Clathrin‐mediated endocytosis is required for ANE 30‐100K‐induced autophagy

Background We identified an autophagy‐inducing areca nut (AN) ingredient (AIAI) in the 30‐100 kDa fraction of AN extract (ANE 30‐100K). This study was to analyze the role of endocytosis in ANE 30‐100K‐induced autophagy. Methods We used benzyl alcohol, dynasore, and shRNA of clathrin and dynamin to assess whether ANE 30‐100K‐induced cytotoxicity and accumulation of microtubule‐associated protein 1 light chain 3 (LC3)‐II were affected in oral (OECM‐1) and esophageal (CE81T/VGH) carcinoma cells. Results Both benzyl alcohol and dynasore effectively reduced ANE 30‐100K‐induced cytotoxicity and LC3‐II accumulation in OECM‐1 and CE81T/VGH cells. Downregulated protein expression of both clathrin and dynamin by their shRNA also significantly attenuated ANE 30‐100K‐induced elevation of LC3‐II levels in CE81T/VGH cells. Conclusions These results indicate that AIAI may be engulfed by cells through clathrin‐mediated endocytosis, which promotes the execution of the following autophagy program.