The apoptotic and necrotic effects of tomatine adjuvant.

Tomatine adjuvant, consisting of tomatine, n-octyl-beta-d-glucopyranoside (OGP), phosphatidylethanolamine and cholesterol is unique in that when combined with soluble protein antigen it elicits a cytotoxic T lymphocyte (CTL) response in immunized animals. The mechanisms underlying this property are unknown. In an attempt to understand how tomatine activates cellular immunity, we examined its potential to induce apoptosis. Thus in the present study, cell death of EL4 thymoma cells induced by whole adjuvant and the surface-active components in the formulation was examined. Cytotoxicity was monitored using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and lactate dehydrogenase release assays, apoptosis and necrosis were quantified by flow cytometry using Annexin V and propidium iodide staining, and morphology was examined by Hoechst 33342 staining. Flow cytometric analysis demonstrated the appearance of the sub-G1 phase in cells treated with these agents and Annexin V/PI staining showed that all three agents induced both apoptosis and necrosis in EL4 cells in a concentration-dependent manner. Tomatine was effective at much lower concentrations than OGP, suggesting that the majority of the effect of whole adjuvant could be attributed to this component. Microscopic examination of EL4 cells after treatment with these agents revealed morphological features of apoptosis, including chromatin condensation and DNA fragmentation. Pretreatment with zVAD-fmk did not block cell death induced by these agents, showing that tomatine adjuvant-induced EL4 cell apoptosis is caspase-independent.