Mechanistic and compositional studies of the autophagy-inducing areca nut ingredient

Areca nut (AN) is a popular chewing carcinogen worldwide causing a variety of diseases such as oral and esophageal carcinomas. We previously found that the partially purified 30-100 kDa fraction of AN extract (ANE 30-100K) induces autophagy in oral carcinoma OECM-1 cells and some other different types of cells. Since autophagy is known to play important roles in tumor establishment and development, the underlying mechanisms of ANE 30-100K-induced autophagy (AIA) is worthy of further investigation. In this study, we further demonstrated that the cytotoxic concentration of ANE 30-100K induces some typical autophagy hallmarks in esophageal carcinoma (CE81T/VGH) cells in an Atg5-dependent manner. Furthermore, the endocytosis inhibitor (methyl-{beta}-cyclodextrin) and two caveolin shRNAs, as well as two proteasome inhibitors (lactacystin and epoxomicin), were shown to attenuate ANE 30-100K-induced cytotoxicity and LC3-II accumulation significantly in OECM-1 and CE81T/VGH cells. Finally, we also analyzed the carbohydrate compositions of ANE 30-100K by phenol-sulfuric acid method and high performance anion exchange chromatography with pulse amperic detector. The results showed that ANE 30-100K contains about 67% carbohydrate and is composed of fucose (5.938%), arabinose (24.631%), glucosamine (8.066%), galactose (26.820%), glucose (21.388%), and mannose (13.157%). Collectively, these results suggest that caveolin-mediated endocytosis and proteasome are required for AIA and the major components of ANE 30-100K are carbohydrates. This study may have provided new knowledges of the action mechanisms and compositions of ANE 30-100K.