Structural influence of isothiocyanates on the antioxidant response element (ARE)-mediated heme oxygenase-1 (HO-1) expression

Abstract

Purpose: Isothiocyanates (ITCs), existing abundantly in cruciferous vegetables, is one class of promising dietary cancer chemopreventive agents that possess strong cancer protective effects by modulation of phase II detoxifying/antioxidant enzyme activities. However, limited studies regarding to the structure-activity relationship (SAR) of ITCs on the induction of phase II detoxifying/antioxidant enzymes are reported. In this study, the effects of ten structurally related isothiocyanates on the antioxidant response element (ARE)-mediated antioxidant enzyme heme oxygenase-1 (HO-1) induction in human hepatoma HepG2-C8 cells were evaluated.

Materials and methods: After exposure of HepG2-C8 cells to ITCs, cell viability, luciferase reporter assay, Western blot analysis and quantitative real-time PCR were conducted.

Results: Treatments with most ITCs significantly activated ARE-mediated luciferase activity with different maximal degree of ARE induction. In addition, ITCs caused a substantial induction of HO-1 protein, which was closely correlated with inductive level of Nrf2 protein. Real-time PCR revealed that the expression of HO-1 mRNA and protein was significantly increased after treatments with ITCs, although not directly correlated. HO-1 induction by ITCs was attenuated in HepG2-C8 cells transiently transfected with a dominant negative mutant of Nrf2 (Nrf2-M4), whereas it was totally absent in Nrf2 -/- mouse embryonic fibroblasts. In addition, ARE activation by ITCs was associated with the depletion of intracellular glutathione.

Conclusion: Collectively, our results demonstrate that the ITC class of compounds activates ARE-mediated HO-1 gene transcription through Nrf2/ARE signaling pathway, however, their inductive effects are quite specific, depending on the chemical structure. These results suggest the possibility that some synthetic ITCs might have superior chemopreventive activity than natural ITCs.