The Nrf2 transcription factor is a positive regulator of myeloid differentiation of acute myeloid leukemia cells

Irene Bobilev; Victoria Novik; Itai Levi; Ofer Shpilberg; Joseph Levy; Yoav Sharoni; George P. Studzinski; Michael Danilenko

doi:10.4161/cbt.11.3.14098

The Nrf2 transcription factor is a positive regulator of myeloid differentiation of acute myeloid leukemia cells

Irene Bobilev, Victoria Novik, Itai Levi, Ofer Shpilberg, Joseph Levy, Yoav Sharoni, George P. Studzinski, Michael Danilenko

Research output: Contribution to journal › Article › peer-review

54 Scopus citations

Abstract

1α,25-dihydroxyvitamin D₃ (1,25D) is a powerful differentiation agent, which has potential for treatment of acute myeloid leukemia (AML), but induces severe hypercalcemia at pharmacologically active doses. We have previously shown that carnosic acid (CA), the polyphenolic antioxidant from rosemary plant, markedly potentiates differentiation induced by low concentrations of 1,25D in human AML cell lines. Here, we demonstrated similar enhanced differentiation responses to the 1,25D/CA combination in primary leukemic cells derived from patients with AML, and determined the role of the Nrf2/antioxidant response element (Nrf2/ARE) pathway in these effects using U937 human monoblastic leukemia cells as the model. CA strongly transactivated the ARE-luciferase reporter gene, induced the ARE-responsive genes, NADP(H)-quinone oxidoreductase and the γ-glutamylcysteine synthetase heavy subunit, and elevated cellular glutathione levels. Interestingly, 1,25D potentiated the effects of CA on these activities. Stable transfection of wild-type (wt) Nrf2 resulted in the enhancement, while transfection of dominant-negative (dn) Nrf2 produced suppression of differentiation induced by the 1,25D/CA combination and, surprisingly, by 1,25D alone. These opposite effects were associated with a corresponding increase or decrease in vitamin D receptor and retinoid X receptor-α protein levels, and in vitamin D responsive element transactivation. Cells transfected with wtNrf2 and dnNrf2 also displayed opposing changes in the levels of the AP-1 family proteins (c-Jun and ATF2) and AP-1 transcriptional activity. Pretreatment with AP-1 decoy oligodeoxynucleotide markedly attenuated the differentiation in wtNrf2-transfected cells, suggesting that the pro-differentiation action of Nrf2 is mediated by functional AP-1. Our findings suggest that the Nrf2/ARE pathway plays an important part in the cooperative induction of myeloid leukemia cell differentiation by 1,25D and a plant polyphenol.

Original language	English
Pages (from-to)	317-329
Number of pages	13
Journal	Cancer Biology and Therapy
Volume	11
Issue number	3
DOIs	https://doi.org/10.4161/cbt.11.3.14098
State	Published - 1 Feb 2011
Externally published	Yes

Keywords

AP-1 transcription factor
Antioxidant response element
Carnosic acid
Vitamin D
Vitamin D receptor

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10.4161/cbt.11.3.14098

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@article{729abaa3582b41ddafad89654e08d87c,

title = "The Nrf2 transcription factor is a positive regulator of myeloid differentiation of acute myeloid leukemia cells",

