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
The nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated signaling pathway is a major cellular defense pathway against oxidative or electrophilic stress (
However, the regulatory mechanisms involved in Nrf2 activation are not yet fully understood. Although it is well established that Nrf2 activity is controlled, at least in part, by the cytosolic protein, Kelch-like ECH-associated protein 1 (Keap1), the mechanisms by which Keap1 acts to repress Nrf2 activity remain to be fully characterized. Previously, it was considered that most of the Nrf2 proteins were bound to Keap1 and sequestered in cytoplasm under homeostatic conditions (
Mangiferin (MA), 2-C-β-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone, is a compound monomer extracted from certain plants of the Anacardiaceae and Gentianaceae families, including
Previous stuides have revealed that MA induces Nrf2-mediated antioxidant response, which provides an explanation for its antioxidant activity (
In this study, we demonstrate that MA increases Nrf2 expression, but not transcription in human HL60 myeloid leukemia cells. We provide evidence that MA prolongs the half-life of the Nrf2 protein by inhibiting its ubiquitination and degradation.
MA (C19H18O11; molecular weight, 422.34) and cycloheximide (CHX, C15H23NO4; molecular weight, 281.4) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MG132 (C26H42N3O5; molecular weight, 475.6) was obtained from Enzo Life Sciences (Farmingdale, NY, USA). MA and MG132 were initially dissolved in dimethyl sulfoxide (DMSO), stored at −20°C and thawed on ice prior to use. CHX was dissolved in ultrapure water, stored at −80°C and thawed on ice prior to use. RNAiso plus, PrimeScript RT Master Mix and SYBR Premix Ex Taq were purchased from Takara Bio, Inc. (Otsu, Japan). The primers used for real-time PCR were designed and synthesized by Takara Bio, Inc.. Rabbit polyclonal antibody against human Nrf2 (C-20) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Mouse monoclonal antibody against human ubiquitin was obtained from Merck Millipore (Billerica, MA, USA). The BCA protein assay kit was from Pierce Biotechnology, Inc. (Rockford, IL, USA). The Image Lab enhanced chemiluminescence (ECL) detection system was from Bio-Rad (Hercules, CA, USA). Cell culture medium, RPMI-1640 and fetal abovine serum (FBS) were from HyClone (Logan, UT, USA).
The human HL60 myeloid leukemia cell line was kindly provided by Professor Jianfeng Zhou (Cancer Biology Research Center, Tongji Hospital, Wuhan, China), and then maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a humidified incubator containing 5% CO2 in air.
Total RNA was isolated using RNAiso Plus according to the manufacturer’s instructions. Total RNA was isolated using RNAiso Plus, and then 400 ng total RNA were reverse transcripted into cDNA. Subsequently, 2 μl cDNA were amplified with SYBR-Green Universal PCR Master mix in triplicate on a real-time PCR system (CFX96, Bio-Rad). Nrf2 mRNA levels related to β-actin levels were calculated using the ΔCt (cycle threshold) method. The primer sequences for human Nrf2 were: forward, 5′-ACTCCGGCATTTCACTAAACACAAG-3′ and reverse, 5′-CTGAGGCCAAGTAGTGTGTCTCCA-3′. The primer sequences for human β-actin were: forward, 5′-GCCCAGTCCTCTCCCAAGTC-3′ and reverse, 5′-GGCACGAAGGTCATCATTC-3′. Control cells were processed in an identical manner apart from MA treatment.
For immunoblotting, whole-cell lysates were prepared using lysis buffer for 30 min on ice. Supernatants were collected as samples following centrifuged at 15,000 rpm for 10 min at 4°C. Protein concentrations were determined using a BCA protein assay kit (Pierce Biotechnology, Inc.). Following denaturation, equal amounts of the protein extracts were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked in phosphate-buffered saline (PBS) containing 5% non-fat milk for 1 h at room temperature. It was subsequently incubated with anti-Nrf2 antibody (dilution 1:1,000) at 4°C overnight, followed by washing and treatment with HRP-labeled secondary antibody (Pierce Biotechnology, Inc.) for 2 h at room temperature. The blots were incubated with ECL reagent for 5 min, and the signals were then detected with a chemiluminescence detection system (Bio-Rad). After stripping, the membrane was re-probed with human anti-β-actin antibody as a control for equal protein loading and protein integrity.
