ANO1-downregulation induced by schisandrathera D: a novel therapeutic target for the treatment of prostate and oral cancers.

Anoctamin 1 (ANO1), a drug target for various cancers, including prostate and oral cancers, is an intracellular calcium-activated chloride ion channel that plays various physiopathological roles, especially in the induction of cancer growth and metastasis. In this study, we tested a novel compound isolated from Schisandra sphenanthera, known as schisandrathera D, for its inhibitory effect on ANO1. Schisandrathera D dose-dependently suppressed the ANO1 activation-mediated decrease in fluorescence of yellow fluorescent protein; however, it did not affect the adenosine triphosphate-induced increase in the intracellular calcium concentration or forskolin-induced cystic fibrosis transmembrane conductance regulator activity. Specifically, schisandrathera D gradually decreased the levels of ANO1 protein and significantly reduced the cell viability in ANO1-expressing cells when compared to those in ANO1-knockout cells. These effects could be attributed to the fact that schisandrathera D displayed better binding capacity to ANO1 protein than the previously known ANO1 inhibitor, Ani9. Finally, schisandrathera D increased the levels of caspase-3 and cleaved poly (ADP-ribose) polymerase 1, thereby indicating that its anticancer effect is mediated through apoptosis. Thus, this study highlights that schisandrathera D, which reduces ANO1 protein levels, has apoptosis-mediated anticancer effects in prostate and oral cancers, and thus, can be further developed into an anticancer agent.

Anticancer effect of verteporfin on non-small cell lung cancer via downregulation of ANO1.

Anoctamin 1 (ANO1) is a calcium-activated chloride channel found in various cell types and is overexpressed in non-small cell lung cancer (NSCLC), a major cause of cancer-related mortality. With the rising interest in development of druggable compounds for NSCLC, there has been a corresponding rise in interest in ANO1, a novel drug target for NSCLC. However, as ANO1 inhibitors that have been discovered simultaneously exhibit both the functions of an inhibition of ANO1 channel as well as a reduction of ANO1 protein levels, it is unclear which of the two functions directly causes the anticancer effect. In this study, verteporfin, a chemical compound that reduces ANO1 protein levels was identified through high-throughput screening. Verteporfin did not inhibit ANO1-induced chloride secretion but reduced ANO1 protein levels in a dose-dependent manner with an IC50 value of ~300 nM. Moreover, verteporfin inhibited neither P2Y receptor-induced intracellular Ca2+ mobilization nor cystic fibrosis transmembrane conductance regulator (CFTR) channel activity, and molecular docking studies revealed that verteporfin bound to specific sites of ANO1 protein. Confirming that verteporfin reduces ANO1 protein levels, we then investigated the molecular mechanisms involved in its effect on NSCLC cells. Interestingly, verteporfin decreased ANO1 protein levels, the EGFR-STAT3 pathway as well as ANO1 mRNA expression. Verteporfin reduced the viability of ANO1-expressing cells (PC9, and gefitinib-resistant PC9) and induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage. However, it did not affect hERG channel activity. These results show that the anticancer mechanism of verteporfin is caused via the down-regulation of ANO1.

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

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.