Abstract
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.
Highlights
Oral squamous cell carcinoma (OSCC) is one of the most common head and neck cancers, accounting for 90% of all oral cancer types found in the mouth, tongue and lips [1].According to GLOBOCAN 2020 reports, OSCC ranks sixth in overall cancer mortality and has a higher age-standardized mortality rate than pancreatic cancer [2]
CAL-27 cells endogenously and highly expressing ANO1 were stably transfected with iodide-sensitive YFP-F46L/H148Q/I152L
The CAL-27 cells were treated with 20 μM test compounds for 24 h and washed with PBS, the effects of the natural products on ANO1 channel activity were measured using YFP fluorescence quenching assay (Figure 1A)
Summary
Oral squamous cell carcinoma (OSCC) is one of the most common head and neck cancers, accounting for 90% of all oral cancer types found in the mouth, tongue and lips [1]. We identified several potent inhibitors of ANO1, such as Ani, Ani9-5f, Ani-D2, luteolin and idebenone, which potently inhibited the chloride channel activity of ANO1, and Ani9-5f, Ani-D2, luteolin and idebenone significantly reduced cell proliferation and migration and induced apoptosis in several cancer cells highly expressing. Fast inhibition of ANO1 chloride channel activity leads to a rapid decrease in blood pressure through the relaxation of vascular smooth muscle and a rapid decrease in gastrointestinal motility via the inhibition of pacemaker activity in interstitial cells of Cajal (ICC) [34,35,36,37] These results suggest that the slow downregulation of ANO1 may be more beneficial in the treatment of cancer patients because it may exhibit anticancer activity without significant changes in blood pressure and gastrointestinal motility by providing sufficient time to compensate for the decreased. We identified a novel natural product, cinobufagin, which induces downregulation of ANO1 without alteration of ANO1 channel activity, and investigated the anticancer effects of cinobufagin on the human OSCC cell line CAL-27
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