMEM16A currents with IC₅₀ values of 12, 58, 1.7 and 1.5 µM, respectively, however, A-9-C and niflumic acid were less efficacious at negative membrane potentials. A-9-C and niflumic acid reduced the rate of TMEM16A tail current deactivation at negative membrane potentials and A-9-C (1 mM) enhanced peak TMEM16A tail current amplitude. In contrast, the inhibitory effects of CACC_inhA01 and T16A_inhA01 were independent of voltage and they did not prolong the rate of TMEM16A tail current deactivation. The effects of niflumic acid and A-9-C on TMEM16A currents were similar to previous observations on CACCs in vascular smooth muscle, strengthening the hypothesis that they are encoded by TMEM16A. However, CACC_inhA01 and T16A_inhA01 were more potent inhibitors of TMEM16A channels and their effects were not diminished at negative membrane potentials making them attractive candidates to interrogate the functional role of TMEM16A channels in future studies.     

MAPK‐mediated upregulation of fibrinogen‐like protein 2 promotes proliferation,migration, and invasion of colorectal cancer cells

Fibrinogen‐like protein 2 (FGL2) has been reported to play a key role in the development of human cancers. However, it is still unmasked whether FGL2 plays a potential role in colorectal carcinogenesis. In this study, the messenger RNA and protein expression levels were measured by quantitative real‐time polymerase chain reaction and western blot. Cell counting kit‐8 assay, transwell migration, and invasion assay were carried out to evaluate the proliferation, migration, and invasion of LOVO and SW620 cells. FGL2 was upregulated in colorectal cancer (CRC) tissues, as well as cell lines. Mitogen‐activated protein kinase (MAPK) signaling was activated in CRC tissues and cell lines. FGL2 was confirmed to be downregulated by MAPK signaling inhibitor U0126. Further, we determined that knockdown of FGL2 caused a reduction of proliferation, migration, and invasion in LOVO and SW620 cells. Consistently, treatment of LOVO and SW620 cells with U0126 led to a decrease in cell proliferation, migration, and invasion. However, these changes initiated by U0126 were abolished by FGL2 overexpression. To conclude, MAPK‐mediated upregulation of FGL2 promotes the proliferation, migration, and invasion of CRC cells.     

Association of the ERK1/2 and p38 kinase pathways with nitric oxide-induced apoptosis and cell cycle arrest in colon cancer cells

To investigate the mechanism by which nitric oxide (NO) induces cell death in colon cancer cells, we compared two types of colon cancer cells with different p53 status: HCT116 (p53 wild-type) cells and SW620 (p53-deficient) cells. We found that S-nitrosoglutathione (GSNO), the NO donor, induced apoptosis in both types of colon cancer cells. However, SW620 cells were much more susceptible than HCT116 cells to apoptotic death by NO. We investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase on NO-induced apoptosis in both types of colon cancer cells. GSNO treatment effectively stimulated activation of the ERK1/2 and p38 kinase in both types of cells. In HCT116 cells, pretreatment with PD98059, an inhibitor of ERK1/2, or SB203580, an inhibitor of p38 kinase, had no marked effect on GSNO-induced apoptosis. However, in SW620 cells, SB203580 significantly reduced the NO-induced apoptosis, whereas PD098059 increases NO-induced apoptosis. Furthermore, we found evidence of cell cycle arrest of the G₀/G₁ phase in SW620 cells but not in HCT116 cells. Inhibition of ERK1/2 with PD098059, or of p38 kinase with SB203580, reduced the GSNO-induced cell cycle arrest of the G₀/G₁ phase in SW620 cells. We therefore conclude that NO-induced apoptosis in colon cancer cells is mediated by a p53-independent mechanism and that the pathways of ERK1/2 and p38 kinase are important in NO-induced apoptosis and in the cell cycle arrest of the G₀/G₁ phase.     

羽扇豆醇通过抑制RhoA-ROCK1信号通路抑制结肠癌细胞增殖

该文探讨了羽扇豆醇(Lupeol)对人结肠癌HCT116和SW620细胞增殖的影响及相关作用机制。使用不同浓度的Lupeol处理HCT116和SW620细胞后,用MTT法检测细胞活性,CCK8法检测细胞增殖能力,平板克隆实验检测细胞克隆形成能力,流式细胞术检测细胞周期和细胞凋亡,(quantitative real-time PCR,qPCR)和Western blot检测相应mRNA和蛋白表达水平,免疫荧光检测β-Catenin蛋白细胞内分布情况。通过构建shRNA敲低两种结肠癌细胞中RhoA,进一步研究Lupeol影响细胞增殖的分子机制。结果显示,Lupeol处理后,HCT116和SW620细胞增殖能力明显下降,克隆形成能力受到抑制,细胞周期阻滞于G0/G1期,细胞内RhoA、ROCK1、β-Catenin、Cyclin D1 mRNA和蛋白表达水平均显著下降,β-Catenin蛋白胞质和胞膜上分布减少。敲低RhoA后抑制了细胞增殖,同时使得RhoA-ROCK1-β-Catenin信号通路蛋白受到抑制,β-Catenin蛋白胞质和胞膜上分布减少。综上所述,Lupeol可通过抑制RhoA-ROCK1信号通路,抑制β-Catenin蛋白表达,进而抑制HCT116和SW620细胞增殖,Lupeol有望成为临床结肠癌治疗的新药物。     

