Mangiferin increases Nrf2 protein stability by inhibiting its ubiquitination and degradation in human HL60 myeloid leukemia cells

The nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant signaling pathway is a key target for cancer chemoprevention. Recent studies have that Nrf2 activation may be the result of an increase in Nrf2 protein stability. Mangiferin (MA), a compound monomer extracted from the mango plant, has antioxidant and cytoprotective activities. Our previous study demonstrated that MA increased Nrf2 expression and activated Nrf2 signaling in hematopoietic cells. Thus, in the present study, we aimed to investigate the mechanisms by which MA increases Nrf2 expression in human HL60 myeloid leukemia cells in vitro. Our western blot analysis results revealed that MA markedly increased Nrf2 expression in dose- and time-dependent manner. However treatment with MA did not affect the Nrf2 mRNA level. The results of cycloheximide (CHX)-chase analysis demonstrated that the Nrf2 protein half-life was prolonged to 58 min when the HL60 cells were pre-incubated with 50 mu M MA for 4 h, whereas its half-life was only 20 min in the non-MA treated control cells. Further experiments revealed that MA mainly enhanced non-ubiquitinated Nrf2 protein levels when increasing Nrf2 protein stability; these effects differed from those induced by the proteasome inhibitor, MG132. Subsequent immunoprecipitation experiments confirmed that MA inhibited Nrf2 ubiquitination in HL60 cells. These results provide evidence that MA increases Nrf2 protein stability by inhibiting its ubiquitination and degradation in hematopoietic cells. This may be one of the mechanisms through which MA activates the Nrf2-mediated antioxidant response and exerts cytoprotective effects.