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.
Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor (GPCR) activated by proteolytic cleavage of its N-terminal domain. Once activated, PAR2 is rapidly desensitized and internalized by phosphorylation and β-arrestin recruitment. Due to its irreversible activation mechanism, some agonists that rapidly desensitized PAR2 have been misconceived as antagonists, and this has impeded a better understanding of the pathophysiological role of PAR2. In the present study, we found that GB83, initially identified as a PAR2 antagonist, is a bona fide agonist of PAR2 that induces unique cellular signaling, distinct from trypsin and PAR2-activating peptide (AP). Activation of PAR2 by GB83 markedly elicited an increase in intracellular calcium levels and phosphorylation of MAPKs, but in a delayed and sustained manner compared to the rapid and transient signals induced by trypsin and PAR2-AP. Interestingly, unlike PAR2-AP, GB83 and trypsin induced sustained receptor endocytosis and PAR2 colocalization with β-arrestin. Moreover, the recovery of the localization and function of PAR2 was significantly delayed after stimulation by GB83, which may be the reason why GB83 is recognized as an antagonist of PAR2. Our results revealed that GB83 is a bona fide agonist of PAR2 that uniquely modulates PAR2-mediated cellular signaling and is a useful pharmacological tool for studying the pathophysiological role of PAR2.
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