Novel ANO1 Inhibitor from Mallotus apelta Extract Exerts Anticancer Activity through Downregulation of ANO1

Figures

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  Figure 1. Chemical structures of compounds 1–6.

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  Figure 2. The key HMBC and COSY correlations of compounds 1 and 2.

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  Figure 3. Identification of novel ANO1 inhibitor, Ani-D2. (A) Inhibitory effect of compounds 1–6 on ANO1 activity was measured in FRT cells expressing human ANO1 and a mutant YFP. The cells were pretreated with compounds 1 (Ani-D1) and 2 (Ani-D2) at 25 µM for 20 min. ANO1 was activated by 100 ATP µM. (B) Representative Western blot analysis of ANO1 in FRT, FRT-ANO1, and PC-3 cells (two independent experiments performed). ANO1 protein expression was measured in FRT, FRT-ANO1, and PC-3 cells. (C) Effect of Ani-D1 and Ani-D2 on the protein expression levels of ANO1 in PC-3 cells. PC-3 cells were treated with 10 μM of Ani-D1 and Ani-D2 for 24 h. (Right) Summary of band intensity. The ANO1 band intensity was normalized to β-actin (mean ± S.E., n = 3). (D) ANO1 mRNA expression levels were determined by real-time PCR in PC-3 cells treated with Ani-D2 for 24 h (mean ± S.E., n = 3). ** p < 0.01.

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  Figure 4. Characterization of Ani-D2. (A) Effect of Ani-D2 on apical membrane current was observed in FRT cells expressing ANO1. Ani-D2 was applied at the indicated concentrations 20 min prior to ANO1 activation by 100 µM ATP. (B) Summary of dose–responses (mean ± S.E., n = 3–4). (C) Effect of Ani-D2 on intracellular calcium concentration was measured by Fluo-4/NW in FRT cells. The cells were pretreated with 0, 10, 30 μM of Ani-D2 for 20 min prior to the treatment of 100 µM ATP. (D) Effect of Ani-D2 on CFTR chloride channel activity was observed in FRT cells expressing human CFTR. CFTR chloride currents were activated by 10 μM forskolin and inhibited by 10 μM CFTRinh-172.

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  Figure 5. Effect of Ani-D2 on protein expression levels of ANO1 and cell viability in PC-3 and CAL-27 cells. (A,B) Representative Western blot analysis of ANO1 in Ani-D2 treated PC-3 and CAL-27 cells expressing ANO1 (three independent experiments performed). Cells were cultured with the indicated concentrations of Ani-D2 for 24 h. ANO1 knockout (KO) cells were established using CRISPR/Cas9 technique. (C,D) Effect of Ani-D2 on cell viability in PC-3, ANO1 KO PC-3, CAL-27, and ANO1 KO CAL-27 cells. Ani-D2 was treated at the indicated concentrations for 72 h, and cell viability was determined using MTS assay kit (mean ± S.E., n = 5). ** p < 0.01, *** p < 0.001.

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  Figure 6. Effect of Ani-D2 on cell migration in PC-3 and CAL-27 cells. (A,B) Wound healing assay was performed in PC-3 cells. Cells were treated with Ani-D2 and representative images were taken at 0 and 24 h post wounding (×10). The wound closure was measured for 48 h (mean ± S.E., n = 3–4). (C,D) Wound healing assay was performed in CAL-27 cells. Cells were treated with Ani-D2 at the indicated concentration and representative images were taken at 0 and 24 h post wounding (×10). The wound closure was measured for 48 h (mean ± S.E., n = 3–4).

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  Figure 7. Effect of Ani-D2 on caspase-3 activity and cleavage of PARP in PC-3 and CAL-27 cells. (A,B) Images were taken after 24 h incubation with Ani-D2. Caspase-3 substrate (green, 2.0 μM) and Hoechst 33342 (blue, 1 μM) were treated for 20 min prior to image acquisition. The white bar represents 200 μm. (C,D) Cells were cultured with Ani-D2 at the indicated concentrations for 24 h, then 2 μM of caspase-3 substrate was treated for 20 min. Caspase-3 activity was inhibited by 10 μM of Ac-DEVD-CHO (mean ± S.E., n = 3–4). (E,F) Cells were cultured with 10 μM of Ani-D2 for 24 h, and expression level of PARP, cleaved PARP, and β-actin were measured by immunoblot analysis (mean ± S.E., n = 3). * p < 0.05, *** p < 0.001.