Developmental expression of the calcium‐activated chloride channels TMEM16A and TMEM16B in the mouse olfactory epithelium

Calcium‐activated chloride channels are involved in several physiological processes including olfactory perception. TMEM16A and TMEM16B, members of the transmembrane protein 16 family (TMEM16), are responsible for calcium‐activated chloride currents in several cells. Both are present in the olfactory epithelium of adult mice, but little is known about their expression during embryonic development. Using immunohistochemistry we studied their expression in the mouse olfactory epithelium at various stages of prenatal development from embryonic day (E) 12.5 to E18.5 as well as in postnatal mice. At E12.5, TMEM16A immunoreactivity was present at the apical surface of the entire olfactory epithelium, but from E16.5 became restricted to a region near the transition zone with the respiratory epithelium, where localized at the apical part of supporting cells and in their microvilli. In contrast, TMEM16B immunoreactivity was present at E14.5 at the apical surface of the entire olfactory epithelium, increased in subsequent days, and localized to the cilia of mature olfactory sensory neurons. These data suggest different functional roles for TMEM16A and TMEM16B in the developing as well as in the postnatal olfactory epithelium. The presence of TMEM16A at the apical part and in microvilli of supporting cells is consistent with a role in the regulation of the chloride ionic composition of the mucus covering the apical surface of the olfactory epithelium, whereas the localization of TMEM16B to the cilia of mature olfactory sensory neurons is consistent with a role in olfactory signal transduction. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 657–675, 2014     

TMEM16A(a)/anoctamin-1 shares a homodimeric architecture with CLC chloride channels

TMEM16A/anoctamin-1 has been identified as a protein with the classic properties of a Ca(2+)-activated chloride channel. Here, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking to assess the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac) splice variants as well as a genetically concatenated TMEM16A(a) homodimer. The constructs carried hexahistidyl (His) tags to allow for their purification using a nondenaturing metal affinity resin. Neither His-tagging nor head-to-tail concatenation of two copies of TMEM16A(a) noticeably affected Ca(2+)-induced measured macroscopic Cl(-) currents compared with the wild-type TMEM16A(a) channel. The digitonin-solubilized, nondenatured TMEM16A(a) protein migrated in the BN-PAGE gel as a homodimer, as judged by comparison with the concatenated TMEM16A(a) homodimer and channel proteins of known oligomeric structures (e.g. the voltage-gated Cl(-) channel CLC-1). Cross-linking with glutaraldehyde corroborated the homodimeric structure of TMEM16A(a). The TMEM16A(a) homodimer detected in Xenopus laevis oocytes and HEK 293 cells dissociated into monomers following denaturation with SDS, and reducing versus nonreducing SDS-PAGE provided no evidence for the presence of intersubunit disulfide bonds. Together, our data demonstrate that the Ca(2+)-activated chloride channel member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel.     

Effect of selenite combined with chemotherapeutic agents on the proliferation of human carcinoma cell lines

Selenite is frequently used in combination with cancer chemotherapeutic agents to reduce side effects. However, the cytoprotective activity of selenite may also reduce the efficacy of chemotherapeutic drugs on tumor cells. This study was designed to examine the effects of selenite combined with cytotoxic agents used in clinical protocols [e.g., doxorubicine, docetaxel, 5-fluorouracil (5-FU), methotrexate (MTX), mafosphamide, mitomycin C, gemcitabine, etoposide, cisplatin, irinotecan, and oxaliplatin] on the proliferation of various carcinoma cell types. The data demonstrated that selenite had no marked effects on the antiproliferative activity of docetaxel, doxorubicine, 5-FU, MTX, and mafosphamide in MDA-MB-231 breast cancer cells. Likewise, no consistent changes were observed in A549 lung cancer cell proliferation when selenite was combined with cisplatin, etoposide, gemcitabine, or mitomycin C. On the other hand, selenite potentiated the cytotoxicity of 5-FU, oxaliplatin, and irinotecan in HCT116 colon cancer cells by approx 1.1-fold, 2.7-fold, and 2.6-fold, respectively. In SW620 colon cancer cells, selenite induced a 1.5-fold and 4.3-fold increase of the antiproliferative activity of 5-FU and oxaliplatin, respectively. Whereas irinotecan showed no effects on SW620 cell growth, a combination with selenite resulted in 23% inhibition. Our results indicate that selenite did not reduce the antiproliferative activity of chemotherapeutic agents in vitro. In addition, selenite was able to increase the inhibitory activity of docetaxel in A549 lung cancer cells, and of 5-FU, oxaliplatin, and irinotecan in HCT116 and SW620 colon cancer cells implying selenite is potentially useful as an adjuvant chemotherapeutic agent.     

