Fig 1.
Identification of inhibitors of TMEM16A.
(A) Principle of cell-based, fluorescence high-throughput screening assay. (B) Chemical structures of ANO1 inhibitors. (C) YFP fluorescence measured in single wells of 96-well plates, showing inhibitory effect of idebenone, miconazole and plumbagin on ANO1 channel activity. Indicated concentrations of idebenone, miconazole and plumbagin were added 20min prior to ANO1 activation by 100 μM ATP.
Fig 2.
Characterization of idebenone, a TMEM16A inhibitor.
(A) Apical membrane currents were measured in FRT-ANO1 cells. Idebenone was added 10 min prior to ANO1 activation by 100 μM ATP. (B) Summary of dose-response (mean ± S.E., n = 3–4). (C) Intracellular calcium concentration was measured using Fluo-4 in HT-29 and FRT cells. 30 μM idebenone (IDE), miconazole (MCZ) and plumbagin (PLB) were pretreated for 20min and then 100 μM ATP was applied. (D) Effect of idebenone on CFTR chloride channel activity was measured in FRT cells expressing human wild-type CFTR. CFTR was activated by 20 μM forskolin and inhibited by 10 μM CFTRinh-172. (E) Effect of idebenone on mouse ANO2 (mANO2) was measured in FRT-mANO2 cells. (F) Effect of Coenzyme Q10 (CoQ10) on ANO1 channel activity was observed in FRT-ANO1 cells. 100 μM CoQ10 was pretreated for 20min and then 100 μM ATP was applied. (right) Summary of peak current (mean ± S.E., n = 3–4). (G) Effect of idebenone on ANO1 activation by Eact in ANO1-expressing FRT cells. 100 μM idebenone (gray line) was pretreated for 20min and ANO1 was activated by 10 μM Eact. The remaining ANO1 currents were inhibited by T16Ainh-A01. (right) Summary of peak current (mean ± S.E., n = 3). (H) Idebenone reversibility. After vanishment of 100 μM ATP-induced ANO1 current, the cells were washed three times for 5 min each and then ANO1 was activated by 10 μM Eact. (right) Summary of peak current (mean ± S.E., n = 3). **P < 0.01, ***P < 0.001, Students’ unpaired t-test.
Fig 3.
Idebenone inhibits ANO1 in FRT-ANO1 and PC3 cells.
(A) (left) Whole-cell ANO1 current recorded at a holding potential at 0 mV and pulsing to voltages between ± 80 mV (in steps of 20 mV) in the absence and presence of 10 μM idebenone. ANO1 was activated by 100 μM ATP. (center) Current/voltage (I/V) plot of mean currents at the middle of each voltage pulse. (right) The bar graphs summarize current density data measured at + 80 mV (mean ± S.E., n = 4). (B) (left) Whole-cell patch clamp recordings of PC3 cells. CaCC currents recorded in the absence and presence of idebenone. CaCC was stimulated by 100 μM ATP. (center) Current/voltage (I/V) plot of mean currents at the middle of each voltage pulse. (right) The bar graphs summarize current density data measured at + 80 mV (mean ± S.E., n = 4). **P < 0.01, Students’ unpaired t-test.
Fig 4.
ANO1 expression in adenocarcinoma cell lines and effect of ANO1 inhibitors on the cell viability and migration.
(A) Immunoblot of ANO1 protein in FRT-ANO1, PC3, CFPAC-1 and A549 cells. Representatives of three sets of studies are shown. (B) Effect of idebenone on CaCCs was measured in CFPAC-1 cells expressing halide sensitive mutant YFP. CaCCs were activated by 100 μM ATP. (C) PC3 and A549 cells were treated with idebenone (30 μM), miconazole (30 μM) and plumbagin (30 μM), and cell proliferation was measured after 2 days using MTS assays (mean ± S.E., n = 6). (D) Wound healing assay in PC3 cells. The cells were treated with T16Ainh-A01 (30 μM) and idebenone. (left) The wound closure was quantified at every 2 h post-wound (mean ± S.E., n = 5). (right) Representative images taken at 0 h and 48 h post wounding (× 10). **P < 0.01, Students’ unpaired t-test.
Fig 5.
Effects of ANO1 inhibition on the cell proliferation and apoptosis in PC3, CFPAC-1 and A549 cells.
(A-C) PC3, CFPAC-1 and A549 cells were seeded in 96 well plates, and after 24 h incubation, they were treated with the indicated concentrations of idebenone (IDE), 100 μM coenzyme Q10 (Q10) and 10 μM T16Ainh-A01 (A01) in MTS assay. IDE (30 μM), coenzyme Q10 (100 μM) and T16Ainh-A01 (10 μM) were applied to the cells in BrdU assay. Cell proliferation was estimated after 2 days via MTS (left) or BrdU (right) assay (mean ± S.E., n = 6). (D-F) PC3, CFPAC-1 and A549 cells were treated with 30 μM idebenone. The cells were stained with TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, green) and DAPI (4,6-diamidino-2-phenylindole, blue). Scale bars represent 20 μm. *P < 0.05, Students’ unpaired t-test.