Different roles of Nrf2 and NFKB in the antioxidant imbalance produced by esculetin or quercetin on NB4 leukemia cells

Accepted Manuscript Different roles of Nrf2 and NFKB in the antioxidant imbalance produced by esculetin or quercetin on NB4 leukemia cells Virginia Rubio, Ana I. García-Pérez, Angel Herráez, José C. Diez PII:

S0009-2797(18)30610-0

DOI:

10.1016/j.cbi.2018.08.015

Reference:

CBI 8389

To appear in:

Chemico-Biological Interactions

Received Date: 1 June 2018 Revised Date:

23 July 2018

Accepted Date: 17 August 2018

Please cite this article as: V. Rubio, A.I. García-Pérez, A. Herráez, José.C. Diez, Different roles of Nrf2 and NFKB in the antioxidant imbalance produced by esculetin or quercetin on NB4 leukemia cells, Chemico-Biological Interactions (2018), doi: 10.1016/j.cbi.2018.08.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Different roles of Nrf2 and NFKB in the antioxidant imbalance

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produced by esculetin or quercetin on NB4 leukemia cells

Virginia Rubio, Ana I. García-Pérez, Angel Herráez and José C. Diez*

Unidad de Bioquímica y Biología Molecular. Departamento de Biología de Sistemas.

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Alcalá. 28871 Alcalá de Henares (Madrid) Spain.

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Facultad de Medicina y Ciencias de la Salud. Campus Universitario. Universidad de

Corresponding author: * José C. Diez. Departamento de Biología de Sistemas. Unidad de Bioquímica y Biología Molecular. Campus Universitario. Universidad de Alcalá. 28871 Alcalá de Henares (Madrid) SPAIN.

Phone number: 34-91-8854582 or 34-91-8854579. Fax number: 34-91-8854585.

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E-mail: [email protected]

Running title: Nrf2 and NFKB in the antioxidant imbalance of NB4 leukemia cells

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Key words: Antioxidants, apoptosis, esculetin, hydrogen peroxide, reactive oxygen

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species, tert-butylhydroperoxide.

Abbreviations

APL, Acute promyelocytic leukaemia; Annexin V-FITC, fluorescein isothiocyanate coupled to annexin V; BSO, buthionine-[S, R]-sulfoximine; DMSO, dimethyl sulfoxide; HEL, human erythromegakaryocytic leukaemia; H2O2, hydrogen peroxide; DHE, hydroethidine; LOX, lipoxygenase; PI, propidium iodide; MTT, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; SOD, Superoxide dismutase; t-BHP, tert-butylhydroperoxide.

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ACCEPTED MANUSCRIPT Abstract Esculetin

(6,7-dihydrocoumarin)

and

the

flavonoid

quercetin

(3,5,7,3',4'

pentahydroxyflavone) are compounds that could change the balance of redox homeostasis. NB4 leukemia cells were treated with 25 µM quercetin for 24 h and with esculetin at either 100 or 500 µM for different times. Quercetin increased the levels of

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pro-inflammatory NFkB p65 in the nucleus correspondingly reducing them in the cytosol. The levels of NFkB p65 decreased in the nucleus at high esculetin concentration treatments for long times (19 h), concomitantly increasing the levels of antiinflammatory NFkB p50 in the nucleus. This could suggest formation of inhibitory p50

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homodimers possibly related with anti-inflammatory response. Lipoxygenase expression was reduced either by esculetin or quercetin. A significant increase of Nrf2 in the nucleus of NB4 cells treated with 100 µM esculetin for 19 hours was observed.

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Quercetin increased the levels of Nrf2 in the cytosol reducing them in the nucleus. Superoxide dismutase expression increased in NB4 cells treated with esculetin in contrast with quercetin. All these data support a relevant differential role for NFkB and Nrf2 in anti-inflammatory and redox response when apoptosis was induced by esculetin

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or quercetin in human leukemia NB4 cells.

