Euonymus alatus (Thunb.) Sieb (EA) is a traditional Korean herbal medicine, commonly used to treat tumors in Korea and China for centuries. Our earlier studies have indicated that EA exhibits antitumor properties, but its mechanism remains to be elucidated. In this study, we evaluated the molecular mechanism of EA in a human uterine leiomyomal smooth muscle cell (ULSMC) line. Water extracts of EA have been reported to not only function as antioxidants but also cause cytotoxic effect. We investigated the mechanism of EA-induced cytotoxicity in human ULSMC. When cells were cultured with 20-200 microg/mL EA for 6 h, caspase-3 was activated and then cells fell into apoptosis. Induction of apoptosis by EA was accompanied with increase of the cytosolic fractions of cytochrome c prior to the activation of caspase-3. The preculture with 5 mM of buthionine sulfoximine, an inhibitor of glutathione synthesis, facilitated EA-induced induction of apoptosis. The preculture with N-benzyloxycarbonyl-valyl-alanyl-aspartyl fluoromethylketone, a pan-caspase inhibitor, partially suppressed the induction of apoptosis. EA showed little toxic effect on peripheral blood mononuclear cells from healthy volunteers. These results indicate that EA acts as a prooxidant and induces caspase-3 activation and apoptosis via mitochondrial pathway.

Glucosamine (GlcN), like N-acetylglucosamine (GlcNAc), is salvaged into the hexosamine pathway and is converted to UDP-GlcNAc. Golgi N-glycan branching enzymes produce N-glycans, using UDP-GlcNAc as a substrate, which attach to the T cell receptor (TCR) and cytotoxic T-lymphocyte antigen-4 (CTLA-4). These findings suggest that GlcN exerts the immunoregulation through TCR signalling, which could be involved not only in cytokine production but also activated T cell apoptosis. In fact, a preliminary study showed that GlcN reduced the number of CD3+ T cells of NC/Nga mice with AD-like skin lesions. Therefore, whether apoptosis of T cells would be one of the potential molecular mechanisms of GlcN-induced immunosuppression was investigated. Cultured human primary along with Jurkat T cells and purified T cells from NC/Nga mice with or without Df-induced AD-like skin lesion were used for the study. Glucosamine treatment increased the number of T cells expressing b1,6GlcNAc-branched N-glycans, with reduced ZAP-70 phosphorylation and enhanced CTLA-4 expression. Glucosamine treatment reduced the number of activated T cells from both the human primary and Jurkat cells and the dermatitis-induced mice. The expression of FasL and activated caspases, particularly caspase-3, was increased, whereas the phosphorylation of PI3K, Akt and NF-jB was decreased by GlcN treatment. Therefore, in addition to downregulating TCR signalling and promoting CTLA-4 expression, GlcN may also suppress T cell function by enhancing apoptosis of activated T cells, through both extrinsic and intrinsic apoptotic signalling pathways, which were regulated by the inhibition of PI3K/Akt and NF-jB phosphorylation.