Inhibition of calcium-activated chloride channel ANO1 suppresses proliferation and induces apoptosis of epithelium originated cancer cells

Abstract

ANO1, a calcium-activated chloride channel, has been reported to be amplified or overexpressed in tissues of several cancers. However, reports on its roles in tumor progression obtained from cancer cell lines are inconsistent, suggesting that the role of ANO1 in tumorigenesis is likely dependent on either its expression level or cell-type expressing ANO1. To investigate the biological roles of ANO1 in different tumor cells, we, in this study, selected several cancer cell lines and a normal HaCaT cell line with high expression levels of ANO1, and examined the function of ANO1 in these cells using approaches of lentiviral knockdown and pharmacological inhibition. We found that ANO1 knockdown significantly inhibited cell proliferation and induced cell apoptosis in either tumor cell lines or normal HaCaT cell line. Moreover, silencing ANO1 arrested cancer cells at G1 phase of cell cycle. Treatment with ANO1 inhibitor CaCCinh-A01 reduced cell viability in a dose-dependent manner. Furthermore, both ANO1 inhibitors CaCCinh-A01 and T16Ainh-A01 significantly suppressed cell migration. Our findings show that ANO1 overexpression promotes cancer cell proliferation and migration; and genetic or pharmacological inhibition of ANO1 induces apoptosis and cell cycle arrest at G1 phase in different types of epithelium-originated cancer cells.

Effects of the Calcium-Activated Chloride Channel Inhibitors T16Ainh-A01 and CaCCinh-A01 on Cardiac Fibroblast Function

“…Several studies have confirmed that T16Ainh-A01 and CaCCinh-A01 exert their inhibitory effects in a dose-dependent manner [18,19,27,28]. T16Ainh-A01 (10 µM) strongly inhibits TMEM16A-mediated iodide influx and completely blocks CaCC conductance in salivary gland cells.…”

“…CaCCinh-A01 can inhibit ANO1-dependent chloride conductance and decrease the proliferation of ANO1-dependent cancer cell lines (Te11 and FaDu cells) by degrading ANO1 protein, whereas T16Ainh-A01 has no effect on ANO1 protein, although it exhibits anti-proliferative effects on cell lines (human airway and intestinal cells) with extremely low ANO1 expression levels [17,18]. Furthermore, CaCCinh-A01 reduces cell viability, whereas T16Ainh-A01 has a minimal effect on the viability of colon cancer cells [19]. The inhibitory effects of T16Ainh-A01 and CaCCinh-A01 on CaCCs might be mediated by either the same or distinct signaling pathways in a cell type-specific manner.…”

Inhibition of ANO1 by luteolin and its cytotoxicity in human prostate cancer PC-3 cells

“…In addition, ANO1 is amplified and highly expressed in a number of cancers, including prostate cancer, breast cancer, gastrointestinal stromal tumor (GIST), head-and-neck squamous cell carcinoma (HNSCC), and esophageal squamous cell carcinoma (ESCC), and involved in cancer cell proliferation, tumorigenesis and cancer progression [6–10]. Recent studies showed that pharmacological or genetic downregulation of ANO1 significantly inhibited cancer cell proliferation, migration and invasion, even though the underlying mechanisms are still uncertain [11–13]. For instance, molecular and electrophysiological studies showed strong and functional expression of ANO1 in human metastatic prostate cancer PC-3 cells, and downregulation of ANO1 expression by shRNA induced significant reduction of cell proliferation, metastasis and invasion [6].…”

Molecular, biophysical, and pharmacological properties of calcium‐activated chloride channels

“…Once exposed to permeant anions, the Ca 2+ dependency of CaCCs will be altered (Qu & Hartzell, 2000;Xiao et al, 2011), suggesting that interactions of CaCCs with different anions can be coupled with calcium-sensing, voltage-sensing, and the gating kinetics of the channel. In comparison to Cl − , bulkier anions, such as NO 3 − , I − , and Br − , are preferred to cross the membrane (Rock, Futtner, & Harfe, 2008); gastrointestinal motility (Hwang et al, 2009); pain sensation (Cho & Oh, 2013;Huang et al, 2013;Takayama, Uta, Furue, & Tominaga, 2015); salivary gland secretion (Romanenko et al, 2010); airway epithelial secretion (Huang et al, 2009(Huang et al, , 2013; luminal pH regulation (Han Y, Shewan AM, & Thorn, 2016); insulin secretion (Crutzen et al, 2016;Edlund, Esguerra, Wendt, Flodstrom-Tullberg, & Eliasson, 2014;Xu et al, 2014); intestinal secretion and contraction (Namkung et al, 2010;Namkung, Phuan, et al, 2011;Namkung, Yao, et al, 2011); primary ciliogenesis (Ruppersburg & Hartzell, 2014); arterial myogenic response (Bulley et al, 2012); cerebral vasoconstriction (Li et al, 2011; pain and itch response (Liu, Cao, et al, 2015;Liu et al, 2010Liu et al, , 2016Liu, Liu, et al, 2015); airway smooth muscle contraction (Danielsson et al, 2015); thyrocyte iodide secretion (Twyffels et al, 2014); mucociliary clearance (Zhang et al, 2014Sondo, Caci, & Galietta, 2014); sweat secretion (Ertongur-Fauth, Hochheimer, Buescher, Rapprich, & Krohn, 2014); proinflammatory cytokine secretion inhibition (Veit et al, 2012); cancer cell proliferation and migration (Britschgi et al, 2013;Duvvuri et al, 2012;Guan et al, 2016;Jia et al, 2015;…”

“…In addition to other members of the Anoctamin family (Table 1) Amplification and overexpression of ANO1 in different cancerous tissues have been confirmed; however, there is no strong evidence on how cell proliferation and migration could be related to ANO1 overexpression. Antagonists of CaCCs can be investigated to see their therapeutic potentials via apoptosis of cancer cells as recently suggested in different studies (Guan, Song, Gao, Gao, & Wang, 2016;Jia, Liu, Guan, Lu, & Wang, 2015;Musrap et al, 2015). Furthermore, the crystallography studies in higher resolutions, in combination with bioinformatics approaches, can help us decipher the proper gating, voltage, and calcium sensitivity of these channels that can be of great value in designing novel therapeutic drugs.…”