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

Chang-Ta Chiu; Che-Yi Lin; Ching-Yu Yen; Meng-Ting Tsai; Huei-Cih Chang; Young-Chau Liu; Mei-Huei Lin

doi:10.1016/j.jds.2020.05.007

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

J Dent Sci. 2020 Dec;15(4):526-535. doi: 10.1016/j.jds.2020.05.007. Epub 2020 Jun 5.

Authors

Chang-Ta Chiu¹, Che-Yi Lin², Ching-Yu Yen^{2

3

4}, Meng-Ting Tsai⁵, Huei-Cih Chang⁵, Young-Chau Liu^{6

7}, Mei-Huei Lin^{5

6}

Affiliations

¹ Department of Dentistry, Tainan Municipal An-Nan Hospital China Medical University, Tainan, Taiwan, Republic of China.
² Oral and Maxillofacial Surgery Section, Chi Mei Medical Center, Tainan, Taiwan, Republic of China.
³ Department of Dentistry, Taipei Medical University, Taipei, Taiwan, Republic of China.
⁴ Department of Dentistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
⁵ Department of Biotechnology, Chia Nan University of Pharmacy, Tainan, Taiwan, Republic of China.
⁶ Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan, Republic of China.
⁷ Division of Natural Science, College of Liberal Education, Shu-Te University, Yanchao District, Kaohsiung City, Taiwan, Republic of China.

Abstract

Background/purpose: We previously found that the partially purified 30-100 kDa fraction of areca-nut-extract (ANE 30-100K) induces autophagy in different types of cells including oral carcinoma OECM-1 cells. This study was to analyze the composition and possible mechanisms of ANE 30-100K-induced autophagy (AIA).

Materials and methods: Phenol-sulfuric acid method and high performance anion exchange chromatography were utilized to analyze the composition of ANE 30-100K. OECM-1 and esophageal CE81T/VGH cells were taken as the experimental models. Microscope and transmission electron microscope were used to observe morphological changes. Cell viability and specific proteins were respectively measured by XTT and Western bot assay. shRNA and chemical inhibitors were applied to assess the involvement of Atg5, caveolin, and proteasome in AIA.

Results: ANE 30-100K contains ∼67% carbohydrate, which is composed of fucose (5.938%), arabinose (24.631%), glucosamine (8.066%), galactose (26.820%), glucose (21.388%), and mannose (13.157%). After ANE 30-100K stimulation, CE81T/VGH cells showed intracellular vacuoles, acidic vesicles, double-membrane vacuoles, and elevated LC3-II level. ANE 30-100K-induced cytotoxicity and LC3-II accumulation were significantly inhibited by Atg5 knockdown. Furthermore, the endocytosis inhibitor (methyl-β-cyclodextrin) and two caveolin shRNAs, as well as two proteasome inhibitors (lactacystin and epoxomicin), were shown to significantly attenuate ANE 30-100K-induced cytotoxicity and LC3-II accumulation in both OECM-1 and CE81T/VGH cells.

Conclusion: The major components of ANE 30-100K are carbohydrates. CE81T/VGH also exhibited autophagic responses to ANE 30-100K. Caveolin-mediated endocytosis and proteasome are involved in AIA. This study may have provided new knowledges of the action mechanisms and compositions of ANE 30-100K.

Keywords: Areca nut; Atg5; Autophagy; Caveolin; Endocytosis; Proteasome.