abstract = "1α,25-dihydroxyvitamin D3 (1,25D) is a powerful differentiation agent, which has potential for treatment of acute myeloid leukemia (AML), but induces severe hypercalcemia at pharmacologically active doses. We have previously shown that carnosic acid (CA), the polyphenolic antioxidant from rosemary plant, markedly potentiates differentiation induced by low concentrations of 1,25D in human AML cell lines. Here, we demonstrated similar enhanced differentiation responses to the 1,25D/CA combination in primary leukemic cells derived from patients with AML, and determined the role of the Nrf2/antioxidant response element (Nrf2/ARE) pathway in these effects using U937 human monoblastic leukemia cells as the model. CA strongly transactivated the ARE-luciferase reporter gene, induced the ARE-responsive genes, NADP(H)-quinone oxidoreductase and the γ-glutamylcysteine synthetase heavy subunit, and elevated cellular glutathione levels. Interestingly, 1,25D potentiated the effects of CA on these activities. Stable transfection of wild-type (wt) Nrf2 resulted in the enhancement, while transfection of dominant-negative (dn) Nrf2 produced suppression of differentiation induced by the 1,25D/CA combination and, surprisingly, by 1,25D alone. These opposite effects were associated with a corresponding increase or decrease in vitamin D receptor and retinoid X receptor-α protein levels, and in vitamin D responsive element transactivation. Cells transfected with wtNrf2 and dnNrf2 also displayed opposing changes in the levels of the AP-1 family proteins (c-Jun and ATF2) and AP-1 transcriptional activity. Pretreatment with AP-1 decoy oligodeoxynucleotide markedly attenuated the differentiation in wtNrf2-transfected cells, suggesting that the pro-differentiation action of Nrf2 is mediated by functional AP-1. Our findings suggest that the Nrf2/ARE pathway plays an important part in the cooperative induction of myeloid leukemia cell differentiation by 1,25D and a plant polyphenol.",

keywords = "AP-1 transcription factor, Antioxidant response element, Carnosic acid, Vitamin D, Vitamin D receptor",

author = "Irene Bobilev and Victoria Novik and Itai Levi and Ofer Shpilberg and Joseph Levy and Yoav Sharoni and Studzinski, {George P.} and Michael Danilenko",