Theaflavin binds to a druggable pocket of TMEM16A channel and inhibits lung adenocarcinoma cell viability

As a calcium-activated chloride channel regulated by the intracellular Ca²⁺ concentration and membrane potential, TMEM16A has attracted considerable attention and has been proposed as a novel anticancer drug target. We have previously reported that the pocket above the ion conductance pore could be a nonselective inhibitor-binding pocket. However, whether this pocket is druggable remains unexplored. In this study, we performed virtual screening to target the presumed inhibitor-binding pocket and identified a highly effective TMEM16A inhibitor, theaflavin (TF: a tea polyphenol in black tea). Molecular dynamics simulations revealed that theaflavin adopts a “wedge insertion mode” to block the ion conduction pore and induces pore closure. Moreover, the binding mode showed that the TF pedestal plays an important role in pore blockade, and R515, R535, T539, K603, E623, and E633 were determined to be most likely to interact directly with the pedestal. Mutagenesis experiment results corroborated the mechanism through which TF binds to this pocket. Combined with the quantitative calculation results, our data indicated that the three hydroxyl groups on the pedestal may be the most crucial pharmacophores for TMEM16A inhibition by TF. Finally, antitumor experiments revealed that TF could target TMEM16A to inhibit the proliferation and migration of LA795 cells, indicating the potential therapeutic effect of TF on the growth of lung adenocarcinoma with high TMEM16A expression. The successful application of drug screening strategies based on this binding pocket highlights new directions for discovering superior modulators and contributes to the development of novel therapeutics for lung adenocarcinoma.     

MG53 inhibits cellular proliferation and tumor progression in colorectal carcinoma

Cancer is the second leading cause of mortality after cardiovascular diseases in the United States. Chemotherapy is widely used to treat cancers. Since the development of drug resistance is a major contributor towards the failure of chemotherapeutic regimens, efforts have been made to develop novel inhibitors that can combat drug resistance and sensitize cancer cells to chemotherapy. Here we investigated the anti-cancer effects of MG53, a TRIM-family protein known for its membrane repair functions. We found that rhMG53 reduced cellular proliferation of both parental and ABCB1 overexpressing colorectal carcinoma cells. Exogenous rhMG53 protein entered SW620 and SW620/AD300 cells without altering the expression of ABCB1 protein. In a mouse SW620/AD300 xenograft model, the combination of rhMG53 and doxorubicin treatment significantly inhibited tumor growth without any apparent weight loss or hematological toxicity in the animals. Our data show that MG53 has anti-proliferative function on colorectal carcinoma, regardless of their nature to drug-resistance. This is important as it supports the broader value for rhMG53 as a potential adjuvant therapeutic to treat cancers even when drug-resistance develops.     

Role of the Ca2+ -activated Cl- channels bestrophin and anoctamin in epithelial cells

Two families of proteins, the bestrophins (Best) and the recently cloned TMEM16 proteins (anoctamin, Ano), recapitulate properties of Ca(2+)-activated Cl(-) currents. Best1 is strongly expressed in the retinal pigment epithelium and could have a function as a Ca(2+)-activated Cl(-) channel as well as a regulator of Ca(2+) signaling. It is also present at much lower levels in other cell types including epithelial cells, where it regulates plasma membrane localized Cl(-) channels by controlling intracellular Ca(2+) levels. Best1 interacts with important Ca(2+)-signaling proteins such as STIM1 and can interact directly with other Ca(2+)-activated Cl(-) channels such as TMEM16A. Best1 is detected in the endoplasmic reticulum (ER) where it shapes the dynamic ER structure and regulates cell proliferation, which could be important for renal cystogenesis. Ca(2+)-activated Cl(-) channels of the anoctamin family (TMEM16A) show biophysical and pharmacological properties that are typical for endogenous Ca(2+)-dependent Cl(-) channels. TMEM16 proteins are abundantly expressed and many reports demonstrate their physiological importance in epithelial as well as non-epithelial cells. These channels are also activated by cell swelling and can therefore control cell volume, proliferation and apoptosis. To fully understand the function and regulation of Ca(2+)-activated Cl(-) currents, it is necessary to appreciate that Best1 and TMEM16A are embedded in a protein network and that they probably operate in functional microdomains.     

NDRG1对人肠癌细胞失巢凋亡的影响

目的:探讨NDRG1对体外培养的人肠癌细胞系失巣凋亡的影响。方法:采用慢病毒系统将NDRG1表达单元转入人肠癌细胞系SW620、HCT8中,建立相应的过表达稳定细胞系;通过siRNA的方法干扰HCT116和LOVO细胞系中NDRG1的表达,分别在非贴壁培养的情况下培养48小时,采用流式细胞术和TUNEL染色检测细胞的凋亡情况。结果:在贴壁培养条件下,NDRG1过表达并没有显著影响肠癌细胞的生长及增殖,而NDRG1特异性siRNA干扰HCT116细胞中NDRG1的表达后,其凋亡率无明显变化(P0.05)。在悬浮培养条件下,NDRG1过表达的肠癌细胞的失巢凋亡率显著低于正常对照组(P0.05),而用三种不同的siRNA干扰HCT116及LOVO细胞中NDRG1的表达后,其失巢凋亡率均显著高于正常对照组(P0.05)。结论:NDRG1在体外可抑制人肠癌细胞的失巢凋亡。