Long-term stimulation of areca nut components results in increased chemoresistance through elevated autophagic activity.

BACKGROUND: We previously demonstrated the autophagy-inducing activity in the crude extract of areca nut (ANE) and its 30-100 kDa fraction (ANE 30-100 K). This study aimed to analyze whether chronic ANE and ANE 30-100 K stimulations lead to higher stress resistance and autophagic activity in oral cells, and whether the resulting autophagic status in stimulated cells correlates with stress resistance. MATERIALS AND METHODS: Malignant cells from the mouth oral epidermoid carcinoma Meng-1 (OECM-1) and blood (Jurkat T) origins were stimulated with non-cytotoxic ANE and ANE 30-100 K for 3 months. Sensitivity to anticancer drugs of and autophagy status in stimulated cells, analyzed respectively by XTT assay and calculating microtubule-associated protein 1 light chain 3-II LC3-II/ß-actin ratios from Western blot, were compared to non-treated cells. Autophagy inhibitors, 3-methyladenine (3-MA) and chloroquine (CQ), were used to assess whether autophagy inhibition interferes the altered chemoresistance. RESULTS: Areca nut extract-stimulated (ANE-s) and ANE 30-100 K-stimulated (30-100 K-s) OECM-1 and Jurkat T cells generally exhibited higher cisplatin and 5-fluorouracil (5-FU) resistances, compared to non-stimulated cells. Most stimulated cells expressed significantly higher levels of LC3-II and Atg4B proteins. Interestingly, these cells also showed stronger tolerances against hypoxia environment and expressed higher LC3-II levels under glucose-deprived and hypoxia conditions. Finally, both 3-MA and CQ alleviated, albeit to different degrees, the increased chemoresistance in ANE-s and/or 30-100 K-s cells. CONCLUSIONS: Chronic stimulations of ANE or ANE 30-100 K may increase tolerance of oral cancer and leukemia T cells to anticancer drugs, as well as to glucose deprivation and hypoxia conditions, and cause an elevation of autophagy activity responsible for increased drug resistance.

Autophagy induction by the 30-100kDa fraction of areca nut in both normal and malignant cells through reactive oxygen species.

Areca nut (AN) is an addictive carcinogen used by about 200-600 million people worldwide. Some AN components are shown to induce apoptosis; however, we previously demonstrated that AN extract (ANE) and the 30-100kDa fraction of ANE (ANE 30-100K) induced autophagy-like responses, such as swollen cell morphology, empty cytoplasm, acidic vesicles, and LC3-II accumulation, in an oral cancer cell line, OECM-1. To further assess the responses of other cell types to ANE 30-100K, we used both normal and malignant cells as the targets of ANE 30-100K and found that normal oral fibroblasts (CMT415), peripheral blood lymphocytes (PBLs), Jurkat leukemia T cells, and esophageal carcinoma cells (CE81T/VGH) exhibited similar responses after ANE 30-100K challenge. ANE 30-100K drastically increased acidic vesicle-containing PBLs isolated from two independent donors (from 0.1% to 92.1% and 2.9% to 64.2%). Furthermore, both ANE- and ANE 30-100K-induced LC3-II accumulation in CMT415 and CE81T/VGH was further increased in the presence of the lysosomal protease inhibitors (pepstatin A, E64d, and leupeptin). On the other hand, ANE 30-100K also increased the level of intracellular reactive oxygen species (ROS), and the ROS scavengers, N-acetylcysteine (NAC) and Tiron, inhibited ANE 30-100K-induced cell death and LC3-II accumulation. Collectively, these results suggest the existence of an autophagy-inducing AN ingredient (AIAI) in ANE 30-100K, which renders ANE as an autophagic flux inducer through ROS in both normal and malignant cells.