Impacts of Autophagy-Inducing Ingredient of Areca Nut on Tumor Cells

Abstract

Areca nut (AN) is a popular carcinogen used by about 0.6-1.2 billion people worldwide. Although AN contains apoptosis-inducing ingredients, we previously demonstrated that both AN extract (ANE) and its 30-100 kDa fraction (ANE 30-100K) predominantly induce autophagic cell death in both normal and malignant cells. In this study, we further explored the action mechanism of ANE 30-100K-induced autophagy (AIA) in Jurkat T lymphocytes and carcinoma cell lines including OECM-1 (mouth), CE81T/VGH (esophagus), SCC25 (tongue), and SCC-15 (tongue). The results showed that chemical- and small hairpin RNA (shRNA)-mediated inhibition of AMP-activated protein kinase (AMPK) resulted in the attenuation of AIA in Jurkat T but not in OECM-1 cells. Knockdown of Atg5 and Beclin 1 expressions ameliorated AIA in OECM-1/CE81T/VGH/Jurkat T and OECM-1/SCC25/SCC-15, respectively. Furthermore, ANE 30-100K could activate caspase-3 after inhibition of Beclin 1 expression in OECM-1/SCC25/SCC15 cells. Meanwhile, AMPK was demonstrated to be the upstream activator of the extracellular-regulated kinase (ERK) in Jurkat T cells, and inhibition of MEK attenuated AIA in Jurkat T/OECM-1/CE81T/VGH cells. Finally, we also found that multiple myeloma RPMI8226, lymphoma U937, and SCC15 cells survived from long-term non-cytotoxic ANE 30-100K treatment exhibited stronger resistance against serum deprivation through upregulated autophagy. Collectively, our studies indicate that Beclin-1 and Atg5 but not AMPK are commonly required for AIA, and MEK/ERK pathway is involved in AIA. Meanwhile, it is also suggested that long-term AN usage might increase the resistance of survived tumor cells against serum-limited conditions.

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

“…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 . Similar increased cisplatin resistance was also observed in oral carcinoma cell lines after 6‐day ANE (3 μg/mL) treatment …”

“…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 . Furthermore, ANE 30‐100K‐induced autophagic activity is dependent on reactive oxygen species, Beclin‐1, and Atg5 . 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.…”

Screening of Crude Drugs Used in Japanese Kampo Formulas for Autophagy-Mediated Cell Survival of the Human Hepatocellular Carcinoma Cell Line

“…These results probably reflect the conclusion that crude drug numbers 102 and 106 induced cell growth by inducing autophagic activity, but further investigations are needed to confirm the findings. In fact, crude drug number 107 (Binroji; areca) has been reported to induce autophagy in several cell lines, such as leukemic Jurkat T cells [39] and oral carcinoma cells (OECM-1), Cal-27, and Scc-9 [40] by activating the AMPK/mTOR signaling pathway after accumulating reactive oxygen species [40]. However, the relationship between autophagy and crude drug numbers 102 (Hishinomi; water chestnut) or 106 (Biwayo; loquat leaf) has not been previously reported.…”

Areca Nut contains both Apoptosis- and Autophagy-inducing Ingredients and its Possible Effects on Cancer Cells

“…Secondly, these treated cells exhibit stronger resistance against hypoxia, anti-cancer drugs, and serum starvation. Finally, upregulated autophagy is illustrated to be responsible for the increased tolerance of chronic ANE-and ANE 30-100K-treated cancer cells against drugs and serum deprivation [48,49]. Additionally, autophagy mediators Beclin 1 and ATG5 are shown to be required for ANE 30-100K-induced autophagy; whereas the role of AMP-activated protein kinase may be cancer-dependent [49].…”

“…Finally, upregulated autophagy is illustrated to be responsible for the increased tolerance of chronic ANE-and ANE 30-100K-treated cancer cells against drugs and serum deprivation [48,49]. Additionally, autophagy mediators Beclin 1 and ATG5 are shown to be required for ANE 30-100K-induced autophagy; whereas the role of AMP-activated protein kinase may be cancer-dependent [49]. Collectively, our studies indicate that the autophagy-stimulating activity of AN may be tumor supportive, i.e., as those of RASdriven cancers, malignant cells developed in AN-addicted users might become autophagy-addicted.…”