note = "Funding Information: In this study, we demonstrate for the first time that the transcrip- tion factor Nrf2 largely mediates the enhancing effect of a plant polyphenol on 1,25D-induced differentiation and is involved in the differentiation effect of 1,25D alone in human AML cells. This pro-differentiation role of Nrf2 was supported by the following {\"U} lines of evidence. -BOEFT#JPTDJFOFDirsFt, the differentiation-enhancing effect of CA in U937 Figure7.Treatment with AP-1 decoy oligonucleotide blocks the differentiation responses to the 1,25D/CA cells correlated with its ability %POPUEJTUSJCVUFcombination, most robustly in wtNrf2-transfected cells. U937 cells stably expressing empty vector (pEF) or to activate the Nrf2/ARE tran-with the indicated agents for an additional 60 h. The expression of CD11b and CD14 was then measured by wtNrf2 were preincubated with 7.5 μM TRE-ODN or the mutant mTRE-ODN for 24 h followed by treatment scription system and to increase the bivariate flow cytometric analysis. (A) Comparative read-outs of CD11b and CD14 cell surface expression de novo synthesis of cellular glu-obtained from ODN-treated pEF #3 and wtNrf2 #18 cells. (B) Averaged CD11b and CD14 expression data tathione in these cells. Moreover, from ODN-treated pEF #3 and pEF-mix cells (pEF-CD11b and pEF-CD14, respectively) and from wtNrf2 #18 1,25D was able to augment these #and wtNrf2 #20 cells (wtNrf2-CD11b and wtNrf2-CD14, respectively). Data are the means ± SE of three in- Nrf2/ARE-related CA activi-dependent experiments *p < 0.05 and p < 0.01 versus response to mTRE-ODN in the same treatment group. ties. Since in our experiments 1,25D did not affect Nrf2 levels, treatment of pEF#3 cells with CA for 24 h increased, to vari-its potentiation of CA-stimulated ARE-Luc reporter activity ous extents, both VDR and RXRα protein levels, as compared in U937 cells could result from post-translational modifica-to untreated control cells. On the other hand, 1,25D elevated tion of Nrf2. For instance, different signaling protein kinases the VDR content but had almost no effect on RXRα levels. The (Fig. 9) which are known to be activated by 1,25D in AML 1,25D/CA combination was slightly more effective than single cells (reviewed in ref. 5 and 49), have also been shown to agents (Fig. 8A and B). Importantly, wtNrf2 overexpression phosphorylate and activate Nrf2.50-52 Interestingly, despite the resulted in a marked increase in both VDR and, particularly, fact that 1,25D alone, even at a high concentration, had only RXRα levels both under basal and treatment conditions (Fig. a slight effect on the ARE-transactivation, including γGCSh-8A). In contrast, transfection of dnNrf2 strongly reduced both ARE4-Luc activation, it was capable of significantly inducing the basal and treatment-induced VDR and RXRα expression γGCSh expression and moderately elevating glutathione levels. (Fig. 8B). These changes in protein expression correlated with This suggests that in addition to the cooperation with CA in the corresponding changes in VDRE transactivation, as deter-the Nrf2/ARE-dependent γGCSh induction, 1,25D may also mined by the VDREx6-Luc reporter assay in transiently trans-activate its expression by an independent mechanism, thereby fected U937 cells (Fig. 8C). In pEF#3 and untransfected cells, increasing the glutathione generating capacity of the 1,25D/CA CA, which alone was practically ineffective, moderately increased system. VDRE activation by a low concentration of 1,25D (2.5 nM) to Second, ectopic expression of wtNrf2 or dnNrf2 in U937 cells the level comparable to that induced by a 100 nM 1,25D (Fig. resulted in a strong enhancement or attenuation, respectively, 8C). wtNrf2 overexpression strongly augmented this potentiating of the differentiation effects of both the 1,25D/CA combina-effect of CA, whereas dnNrf2 expression abrogated it (Fig. 8C). tion and, unexpectedly, 1,25D alone. The latter finding implies Importantly, the VDRE activation induced by 100 nM 1,25D3 that while 1,25D alone had minimal effects on Nrf2/ARE, this transcriptional pathway or Nrf2 itself plays an important regulatory role in the differentiation-inducing activity of 1,25D in at least some subtypes of AML cells. Third, the opposite consequences of wtNrf2 or dnNrf2 transfection on dif- ferentiation responses in U937 cells were associated with concomitant changes in the AP-1 and VDR transcription factor modules, the critical regulators of dif- ferentiation in AML cells. Moreover, the fact that AP-1 decoy ODN inhib- ited the wtNrf2-augmented differentia- tion responses further suggests that AP-1 can mediate, at least in part, the