Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1

Figures

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  Figure 1. High-throughput YFP reduction assay used to screen for ANO1 inhibitors. (A) Cell-based high-throughput YFP reduction assay. ATP-induced P2Y receptor increased calcium levels, which in turn activated ANO1, causing an influx of iodide and a decrease in YFP. (B) YFP fluorescence monitored using 96-well plates showing the inhibitory effect of strong inhibitors. (C) Chemical structure of the ANO1 inhibitor, diethylstilbestrol (DES).

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  Figure 2. Characterization of the ANO1 inhibitor, diethylstilbestrol (DES). (A) Inhibitory effect of DES exerted on ANO1 channel activity determined using YFP fluorescence. (B) Summary of dose-response (mean ± S.D., n = 5). (C) Cytosolic Ca2+ level was monitored using Fluo-4 NW in CHO-K1 cells. The indicated concentrations of 10 and 30 μM DES were added for 20 min, and then treatment with 100 μM ATP was performed. (D,E) Effect of DES on human CFTR activity was measured in FRT-hCFTR-YFP cells using YFP fluorescence and an Ussing chamber. The CFTR was activated in the presence of 10 μM forskolin, whereas its activity was blocked in the presence of 10 μM CFTRinh-172. (F) Apical membrane currents in FRT-hANO1 cells treated with DES for 10 min prior to ATP-induced ANO1 activation.

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  Figure 3. DES and Ani9 inhibit ANO1-induced currents in ANO1-expressing HEK293 cells. (A) Current/voltage (I/V) plot of mean currents. (B) Bar graph summarizing current densities measured at +80 mV (mean ± S.D., n = 5–8). ** p < 0.01, *** p < 0.001, Students’ unpaired t-test.

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  Figure 4. ANO1 expression levels in lung cancer cell lines, and the effect of an ANO1 activator (Eact) and inhibitors (Ani9 and DES) on the PC9 lung cancer cell line. (A) Western blot results of ANO1 protein expressed in FRT, FRT-hANO1, A549, H1993, HCC827, H1975 and PC9 cells. (B) Relative ANO1 protein levels (mean ± S.D., n = 5). (C) PC9 cells were subjected to treatment with Eact, Ani9, or DES; ANO1 protein levels were measured after 72 h. (D) ANO1 protein intensities were normalized to β-actin (mean ± S.D., n = 5). ** p < 0.01, *** p < 0.001.

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  Figure 5. Effect of DES, Eact and Ani9 on cell viability and migration. (A,B) H1975 cells expressing low levels of ANO1 and PC9 cells expressing high levels of ANO1 were subjected to treatment with DES, Eact or Ani9 at the indicated concentrations; cell viability was determined after 72 h using the CCK-8 assay (mean ± S.D., n = 5). (C,D) Wound-healing assay was performed using PC9 cells expressing high levels of ANO1. The cells were subjected to treatment with 3 μM DES, 10 μM DES, and 10 μM Ani9, and representative images were acquired at 0 and 48 h post wound infliction. (right) The wound closure was quantified at 48 h (mean ± S.D., n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 6. Effect of ANO1 activator (Eact) and inhibitors (Ani9, DES) on ANO1, p-EGFR and p-ERK1/2 protein levels. (A) Immunoblot-mediated detection of ANO1 in PC9 cells expressing high levels of ANO1. The cells were subjected to treatment with the indicated concentrations of Eact, Ani9, or DES for 72 h. (right) ANO1 protein intensities were normalized to those of β-actin (mean ± S.D., n = 5). (B) The levels of phosphorylation of EGFR in the presence of ANO1 modulators. (right) The p-EGFR band intensities were normalized to those of t-EGFR (mean ± S.D., n = 5). (C) The levels of phosphorylation of ERK1/2 in the presence of ANO1 modulators. (right) The p-ERK1/2 levels were normalized to those of t-ERK1/2 (mean ± S.D., n = 5). *** p < 0.001.

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  Figure 7. Apoptotic effect and hepatocytotoxicity of DES. (A) PC9 cells cultured with DES, Eact and Ani9 at the indicated concentrations for 24 h, with subsequent assessment of caspase-3 activity (mean ± S.D., n = 5). (B) The cells were cultured with the indicated concentrations of compounds for 24 h, and then cleaved PARP-1 activity was measured (mean ± S.D., n = 5). (C) DES concentration-dependent relative viability of HepG2 cells (red) was measured by quantifying resorufin derived from resazurin by intracellular reductases. Control experiments (Eact and Ani9) are marked for comparative analysis (gray) (mean ± S.D., n = 8). ** p < 0.01, *** p < 0.001.