Mangiferin Regulates Nrf2Ubiquitination And Degradation In Human HL-60Acute Myeloid Leukemia Cells

Purpose: To investigate the regulative effect of mangiferin (MA) on Nrf2proteinexpression in human HL-60acute myeloid leukemia cells. And to further explore themechanisms of interfering Nrf2ubiquitination and degradation by MA. Methods: HL-60cells were treated as follows:①treated with MA in a dose-course(0,50,100,200μM MA) and a time-course (0,1,4,12,24h) experiments;②treatedwith100μg/ml cycloheximide (CHX) or pretreated with50μM MA for4h followedby CHX over a1-h time-course period;③treated with MA, MG-132, MA andMG-132for4h. The treated cells were lysated and prepared. Nrf2protein levels wereanalyzed by Western blotting. Nrf2-mRNA levels were analyzed by Real-time PCR.The ubiquitinated Nrf2was detected by immunoprecipitation (IP).Results:①MA treatment increased Nrf2protein level in human HL-60myeloidleukemia cells in a dose-dependent and a time-dependent manner.②MA treatmentdid not affect the transcription of Nrf2.③MA treatment prolonged the half-time ofNrf2protein.④MA treatment mainly enhanced unubiquitinated Nrf2protein level,while enhanced and proteasome inhibitor MG-132treatment increased bothunubiquitinated and ubiquitinated Nrf2protein levels.⑤MA inhibited binding ofNrf2protein to ubiquitin in HL-60cells.Conclusion: MA up-regulates Nrf2protein expression in human HL-60acutemyeloid leukemia cells. Its molecular mechanism seems not involve Nrf2genetranscription and protein synthesis. However, MA increases protein stability andprolongs half-time of Nrf2which may lead to accumulation of Nrf2protein in HL-60...