pro- differentiation activity of Nrf2. The possible modes of Nrf2 involve- ment in the differentiation of human AML cells are represented in Figure 9. Our observations that transfection of dnNrf2, which lacks the transactivation domain of the intact Nrf2,35 produced negative effects on the induction of dif- ferentiation markers and on AP-1 and VDR/RXRα expression and activity indicates that the tra{\"U}nscriptionally active -BOEFT#JPTDJFODF Nrf2 is required for the upregulation of sion of wtNrf2 or dnNrf2. The indicated clones of U937 cells (1 x 105 cells/ml) stably transfected with these systems. In support of the involve-empty vector (pEF#3; A and B), wtNrf2 (A) or dnNrf2 (B) were treated with vehicle (control) or the ment of the functional Nrf2/ARE t%ran-POPUEJTUSJCVUFagents indicated in (C), for 24 h. Whole cell lysates were analyzed by western blotting. Calreticulin scription pathway in the differentiation are shown. The identification numbers of wtNrf2 and dnNrf2 clones are indicated next to the cor-(CRN) was used as a protein loading control. Representative blots of three similar experiments of AML cells, we have previously shown responding blots. Note that the exposure of the RXRαblot from dnNrf2 #13 cells (B) to X-ray film that treatment of HL60 cells with buthi-was longer than in the rest of the RXRα blots (A and B) in order to allow visualization of lower RXRα onine sulfoximine, the specific inhibitor expression in this dnNrf2 clone compared to that in the pEF #3 clone. (C) VDRE-Luc reporter activ- of γGCS, resulted in a marked inhibition ity was determined in untransfected U937 cells (U937) and in the indicated stable clones following of 1,25D/CA-and 1,25D alone-induced or indicated test agent for 24 h. The relative VDRE-Luc activity (means ± SE) was calculated from transient transfection with VDREx6-Luc and Renilla luciferase and treatment with vehicle (control) differentiation which paralleled gluta-the data of three individual experiments performed in triplicate. The treatment groups in (A and B) thionedepletion.12 Another glutathione-are designated “1” to “5” as indicated in (C). §p < 0.05; ¶p < 0.01 and ‡p < 0.001 versus corresponding depleting compound, diethyl maleate, values of the pEF#3 cells. has been shown to block TPA-induced differentiation in HL60 cells and KG-1 human AML cells.53 fibroblasts isolated from Nrf2-/- mice had much lower cJun and We have previously shown that CA and 1,25D, alone and cFos levels compared to their wild-type counterparts and that in combination, decrease the levels or reactive oxygen species treatment with the Nrf2 activator tBHQ resulted in a marked in human and murine AML cell lines.12,14 This supports the increase in cJun and cFos levels in the wild-type cells but not in hypothesis that generation of reducing conditions through ele-Nrf2-/-cells. vation of intracellular glutathione and the induction of other AP-1 has been found to play an important role in myeloid Nrf2/ARE-responsive cellular antioxidants, such as thioredoxin differentiation of hematopoietic progenitors and different types (Fig. 9), may represent one possible mechanism by which CA of AML cells induced by various agents,48,57,58 including 1,25D,38 and, probably, other Nrf2-activating agents can potentiate and downregulation of the Jun family proteins has been asso-1,25D-differentiation of AML cells. Reducing intracellular envi-ciated with the pathogenesis of myeloproliferative disorders, ronment can lead to increased activity of AP-1 through main-chronic myelogenous leukemia and AML.58,59 Although the taining the cysteine residues in the DNA-binding sites of Jun/ precise mechanism of the pro-differentiation activity of AP-1 Fos proteins in their active reduced form.54,55 However, as indi-in AML cells remains unclear, one possibility is that AP-1 may cated by our data, activated Nrf2 may also contribute to AP-1 transcriptionally enhance VDR expression. The VDR promoter activation by upregulating the expression of at least some of the contains two AP-1 response elements.60 The putative role of AP-1 AP-1 family proteins, such as cJun and ATF-2 in AML cells. in the regulation of VDR expression is supported by our previous This is supported by the earlier published results56 showing that finding that the enhancement of 1,25D-induced differentiation have been implicated in cancer prevention, our evidence for the cooperation between 1,25D and a plant polyphenol in the activation of the Nrf2/ ARE pathway may provide a basis for the mechanism-based clinical and epidemiological chemoprevention studies of these combinations in AML and other neoplastic diseases.",