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ACCEPTED MANUSCRIPT 1. Introduction The levels of Reactive Oxygen (ROS) and Nitrogen species (RNS) in cells with high proliferative capacity can be used for the development of new therapeutic strategies using ROS as targets [1,2,3]. Cell signalling pathways respond accordingly to redox signals and correlate with the cell antioxidant response through the transcription factor

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Nrf2, that activates genes with antioxidant response element (ARE) [4]. Thus, it controls the expression of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase (GR) glutathione S-transferases (GSTs), NADPH:quinone oxidoreductase and hemo oxygenase-1 [4, 5]. Several studies have

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shown that activation of Nrf2 protects against the toxicity induced by oxidative damage (H2O2) [6-9] . Nrf2 supports cell survival, promoting NFkB p-50 translocation to the nucleus with activation of survival genes. Additionally, it controls cell death by caspases Activation of NFkB in the nucleus represents the final step of

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modulation [6-9].

activation of signalling cascades induced by oxidative stress. NFkB is a critical survival factor in lymphopoiesis [10,11]. In several hematologic diseases, constitutive activation of NFkB contributes to abnormal proliferation and survival of transformed cells [11] . NF-kB is sensitive to intracellular redox state and it is modulated by RNS and NOS [12].

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It has also been demonstrated that inflammation favours the development of cancer and metastasis. Inflammation represents the first immune response to an exogenous or endogenous damage. It includes release of inflammatory cytokines and mediators such as IL-1, IL-6, IL10, TNF-α, NFkB, NO, iNOS and COX) [13]. As a

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matter of fact, some CLL (chronic lymphoid leukemia) patients showed an increase of these factors in their sera what is related to leukemia progression. A great number of studies demonstrated the antiinflammatory properties of polyphenolic compounds as

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inflammatory mediators [14, 15]. Several antioxidants such as the flavonoid quercetin at low concentrations produce a benefitial effect though the activation of Nrf2/ARE, promoting the expression of antioxidant enzymes like superoxide dismutase and survival pathways inhibiting the expression of proapoptotic and proinflammatory genes regulated by NFkB [16, 17]. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is persistently activated in many human tumors including acute myeloid leukemia (AML). Therefore, inhibition of Nrf2 activity may be a promising target in leukemia therapy [18]. Acute myeloid leukemia (AML) cells possess high constitutive nuclear levels of Nrf2. Elevated Nrf2 was reduced by NF-κB inhibitors. NF-κB subunits p50 and p65 induce

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ACCEPTED MANUSCRIPT transcription of Nrf2 in AML cells and improved their chemotherapeutic responsiveness [10]. A constitutive activation of the nuclear factor-κB (NF-κB) transcription factor was detected in AML blasts and other hematopoietic cancers as well as in various solid tumors. Transcription factors of the NF-κB family are regulators of cell proliferation and

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survival and control expression of several genes relevant to the tumorigenic process. For instance, NF-κB promotes cell survival through expression of genes coding for antiapoptotic proteins (cellular inhibitor of apoptosis protein-1 [c-IAP1], c-IAP2, bfl-1, and bcl-xl). NF-κB is also known to stimulate cell proliferation via induction of growth

CSF]) or cell cycle regulators (cyclin D1, c-myc) [19].

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factors (interleukin-2 [IL-2], granulocyte-macrophage colony-stimulating factor [GM-

Furthermore, the antioxidant action of phenolic compounds can be applied

hepatoprotective effects [16,17,20].

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pharmacologically as antiinflammatory or antitumor drugs with neuro- and

It has been shown that quercetin shows inhibitory effects on the growth of malignant cells in leukemia, ovary, liver, bladder or colorectal cancer [21, 22]. Furthermore, quercetin produces apoptosis in leukemia P39 cells, with reduction of Bcl-

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2, Bcl-xL y Mcl-1, increase of Bax and release of cytochrome, activation of caspases with expression of FasL [23]. Additionally, in breast cancer cells treated with quercetin there was a significant decrease in the expression of Nrf2 mRNA and protein levels and the nuclear/cytosolic Nrf2 ratio was reduced in lung, colorectal and ovarian cancer cells

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[24]. The inductions of antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in PC-12 cells exposed to H2O2 were significantly reduced by pre-treatment with quercetin. In addition, quercetin pre-

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treatment significantly increased Bcl-2 expression, and reduced Bax, cleaved caspase-3 and p53 expressions [25]. Esculetin is present in many plants traditionally used as natural medicines,

showing multiple biological activities such as lipoxygenase and tyrosinase inhibitor [26,27], modulator of different enzymes such as a cyclooxygenase (COX), lipoxygenase (LOX) or inducible nitric oxide synthase (iNOS) related to cytoprotective properties [26, 28-30] and inducer of apoptosis in other cell lines such as adipocyte 3T3-L1 cells [31], human leukemia U937 cells [32,33] and HL-60 cells [34].

