Molecular mechanism of dihydropyrazine-induced cytotoxicity: the possibility of an independent pathway from the receptor for advanced glycation end products

Abstract

Dihydropyrazines (DHPs) are one of glycation products that are non-enzymatically generated in vivo and in food. We had previously revealed that 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), a methyl-substituted DHP, elicited redox imbalance and cytotoxicity in cultured cells. However, the molecular mechanisms underlying DHP-3-induced cytotoxicity remain unclear. To address this issue, we examined the involvement of the receptor for advanced glycation end products (RAGE) in DHP-3-induced cytotoxicity. To evaluate the role of RAGE, we prepared HeLa cells that constitutively expressed RAGE and its deletion mutant, which lacks the cytoplasmic domain (RAGE_Δcyto), using an episomal vector. After transfection with the vector, cells were selected following incubation with multiple concentrations of hygromycin to remove non-transfected cells. The expression of RAGE and RAGE_Δcyto in the cells was confirmed by immunoblotting. RAGE and RAGE_Δcyto were apparently expressed in transfected cells; however, there were no significant differences in DHP-3-induced cytotoxicity between these cells and mock vector-transfected cells. These results suggested that DHP-3 elicits cytotoxicity in a RAGE-independent manner.