year = "2011",

month = feb,

day = "1",

doi = "10.4161/cbt.11.3.14098",

language = "אנגלית",

volume = "11",

pages = "317--329",

journal = "Cancer Biology and Therapy",

issn = "1538-4047",

publisher = "Landes Bioscience",

number = "3",

}

TY - JOUR

T1 - The Nrf2 transcription factor is a positive regulator of myeloid differentiation of acute myeloid leukemia cells

AU - Bobilev, Irene

AU - Novik, Victoria

AU - Levi, Itai

AU - Shpilberg, Ofer

AU - Levy, Joseph

AU - Sharoni, Yoav

AU - Studzinski, George P.

AU - Danilenko, Michael

N1 - Funding Information: In this study, we demonstrate for the first time that the transcrip- tion factor Nrf2 largely mediates the enhancing effect of a plant polyphenol on 1,25D-induced differentiation and is involved in the differentiation effect of 1,25D alone in human AML cells. This pro-differentiation role of Nrf2 was supported by the following Ü lines of evidence. -BOEFT#JPTDJFOFDirsFt, the differentiation-enhancing effect of CA in U937 Figure7.Treatment with AP-1 decoy oligonucleotide blocks the differentiation responses to the 1,25D/CA cells correlated with its ability %POPUEJTUSJCVUFcombination, most robustly in wtNrf2-transfected cells. U937 cells stably expressing empty vector (pEF) or to activate the Nrf2/ARE tran-with the indicated agents for an additional 60 h. The expression of CD11b and CD14 was then measured by wtNrf2 were preincubated with 7.5 μM TRE-ODN or the mutant mTRE-ODN for 24 h followed by treatment scription system and to increase the bivariate flow cytometric analysis. (A) Comparative read-outs of CD11b and CD14 cell surface expression de novo synthesis of cellular glu-obtained from ODN-treated pEF #3 and wtNrf2 #18 cells. (B) Averaged CD11b and CD14 expression data tathione in these cells. Moreover, from ODN-treated pEF #3 and pEF-mix cells (pEF-CD11b and pEF-CD14, respectively) and from wtNrf2 #18 1,25D was able to augment these #and wtNrf2 #20 cells (wtNrf2-CD11b and wtNrf2-CD14, respectively). Data are the means ± SE of three in- Nrf2/ARE-related CA activi-dependent experiments *p < 0.05 and p < 0.01 versus response to mTRE-ODN in the same treatment group. ties. Since in our experiments 1,25D did not affect Nrf2 levels, treatment of pEF#3 cells with CA for 24 h increased, to vari-its potentiation of CA-stimulated ARE-Luc reporter activity ous extents, both VDR and RXRα protein levels, as compared in U937 cells could result from post-translational modifica-to untreated control cells. On the other hand, 1,25D elevated tion of Nrf2. For instance, different signaling protein kinases the VDR content but had almost no effect on RXRα levels. The (Fig. 9) which are known to be activated by 1,25D in AML 1,25D/CA combination was slightly more effective than single cells (reviewed in ref. 5 and 49), have also been shown to agents (Fig. 8A and B). Importantly, wtNrf2 overexpression phosphorylate and activate Nrf2.50-52 Interestingly, despite the resulted in a marked increase in both VDR and, particularly, fact that 1,25D alone, even at a high concentration, had only RXRα levels both under basal and treatment conditions (Fig. a slight effect on the ARE-transactivation, including γGCSh-8A). In contrast, transfection of dnNrf2 strongly reduced both ARE4-Luc activation, it was capable of significantly inducing the basal and treatment-induced VDR and RXRα expression γGCSh expression and moderately elevating glutathione levels. (Fig. 8B). These changes in protein expression correlated with This suggests that in addition to the cooperation with CA in the corresponding changes in VDRE transactivation, as deter-the Nrf2/ARE-dependent γGCSh induction, 1,25D may also mined by the VDREx6-Luc reporter assay in transiently trans-activate its expression by an independent mechanism, thereby fected U937 cells (Fig. 8C). In pEF#3 and untransfected cells, increasing the glutathione generating capacity of the 1,25D/CA CA, which alone was practically ineffective, moderately increased system. VDRE activation by a low concentration of 1,25D (2.5 nM) to Second, ectopic expression of wtNrf2 or dnNrf2 in U937 cells the level comparable to that induced by a 100 nM 1,25D (Fig. resulted in a strong enhancement or attenuation, respectively, 8C). wtNrf2 overexpression strongly augmented this potentiating of the differentiation effects of both the 1,25D/CA combina-effect of CA, whereas dnNrf2 expression abrogated it (Fig. 8C). tion and, unexpectedly, 1,25D alone. The latter finding implies Importantly, the VDRE activation induced by 100 nM 1,25D3 that while 1,25D alone had minimal effects on Nrf2/ARE, this transcriptional pathway or Nrf2 itself plays an important regulatory role in the differentiation-inducing activity of 1,25D in at least some subtypes of AML cells. Third, the opposite consequences of wtNrf2 or dnNrf2 transfection on dif- ferentiation responses in U937 cells were associated with concomitant