Fig 1.
Identification of a novel inhibitor of ANO1, luteolin.
A) Chemical structure of luteolin. B) ANO1 activity was measured in FRT cells expressing human ANO1 and a halide sensor YFP. ANO1 activity was inhibited by the indicated concentrations of luteolin. C) Apical membrane currents were recorded from FRT cells expressing ANO1. Representative current traces showing luteolin induced-ANO1 inhibition at the indicated concentration. ANO1 was activated by100 μM ATP. D) Summary of dose-response (mean ± S.E., n = 3–4).
Fig 2.
ANO1 inhibition by analogs of luteolin.
IC50 values were determined using YFP fluorescence quenching assay in FRT cells expressing ANO1 (mean ± S.E., n = 3).
Fig 3.
A) Intracellular calcium concentration was measured using Fluo-4 in FRT cells. The cells were pretreated with the indicated concentrations of luteolin for 20 min and then 100 μM ATP was applied. B) Effect of luteolin on ANO2 activity was observed in FRT cells expressing mouse ANO2 (mANO2) and a halide sensor YFP. Treatment with the indicated concentrations of luteolin inhibited ATP-induced activation of mANO2. (right) Summary of dose-response (mean ± S.E., n = 3). C) Effect of luteolin on protease-activated receptor 2 (PAR2)-induced ANO1 activation in FRT-ANO1 cells. The cells were pretreated for 20 min with luteolin (100 μM) and ANO1 was activated by PAR2 activating peptide (PAR2-AP, 50 μM). (right) Summary of peak currents (mean ± S.E., n = 3–4). D) Luteolin reversibility. After the extinction of 100 μM ATP-induced ANO1 currents, the cells were washed 3 times for 5 min each and then ANO1 was activated by 50 μM PAR2-AP. (middle, right) Summary of peak current (mean ± S.E., n = 3–4). E) Whole-cell ANO1 currents were recorded at a holding potential of 0 mV and pulsed to voltages between ± 80 mV (in steps of 20 mV) in the absence and presence of 10 μM and 100 μM luteolin in FRT-ANO1 cells. ANO1 was activated by 100 μM ATP. F) Current/voltage (I/V) plot of mean currents at the middle of each voltage pulse. G) The bar graphs summarize the current density data measured at + 80 mV (mean ± S.E., n = 5). **P < 0.01, ***P < 0.001.
Fig 4.
Effect of luteolin and kaempferol on the ANO1 activity of PC-3 cells.
A) Immunoblot of ANO1 protein in FRT, FRT-ANO1 and PC-3 cells expressing high and low levels of ANO1. Representatives of three sets of studies are shown. B, C) ANO1 activity was measured in PC-3 cells expressing high and low levels of ANO1. ANO1 activity was inhibited by the indicated concentrations of luteolin and Ani9 (1 μM), a specific ANO1 inhibitor. D) Effect of kaempferol on ANO1 channel activity was observed in FRT-ANO1 cells. Indicated concentration of kaempferol was pretreated for 20 min and then 100 μM ATP was applied to activate ANO1. E) Effect of kaempferol on ANO1 activity in PC-3 cells expressing high levels of ANO1. F) Effect of luteolin and kaempferol on calcium signaling was measured using Fluo-4 in PC-3 cells.
Fig 5.
Effect of luteolin and kaempferol on the cell viability and migration of PC-3 cells.
A, B) PC-3 cells expressing high and low levels of ANO1 were treated with luteolin and kaempferol at the indicated concentration, and cell proliferation was measured after 24 hour incubation using MTS assay (mean ± S.E., n = 6). C, D) Wound healing assay was performed on PC-3 cells expressing high and low levels of ANO1. The cells were treated with 10 and 30 μM luteolin, and representative images were taken at 0 h and 48 h post wounding (× 10). (right) The wound closure was quantified at 48 h post-wound (mean ± S.E., n = 5). **P < 0.01, ***P < 0.001.
Fig 6.
Effect of luteolin and kaempferol on the expression levels of ANO1 in PC-3 cells.
A) Immunoblot analysis of ANO1 in PC-3 cells expressing high levels of ANO1. Cells were incubated with indicated concentration of luteolin and kaempferol for 24 hour. B) The ANO1 band intensity was normalized to β-actin (mean ± S.E., n = 3–4). C) Effect of luteolin and kaempferol on gene expression levels of ANO1 in the PC-3 cells (mean ± S.E., n = 3). D) Luteolin-induced time-dependent changes of ANO1 protein levels in the PC-3 cells. E) The ANO1 band intensity was normalized to β-actin (mean ± S.E., n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.