Nrf2 protein degradation was analyzed by CHX-chase analysis. The HL60 cells were pre-incubated with or without 50 μM MA for 4 h. Subsequently, 100 μg/ml CHX were added to inhibit protein synthesis. The cells were collected at 0, 5, 15, 30, 45 and 60 min after following treatment with CHX, as previously described (
The HL60 cells were treated with 50 μM MA, 10 μM MG132, or a combination of 50 μM MA and 10 μM MG132 for 4 h. The cells were washed twice with ice-cold PBS. The cells were then prepared in lysis/IP buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% DOC, 0.1% SDS, 50 mM NaF, 1 mM Na3VO4, 20 mM β-glycerophosphate and 1 mM okadaic acid] on ice for 30 min, then centrifuged at 14,000 rpm for 15 min. Cell lysates were incubated with anti-Nrf2 antibody at 4°C for 16 h. The immune complexes were then precipitated with protein A-Sepharose beads at 4°C for an additional 2 h. Subsequently, the precipitates were washed extensively with IP buffer, fractionated by SDS-PAGE and immunobloted with anti-ubiquitin antibody, as previously described (
Data are expressed as the mean ± SD of at least 3 independent experiments and processed by SPSS 17.0 statistical software for Windows. One-way ANOVA and Student-Newman-Keuls tests were applied for comparisons between each group. A value of P<0.05 was considered to indicate a statistically significant difference.
To determine the effects of MA on Nrf2 expression, the HL60 cells were treated with MA in dose-and time-course experiments. The Nrf2 protein levels in whole cell lysate were then detected by western blot analysis. As shown in
To determine the effects of MA on Nrf2 transcription, the HL60 cells were treated with MA in dose-and time-course experiments as described above, and the Nrf2 mRNA levels were then detected by real-time PCR. As shown in
To determine the effects of MA on Nrf2 protein stability, the half-life of Nrf2 protein was calculated in the MA-treated and non-MA-treated HL60 cells. As shown in
To investigate the mechanisms by which MA increases Nrf2 protein stability, the HL60 cells were treated with 50 μM MA, 10 μM of the proteasome inhibitor, MG132, or a combination of 50 μM MA and 10 μM MG132 for 4 h. The cells were then prepared for the analysis of the Nrf2 level by western blot analysis. The molecular weights of Nrf2 and poly-ubiquitinated Nrf2 are 57 and 100 kDa, respectively, according to the Nrf2 antibody data sheet (Santa Cruz Biotechnology). As shown in
To investigate whether MA suppresses Nrf2 ubiquitination, we performed IP experiments to pull down Nrf2 from the cell lysis of HL60 cells, and then identified ubiquitinated Nrf2 by immunoblot analysis with anti-ubiquitin monoclonal antibody. As shown in
The present study demonstrates that the natural antioxidant, mangiferin (MA), increases Nrf2 expression through post-translational mechanisms in hematopoietic cells. MA enhances the Nrf2 protein level, but does not affect its transcription. MA reduces protein degradation and prolongs the half-life of Nrf2 by inhibiting its ubiquitination, which leads to its intracellular accumulation. These results provide evidence that MA enhances Nrf2 expression by interfering with the ubiquitin-proteasome protein degradation pathway and increasing its protein stability. As mentioned above, MA activates Nrf2 and this activation may result from the increased protein stability and subsequent intracellular accumulation (
Previous studies have confirmed that Nrf2 is targeted for rapid degradation by the ubiquitin-proteasome pathway. Nrf2 is a highly unstable protein and its half-life is only 15–30 min in unstressed cells (
In the present study, MA markedly inhibited Nrf2 ubquitination, which may explain the increase in Nrf2 stability. Certain natural or synthetic Nrf2 inducers/activators can also increase Nrf2 protein stability; however, the mechanisms involved are not completely similar to those of MA (
As a newly identified Nrf2 activator, MA may be a potential cytoprotective agent for hematopoietic cells, as well as a chemopreventive agent against leukemia. It has been well documented that Nrf2 activators/inducers exert cytoprotective effects through antioxidant mechanisms (
In conclusion, our study confirms that MA inhibits Nrf2 ubiquitination and increases its stability. This may be one of the mechanims through which it induces Nrf2 cellular accumulation and activates Nrf2-mediated signaling. MA may be a potential cytoprotective agent for hematopoietic cells and a chemopreventive agent against leukemia, which warrants further study.