changes in the AP-1 and VDR transcription factor modules, the critical regulators of dif- ferentiation in AML cells. Moreover, the fact that AP-1 decoy ODN inhib- ited the wtNrf2-augmented differentia- tion responses further suggests that AP-1 can mediate, at least in part, the pro- differentiation activity of Nrf2. The possible modes of Nrf2 involve- ment in the differentiation of human AML cells are represented in Figure 9. Our observations that transfection of dnNrf2, which lacks the transactivation domain of the intact Nrf2,35 produced negative effects on the induction of dif- ferentiation markers and on AP-1 and VDR/RXRα expression and activity indicates that the traÜnscriptionally active -BOEFT#JPTDJFODF Nrf2 is required for the upregulation of sion of wtNrf2 or dnNrf2. The indicated clones of U937 cells (1 x 105 cells/ml) stably transfected with these systems. In support of the involve-empty vector (pEF#3; A and B), wtNrf2 (A) or dnNrf2 (B) were treated with vehicle (control) or the ment of the functional Nrf2/ARE t%ran-POPUEJTUSJCVUFagents indicated in (C), for 24 h. Whole cell lysates were analyzed by western blotting. Calreticulin scription pathway in the differentiation are shown. The identification numbers of wtNrf2 and dnNrf2 clones are indicated next to the cor-(CRN) was used as a protein loading control. Representative blots of three similar experiments of AML cells, we have previously shown responding blots. Note that the exposure of the RXRαblot from dnNrf2 #13 cells (B) to X-ray film that treatment of HL60 cells with buthi-was longer than in the rest of the RXRα blots (A and B) in order to allow visualization of lower RXRα onine sulfoximine, the specific inhibitor expression in this dnNrf2 clone compared to that in the pEF #3 clone. (C) VDRE-Luc reporter activ- of γGCS, resulted in a marked inhibition ity was determined in untransfected U937 cells (U937) and in the indicated stable clones following of 1,25D/CA-and 1,25D alone-induced or indicated test agent for 24 h. The relative VDRE-Luc activity (means ± SE) was calculated from transient transfection with VDREx6-Luc and Renilla luciferase and treatment with vehicle (control) differentiation which paralleled gluta-the data of three individual experiments performed in triplicate. The treatment groups in (A and B) thionedepletion.12 Another glutathione-are designated “1” to “5” as indicated in (C). §p < 0.05; ¶p < 0.01 and ‡p < 0.001 versus corresponding depleting compound, diethyl maleate, values of the pEF#3 cells. has been shown to block TPA-induced differentiation in HL60 cells and KG-1 human AML cells.53 fibroblasts isolated from Nrf2-/- mice had much lower cJun and We have previously shown that CA and 1,25D, alone and cFos levels compared to their wild-type counterparts and that in combination, decrease the levels or reactive oxygen species treatment with the Nrf2 activator tBHQ resulted in a marked in human and murine AML cell lines.12,14 This supports the increase in cJun and cFos levels in the wild-type cells but not in hypothesis that generation of reducing conditions through ele-Nrf2-/-cells. vation of intracellular glutathione and the induction of other AP-1 has been found to play an important role in myeloid Nrf2/ARE-responsive cellular antioxidants, such as thioredoxin differentiation of hematopoietic progenitors and different types (Fig. 9), may represent one possible mechanism by which CA of AML cells induced by various agents,48,57,58 including 1,25D,38 and, probably, other Nrf2-activating agents can potentiate and downregulation of the Jun family proteins has been asso-1,25D-differentiation of AML cells. Reducing intracellular envi-ciated with the pathogenesis of myeloproliferative disorders, ronment can lead to increased activity of AP-1 through main-chronic myelogenous leukemia and AML.58,59 Although the taining the cysteine residues in the DNA-binding sites of Jun/ precise mechanism of the pro-differentiation activity of AP-1 Fos proteins in their active reduced form.54,55 However, as indi-in AML cells remains unclear, one possibility is that AP-1 may cated by our data, activated Nrf2 may also contribute to AP-1 transcriptionally enhance VDR expression. The VDR promoter activation by upregulating the expression of at least some of the contains two AP-1 response elements.60 The putative role of AP-1 AP-1 family proteins, such as cJun and ATF-2 in AML cells. in the regulation of VDR expression is supported by our previous This is supported by the earlier published results56 showing that finding that the enhancement of 1,25D-induced differentiation have been implicated in cancer prevention, our evidence for the cooperation between 1,25D and a plant polyphenol in the activation of the Nrf2/ ARE pathway may provide a basis for the mechanism-based clinical and epidemiological chemoprevention studies of these combinations in AML and other neoplastic diseases.

