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.

Arecoline and the 30-100 kDa fraction of areca nut extract differentially regulate mTOR and respectively induce apoptosis and autophagy: a pilot study.

Areca nut (AN) is recognized as a human carcinogen; however, few studies of the cytotoxic effects of AN ingredients on cells have been reported. In Taiwan, AN, lime and inflorescence of Piper betle are the common components of betel quid (BQ). We recently noticed that extract of AN (ANE), but not those of lime and inflorescence of Piper betle, induces rounding cell morphology and nuclear shrinkage in different types of carcinoma cells. In this study, the rounding cell activity was first traced to the partially purified >or=10 kDa fraction (ANE >or= 10 K) and subsequently to the 30-100 kDa fraction (ANE 30-100 K). ANE and ANE >or=10 K stimulated nuclear shrinkage (P < 0.001 in both cases) and the clearance of the cytoplasm. ANE, ANE >or= 10 K, and ANE 30-100 K induced the cleavage of LC3-I (P < 0.05, 0.01, and 0.05, respectively) and the emergence of autophagic vacuoles (AVs) and acidic vesicles. On the other hand, arecoline (Are, the major alkaloid of AN) triggered caspase-3 activation, peri-nuclear chromatin condensation, and micronucleation. Meanwhile, ANE 30-100 K, but not Are, inhibited the phosphorylation of the mammalian target of rapamycin (mTOR)-Ser(2448). In conclusion, this study demonstrates that different AN ingredients exerting differential impact on mTOR-Ser(2448) phosphorylation are capable of triggering apoptosis and autophagy.