This study was supported by grants from the National Natural Science Foundation of China (no. 30900632 and no. 81372541). The authors would like to thank the Department of Central Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, for providing relevant experimental facilities and technical support.
Mangiferin (MA) increases Nrf2 expression in HL60 cells. (A) Cells were treated with 0, 50, 100, 200 μM MA and DMSO for 24 h. (B) Cells were treated with 50 μM MA for 0, 1, 4, 12, 24 h. Total cell lysates were subjected to western blot analysis with anti-Nrf2 and anti-β-actin antibodies. β-actin was used as the control for equal protein loading and protein integrity. (C) The results in (A) were quantified by densitometry. (D) The results in (B) were quantified by densitometry. Nrf2 values were plotted after normalization to those of β-actin. Data represent the means ± SD of 3 independent experiments (*P<0.05, compared with the untreated control group).
Mangiferin (MA) does not affect Nrf2 transcription in HL60 cells. (A) Cells were treated with 0, 50, 100, 200 μM MA and DMSO for 24 h. (B) Cells were treated with 50 μM MA for 0, 1, 4, 12, 24 h. Total RNA was extracted and reverse-transcribed into cDNA, then cDNA was subjected to real-time PCR for the detection of Nrf2 mRNA. Data represent the means ± SD of 3 independent experiments.
Mangiferin (MA) prolongs Nrf2 protein half-life in HL60 cells. (A) Cells were treated with 100 μg/ml cycloheximide (CHX) over a 1-h time period. (B) Cells were treated with 50 μM MA for 4 h followed by 100 μg/ml CHX over a 1 h time period. Cells were lysed at the indicated time points. Cell lysates were subjected to western blot analysis with anti-Nrf2 and anti-β-actin antibodies. β-actin was used as the control for equal protein loading and protein integrity. (C) The results in (A and B) were quantified by densitometry and plotted on a semi-log graph. The value of cells not treated with CHX was set as 1. Data represent the means ± SD of 3 independent experiments.
Mangiferin (MA) interferes with the ubiquitin-proteasome degradation of Nrf2 protein in HL60 cells. (A) Cells were treated with 50 μM MA, 10 μM MG132, or a combination of MA and MG132 for 4 h. Total cell lysates were subjected to western blot analysis with anti-Nrf2 and anti-β-actin antibodies. β-actin was used as the control for equal protein loading and protein integrity. (B) The results in (A) were quantified by densitometry. Nrf2 values were plotted after normalization to those of β-actin. Data represent the means ± SD of 3 independent experiments (*P<0.05, compared with the non-treated control group).
Mangiferin (MA) inhibits Nrf2 ubiquitination in HL60 cells. Cells were treated with 50 μM MA, 10 μM MG132, or a combination of MA and MG132 for 4 h. Total cell lysates were immunoprecipitated with anti-Nrf2 antibody, and then immunoblotted with anti-ubiquitin antibody. *Represented ubiquitinated Nrf2.