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ACCEPTED MANUSCRIPT Additionally, we have previously shown that esculetin (6,7-dihydroxycumarin) induced apoptosis in acute promyelocytic leukemia (APL) NB4 cells

producing

imbalance in superoxide and peroxide anion [35-37]. On the basis of this rationale, we have studied the modulation of the transcription factors NFkB and Nrf2 by esculetin and quercetin in relation to their antioxidant actions

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and the ROS imbalance that they induced in APL human NB4 cells.

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ACCEPTED MANUSCRIPT 2. Material and Methods

2.1. Reagents Esculetin (6,7- dihydroxycoumarin, 98% purity) and Quercetin were obtained from Sigma-Aldrich (Steinheim, Germany) and prepared as 196 mM and 100 mM stock

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solutions respectively in dimethyl sulfoxide (DMSO) and stored at -20 ºC. Fluorescent probes hydroethidine and 2',7'-dichlorodihydrofluorescein diacetate were obtained from Molecular Probes (Eugene, Oregon, USA). Primary antibodies to Nrf2, NF-kB p150, NF-kB p50, NF-kB p65, anti-α-tubulin and anti-nucleoporin p62 were purchased from

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Santa Cruz Biotechnology, p-Jnk and p-p38 were obtained from Cell Signaling. The IRDye680 conjugated goat anti-rabbit IgG and IRDye800CW Conjugated Goat Antimousse IgG secondary antibodies were purchased from Li-Cor (Nebraska, USA) for

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fluorometry analyses.

2.2. Cell culture

The human NB4 leukemia cell line was maintained in culture at a density of 3 x 105 cells/ml in RPMI medium (Gibco-Life Technologies) supplemented with 10% fetal

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bovine serum (FBS), 1% penicillin/streptomycin and 0,02 mg/ml gentamicin at 37 ºC in a humidified 5% CO2 atmosphere. 2.3. MTT assay method

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The cell proliferation was determined by colorimetric MTT assay kit (Roche). The cells were seeded in 96-well microplates and after treatments were incubated with 10 µl

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MTT Labeling Reagent for 4 h and then 100 µl Solubilization Solution was added. The absorbance was measured using an ELISA reader. The absorbance revealed directly correlates to the number of viable cells.

2.4. Cell viability studies Cell viability of NB4 cells treated with esculetin or quercetin was determined by measuring the level of impermeability to propidium iodide (PI) by flow cytometry. After treatments, the cells (2,5 x 105 cells) were washed with 500 µl phosphate buffered saline (PBS) and resuspended in 300 µl PBS, then 15 µl propidium iodide (Calbiochem) were added and the fluorescence of each well was measured using a Becton Dickinson FACScalibur flow Cytometer ( San José, CA, USA). 6

ACCEPTED MANUSCRIPT 2.5. Analysis of apoptosis by Annexin-V-FITC cytometry assay. The level of apoptosis in NB4 cells was quantified by the presence of phosphatidylserine on the outer side of the membrane, thus, apoptotic cells were determined by Annexin V-FITC Apoptosis Detection Kit (BioVision). After treatments with esculetin, the cells (2.5 x 105 cells) were centrifuged at 1200 rpm for 5 minutes and

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incubated with 500 µl 1X Annexin V Binding Buffer and 1 µl Anexina V-FITC for 5 min at room temperature in the dark. Then, 10 µl of PI was added and the cells apoptotic were measured by the fluorescence using a Becton Dickinson FACScalibur flow

2.6. Cell cycle study. Analysis of cell DNA content

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cytometer. The results were analized by WinMDI 2.8 software.

After treatments with esculetin, the cell pellets (2,5 x 105 cells) were washed with 500

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µl PBS then 300 µl PBS, 0,1 % NP-40 and 0,5 mg/ml Ribonuclease A (Sigma Chemical) were added, respectively, and immediately before measuring the fluorescence, 0.05 mg/ml PI was added. We used a Becton Dickinson FACScalibur flow cytometer and the results were analized by WinMDI 2.8 software.

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2.7. Measurement of intracellular ROS levels

Intracellular ROS levels were detected using fluorescent probes, H2DCFDA (2',7'dichlorodihydrofluorescein diacetate) and DHE (hydroethidine). H2DCFDA is a nonfluorescent molecule which accumulates intracellulary and reacts with reactive oxygen especially

hydrogen

peroxide,

becoming

green

fluorescent

2',7'-

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species,

dichlorofluorescein (DCF). After treatment, cells were incubated with 10 µM of

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H2DCFDA for 30 minutes at 37ºC, then cells were washed with PBS and fluorescence intensity was measured using flow cytometry FACScalibur. To measure intracellular superoxide, after treatment, cells were incubated with 2 µM DHE during the last 15 min the esculetin treatment. The fluorescence intensity was measured by flow cytometry.

