Anoctamin1 (ANO1), a calcium-activated chloride channel, is involved in the proliferation, migration, and invasion of various cancer cells including head and neck squamous cell carcinoma, lung cancer, and prostate cancer. Inhibition of ANO1 activity or downregulation of ANO1 expression in these cancer cells is known to exhibit anticancer effects. Resveratrol, a natural polyphenol abundant in wines, grapes, berries, soybeans, and peanuts, shows a wide variety of biological effects including anti-inflammatory, antioxidant, and anticancer activities. In this study, we investigated the effects of two stereoisomers of resveratrol on ANO1 activity and found that cis- and trans-resveratrol inhibited ANO1 activity with different potencies. Cis- and trans-resveratrol inhibited ANO1 channel activity with IC₅₀ values of 10.6 and 102 μM, respectively, and had no significant effect on intracellular calcium signaling at 10 and 100 μM, respectively. In addition, cis-resveratrol downregulated mRNA and protein expression levels of ANO1 more potently than trans-resveratrol in PC-3 prostate cancer cells. Cis- and trans-resveratrol significantly reduced cell proliferation and cell migration in an ANO1-dependent manner, and both resveratrol isomers strongly increased caspase-3 activity, PARP cleavage, and apoptotic sub-G1 phase ratio in PC-3 cells. These results revealed that cis-resveratrol is a potent inhibitor of ANO1 and exhibits ANO1-dependent anticancer activity against human metastatic prostate cancer PC-3 cells.

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.

Anoctamin1 (ANO1), a calcium-activated chloride channel, is frequently overexpressed in several cancers, including human prostate cancer and oral squamous cell carcinomas. ANO1 plays a critical role in tumor growth and maintenance of these cancers. In this study, we have isolated two new compounds (1 and 2) and four known compounds (3–6) from Mallotus apelta. These compounds were evaluated for their inhibitory effects on ANO1 channel activity and their cytotoxic effects on PC-3 prostate cancer cells. Interestingly, compounds 1 and 2 significantly reduced both ANO1 channel activity and cell viability. Electrophysiological study revealed that compound 2 (Ani-D2) is a potent and selective ANO1 inhibitor, with an IC₅₀ value of 2.64 μM. Ani-D2 had minimal effect on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity and intracellular calcium signaling. Notably, Ani-D2 significantly reduced ANO1 protein expression levels and cell viability in an ANO1-dependent manner in PC-3 and oral squamous cell carcinoma CAL-27 cells. In addition, Ani-D2 strongly reduced cell migration and induced activation of caspase-3 and cleavage of PARP in PC-3 and CAL-27 cells. This study revealed that a novel ANO1 inhibitor, Ani-D2, has therapeutic potential for the treatment of several cancers that overexpress ANO1, such as prostate cancer and oral squamous cell carcinoma.

Anoctamin1 (ANO1), a calcium-activated chloride channel, is frequently overexpressed in several cancers, including oral squamous cell carcinoma (OSCC). OSCC is a highly aggressive cancer and the most common oral malignancy. ANO1 has been proposed as a potential candidate for targeted anticancer therapy. In this study, we performed a cell-based screening to identify novel regulators leading to the downregulation of ANO1, and discovered cinobufagin, which downregulated ANO1 expression in oral squamous cell carcinoma CAL-27 cells. ANO1 protein levels were significantly reduced by cinobufagin in a dose-dependent manner with an IC₅₀ value of ~26 nM. Unlike previous ANO1 inhibitors, short-term (≤10 min) exposure to cinobufagin did not alter ANO1 chloride channel activity and ANO1-dependent intestinal smooth muscle contraction, whereas long-term (24 h) exposure to cinobufagin significantly reduced phosphorylation of STAT3 and mRNA expression of ANO1 in CAL-27 cells. Notably, cinobufagin inhibited cell proliferation of CAL-27 cells expressing high levels of ANO1 more potently than that of ANO1 knockout CAL-27 cells. In addition, cinobufagin significantly reduced cell migration and induced caspase-3 activation and PARP cleavage in CAL-27 cells. These results suggest that downregulation of ANO1 by cinobufagin is a potential mechanism for the anticancer effect of cinobufagin in OSCC.

A highly efficient approach to a new indolizine scaffold fused with pyrrolo[1,2-c]pyrimidine was achieved via one-pot three-component coupling followed by an oxidative cyclization reaction. The simple two-step sequence allowed rapid access to various tetracyclic compounds from commercially available starting materials with the formation of five new bonds. Here, we observed the effects of these compounds on cell viability in HepG2, H1299, HT29, AGS, and A549 cancer cell lines. Interestingly, this fused scaffold had more potent anticancer activity in hepatocellular carcinoma HepG2 and Huh7 cells than other cancer cells. In particular, 5r strongly decreased cell viability in HepG2 and Huh7 cells with an IC₅₀ value of 0.22 ± 0.08 and 0.10 ± 0.11 µM, respectively, but had a very weak inhibitory effect on the cell viability of other cancer cell lines. In addition, 5r significantly inhibited cell migration and induced apoptosis in HepG2 and Huh7 cells via the activation of caspase-3 and cleavage of PARP in a dose-dependent manner. Notably, the co-treatment of 5r with gemcitabine resulted in the significant additional inhibition of cell viability in HepG2 and Huh7 cells. Our results suggest that 5r could be used to develop new chemotype anticancer agents against liver cancers.

Chloroquine (CQ) is an antimalaria drug that has been widely used for decades. However, CQ-induced pruritus remains one of the major obstacles in CQ treatment for uncomplicated malaria. Recent studies have revealed that MrgprX1 plays an essential role in CQ-induced itch. To date, a few MrgprX1 antagonists have been discovered, but they are clinically unavailable or lack selectivity. Here, a cell-based high-throughput screening was performed to identify novel antagonists of MrgprX1, and the screening of 2543 compounds revealed two novel MrgprX1 inhibitors, berbamine and closantel. Notably, berbamine potently inhibited CQ-mediated MrgprX1 activation (IC₅₀ = 1.6 μM) but did not alter the activity of other pruritogenic GPCRs. In addition, berbamine suppressed the CQ-mediated phosphorylation of ERK1/2. Interestingly, CQ-induced pruritus was significantly reduced by berbamine in a dose-dependent manner, but berbamine had no effect on histamine-induced, protease-activated receptors 2-activating peptide-induced, and deoxycholic acid-induced itch in mice. These results suggest that berbamine is a novel, potent, and selective antagonist of MrgprX1 and may be a potential drug candidate for the development of therapeutic agents to treat CQ-induced pruritus.