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

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Betel quid (BQ) is a popular carcinogen used by 200‐600 million people worldwide. BQ recipes vary in different areas, including areca nut (AN, Areca catechu L.), lime, Piper betle leaf, Piper betle inflorescence, or clove.1 Among these diversified components, AN is the indispensable constituent of BQ, which is also regarded as a human carcinogen.2 In addition to oral submucous fibrosis,3 a recent systematic review and meta‐analysis studies showed that AN usage is positively correlated with oral cancer.4 Thus, mechanisms of AN‐mediated cell responses are important to uncover the pathology of AN‐associated diseases and worthy to be investigated.
Areca nut (AN) is firstly demonstrated to contain the apoptosis‐inducing ingredients such as arecoline and oligomeric procyanidins.5 In our studies, however, we found that both normal and malignant cells underwent autophagic cell death after treatment with the crude extract of AN (ANE) and the 30‐100 kDa fraction of ANE (designated as ANE 30‐100K).7 Autophagy has been classified into macroautophagy, microautophagy, and chaperon‐mediated autophagy and is a conserved lysosomal degradation pathway for cellular components, which maintains cell viability and metabolic homeostasis.9 Among these three types of autophagy, macroautophagy (referred to as autophagy hereafter) have received the most extensive studies in the past two decades and been implicated in pathological conditions such as cancers.10 Autophagy acts as a double‐edged sword that inhibits tumor formation by preventing accumulation of damaged proteins and organelles at one hand, but facilitates the survival of established tumors by providing metabolic substrates. Thus, confirmation of the role of autophagy in individual cancer is important to design an autophagy‐modulated therapy.11
We also found that the activity of autophagy‐inducing AN ingredient (AIAI) present in ANE 30‐100K is sensitive to both cellulase and proteinase K, suggesting this ingredient to be a proteoglycan or glycoprotein.12 Furthermore, ANE 30‐100K‐induced autophagic activity is dependent on reactive oxygen species, Beclin‐1, and Atg5.7 Although cytotoxic and autophagic effects of ANE and ANE 30‐100K were revealed, it is thought that oral tumor cells receiving sublethal concentrations of these AN ingredients might exhibit higher but cytoprotective autophagy activities against stressed conditions in AN chewers. This speculation is further verified in our recent studies that long‐term treatment of malignant cells with non‐cytotoxic concentrations of ANE or ANE 30‐100K (both ≤1.25 μg/mL) result in increased resistance against stressed conditions such as cisplatin and serum deprivation through elevated autophagic activities.13 Similar increased cisplatin resistance was also observed in oral carcinoma cell lines after 6‐day ANE (3 μg/mL) treatment.15
In addition to our studies, ANE‐induced autophagy has also been demonstrated to be mediated through the activation of p38, mitogen‐activated protein kinase phosphatase 1, and hypoxia‐inducible factor.16 There are also evidences demonstrating the correlations of elevated LC3 and Beclin 1 expression respectively with oral submucous fibrosis and oral cancer progression.17 Collectively, the induction of autophagy by AN ingredient is a new field of AN‐mediated cellular response and worthy of studying the underlying mechanisms.
Recent investigations indicate that the endocytic pathway can promote efficient autophagy by promoting phagophore formation and expansion. In addition, the early endosomes, multivesicular bodies, or late endosomes can fuse with autophagosomes before their fusion with lysosomes.19 It is, therefore, speculated that endocytosis might play a role in ANE 30‐100K‐induced autophagy. Among different types of endocytosis, clathrin‐mediated endocytosis, a dynamin‐dependent process, can basically take place in all types of cells. We, therefore, focused on assessing the effects of this specific type of endocytosis on ANE 30‐100K‐induced autophagy in this study. We used benzyl alcohol (a membrane fluidity blocker), dynasore (a specific dynamin inhibitor),21 and shRNAs of dynamin and clathrin genes to inhibit endocytosis, followed by analyzing whether ANE 30‐100K‐induced cytotoxicity and autophagy were interfered.
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