2.8. Protein extraction and Western blot analyses For detection of p-JNK and p-p38 proteins, NB4 cells (3 x 106 cells/ml) were seeded and treated with esculetin (final concentration of 100 µM or 500 µM) and cells without treatment as negative control for 0.5, 3, 6 and 19 hours. After treatments, cells were

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ACCEPTED MANUSCRIPT harvested and centrifuged at 2000 rpm for 10 min at 4 ºC, then the precipitates were resuspended in 100 µl lysis buffer (50 mM Tris-HCl, pH 8,0, 5 mM EDTA, 150 mM NaCl, 0,5 % Nonidet P-40, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml bestastatin, 10 µg/ml aprotinin and 10 µg/ml antipain) for 30 min at 4ºC., then cells were sonicated for 20 s (duty cycle 100 %, output control 50%). After centrifugation for 5 min at 14000

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rpm at 4ºC, supernatants were collected and protein concentration was determined by the method of Bradford using the Bio-Rad protein assay and bovine serum albumin as a standard. An equal amount of total proteins (20-40 µg/ well) plus 5 % sample buffer (300 µM Tris-HCl, pH 6,8 , 50 % glycerol, 50 % sodium dodecylsulfate (SDS) and 10%

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bromophenol blue) were heated at 100 ºC for 5 min and electrophoresed in 10%-15% SDS-polyacrylamide gels with Precision Plus Protein Standards (Bio-Rad), then the proteins were transferred to nitrocellulose membranes (Pure Nitrocellulose Membrane;

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0,45 µm; Biorad). The membranes were blocked with 5 % (w/v) nonfat dry milk in TTBS buffer (50 mM Tris-HCl, pH 7,2, 140 mM NaCl, 0.06%-1% Tween 20 ) for 1 h and then incubated with primary antibodies for 1 h at room temperature or overnight at 4º C. We also used the following antibodies at the indicated dilutions: PhosphoSAPK/JNK (Thr183/Tyr185) (G9) Mouse mAb #9255 monoclonal antibody, 1:200;

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Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mAb #9216 Monoclonal Antibody, 1:200 were supplied by Cell Signaling. For detection of superoxide dismutase and lipoxygenase the cells were collected and centrifuged at 2000 rpm for 10 min at 4ºC. The supernatant was discarded and the pellet

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was suspended in 150 µl of lysis buffer (50 mM Tris pH=8.0; 5 mM EDTA; 150 mM NaCl 150 mM; 0.5% Nonidet P-40 and 1 mM PMSF). A mixture of protease inhibitor

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containing 1 µg/µl leupeptin, 1 µg/µl bestastatin, 10 µg/µl aprotinin, and 10 µg/µl antipain (All of them obtained from Sigma-Aldrich) was added and the cells were incubated for 30 min. at 4ºC. Afterwards, the samples were sonicated for 20 s. and centrifuged at 14000 rpm for 5 min. a 4ºC and the supernatants were collected. Anti-SOD-1 (72B1) sc-58421 mouse monoclonal IgG1 antibodies (1:2000) and Anti-5LOX (33) sc-136195 mouse monoclonal IgG1 antibodies 50µg/0.5ml (1: 500) were purchased from Santa Cruz Biotechnology. For detection of Nrf2 and NF-kB proteins in cytosol or nucleus, NB4 cells (1 x 107 cells/ml) were seeded and treated with esculetin (final concentration of 100 µM or 500 µM) or quercetin (final concentration of 25 µM) and cells without treatment as negative

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ACCEPTED MANUSCRIPT control for 0.5, 3, 6, 19 and 24 hours. After treatments, we used the Cellytic Nuclear Extraction Kit to extraction of nuclear and cytoplasmic proteins. Then nuclear and cytoplasmic proteins were collected, protein concentration was determined and the samples were used for Western blot as above. We used Nrf2 (H-300) sc-13032 rabbit polyclonal antibody at dilution 1:2500, NF-kB

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p105/p50 (E-10) sc-8414 mouse monoclonal and NF-kB p65 (F-6) sc-8008 mouse monoclonal antibodies at dilution 1:1000 from Santa Cruz Biotechnologies. Also, Antinucleoporin p62 (E-4) sc-48389 antibodies supplied by Santa Cruz Biotechnology were purchased from Santa Cruz Biotechnologies. Mouse monoclonal anti-α-tubulin T-8203

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anti-β-tubulin antibodies T4026 were supplied by Sigma-Aldrich. Goat-anti-mouse and goat-anti-rabbit IDDye 800cw antibodies (Bonsai Advanced Technologies; S.L.) at a dilution of 1:15000 were also used as secondary antibodies. IR light emission was

2.9. Determination of SOD activity.

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detected by LI-COR Odyssey method.