PY - 2011/2/1

Y1 - 2011/2/1

N2 - 1α,25-dihydroxyvitamin D3 (1,25D) is a powerful differentiation agent, which has potential for treatment of acute myeloid leukemia (AML), but induces severe hypercalcemia at pharmacologically active doses. We have previously shown that carnosic acid (CA), the polyphenolic antioxidant from rosemary plant, markedly potentiates differentiation induced by low concentrations of 1,25D in human AML cell lines. Here, we demonstrated similar enhanced differentiation responses to the 1,25D/CA combination in primary leukemic cells derived from patients with AML, and determined the role of the Nrf2/antioxidant response element (Nrf2/ARE) pathway in these effects using U937 human monoblastic leukemia cells as the model. CA strongly transactivated the ARE-luciferase reporter gene, induced the ARE-responsive genes, NADP(H)-quinone oxidoreductase and the γ-glutamylcysteine synthetase heavy subunit, and elevated cellular glutathione levels. Interestingly, 1,25D potentiated the effects of CA on these activities. Stable transfection of wild-type (wt) Nrf2 resulted in the enhancement, while transfection of dominant-negative (dn) Nrf2 produced suppression of differentiation induced by the 1,25D/CA combination and, surprisingly, by 1,25D alone. These opposite effects were associated with a corresponding increase or decrease in vitamin D receptor and retinoid X receptor-α protein levels, and in vitamin D responsive element transactivation. Cells transfected with wtNrf2 and dnNrf2 also displayed opposing changes in the levels of the AP-1 family proteins (c-Jun and ATF2) and AP-1 transcriptional activity. Pretreatment with AP-1 decoy oligodeoxynucleotide markedly attenuated the differentiation in wtNrf2-transfected cells, suggesting that the pro-differentiation action of Nrf2 is mediated by functional AP-1. Our findings suggest that the Nrf2/ARE pathway plays an important part in the cooperative induction of myeloid leukemia cell differentiation by 1,25D and a plant polyphenol.

AB - 1α,25-dihydroxyvitamin D3 (1,25D) is a powerful differentiation agent, which has potential for treatment of acute myeloid leukemia (AML), but induces severe hypercalcemia at pharmacologically active doses. We have previously shown that carnosic acid (CA), the polyphenolic antioxidant from rosemary plant, markedly potentiates differentiation induced by low concentrations of 1,25D in human AML cell lines. Here, we demonstrated similar enhanced differentiation responses to the 1,25D/CA combination in primary leukemic cells derived from patients with AML, and determined the role of the Nrf2/antioxidant response element (Nrf2/ARE) pathway in these effects using U937 human monoblastic leukemia cells as the model. CA strongly transactivated the ARE-luciferase reporter gene, induced the ARE-responsive genes, NADP(H)-quinone oxidoreductase and the γ-glutamylcysteine synthetase heavy subunit, and elevated cellular glutathione levels. Interestingly, 1,25D potentiated the effects of CA on these activities. Stable transfection of wild-type (wt) Nrf2 resulted in the enhancement, while transfection of dominant-negative (dn) Nrf2 produced suppression of differentiation induced by the 1,25D/CA combination and, surprisingly, by 1,25D alone. These opposite effects were associated with a corresponding increase or decrease in vitamin D receptor and retinoid X receptor-α protein levels, and in vitamin D responsive element transactivation. Cells transfected with wtNrf2 and dnNrf2 also displayed opposing changes in the levels of the AP-1 family proteins (c-Jun and ATF2) and AP-1 transcriptional activity. Pretreatment with AP-1 decoy oligodeoxynucleotide markedly attenuated the differentiation in wtNrf2-transfected cells, suggesting that the pro-differentiation action of Nrf2 is mediated by functional AP-1. Our findings suggest that the Nrf2/ARE pathway plays an important part in the cooperative induction of myeloid leukemia cell differentiation by 1,25D and a plant polyphenol.

KW - AP-1 transcription factor

KW - Antioxidant response element

KW - Carnosic acid

KW - Vitamin D

KW - Vitamin D receptor

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U2 - 10.4161/cbt.11.3.14098

DO - 10.4161/cbt.11.3.14098

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C2 - 21099366

AN - SCOPUS:79951527854

SN - 1538-4047

VL - 11

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EP - 329

JO - Cancer Biology and Therapy

JF - Cancer Biology and Therapy

IS - 3

ER -

The Nrf2 transcription factor is a positive regulator of myeloid differentiation of acute myeloid leukemia cells

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