After treatments, cells were lysed by freezing-thawing cycles, then the protein concentration was determined by the method of Bradford and the enzyme activity was

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measured by a 19160 SOD determination kit (Sigma) following the manufacturer’s instructions. The SOD activity was expressed as SOD activity/µg protein.

3.0. Statistical analysis

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Data are expressed as the mean ± standard error of the mean from at least three independent experiments. The differences between the control and treatments groups were determined using the Student’s t-test and P<0.05 was considered statistically

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significant. The asterisk * indicates P<0.05, ** indicates P<0.01 and *** indicates P<0.001.

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ACCEPTED MANUSCRIPT 3. Results Previously, we have described the action of esculetin on human leukemia NB4 cells [35-37]. The flavonoid antioxidant quercetin, that possesses growth-suppressive effects in leukemic cells, has been used as a reference to study esculetin effects. It has been described that 25 µM quercetin caused a marked inhibition of K562 leukemia cells

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growth together with a mild cytotoxicity at 24 or 48 of incubation and induced apoptosis induction [38].

We have compared these effects with those produced by quercetin, an antioxidant compound, or esculetin on these leukemia cells (Figure 1). MTT analyses of NB4 cells

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treated with 25 µM quercetin for 24 or 48 hours indicated a significant reduction of metabolic activity (Figure 1). At 48 h. only a level of 10% of the metabolic activity

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remains. This was the reason to study the action of quercetin at a time of 24 h. where about 50% of the initial metabolic activity was observed (Figure 1). MTT analyses of NB4 cells treated with different esculetin concentrations (20, 50, 100, 250 or 500 µM) for 5, 14, 19, 24 and 48 hours (Figure 1). Treatments with 100 µM esculetin for 24 h. produced a 60% reduction of metabolic activity in NB4 treated cells (Figure 1). Cell viability of NB4 cells treated with quercetin was determined measuring the

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impermeability to PI by flow cytometry with a slight increase of dead cells after 24 h (Figure 2A). Also, apoptosis was produced by treatment with 25 µM quercetin for 24 hours (15% accounting for early and late apoptosis as measured by staining with FITCconjugated Annexin V and PI and flow cytometry) (Figure 2B). Cell cycle analyses of

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NB4 cells treated with 25 µM for 24 hours showed a high increase in the cells in G2/M phase (Figure 2C).

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Since both esculetin and quercetin are considered as antioxidant agents, we

studied changes in ROS in NB4 cells treated with either of these two compounds (Figure 3) using fluorescent probes (DHE and H2DCFDA) for measuring intracellular superoxide and peroxides levels in treated cells. Previous results showed that esculetin induces an increase in superoxide anion at long times of treatment and a reduction of peroxides at short times (1 h) with an increase at 2-4 h of treatment ]36]. Our results show that quercetin increased significantly superoxide levels mainly at 48 h (Figure 3A). Interestingly, a reduction of the peroxide levels was found at 24 h. of quercetin treatment (Figure 3B).

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ACCEPTED MANUSCRIPT Expression levels of Nrf2 protein related to oxidative stress in NB4 cells treated with esculetin o quercetin were studied (Figure 4). Cells (1 x 107 cells/ml) were incubated with 25 µM of quercetin for 24 hours or 100 µM of esculetin for 0.5, 3, 6 or 19 hours. Nrf2 expression in cytosol or in the nucleus was examined by Western blot. αtubulin or nucleoporin, respectively, were used as internal controls. The effects produced

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by treatments with esculetin are shown in Figure 4. A significant increase of Nrf2 in the nucleus at 19 hours was observed. 25 µM quercetin increases Nrf2 in the cytosol with a clear reduction in the nucleus (Figure 4).

The transcription factor NF-kB may be related with the antiinflammatory

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properties of esculetin. Figure 5 shows that treatment with 100 µM or 500 µM esculetin for different time periods induced a decrease in the levels of NF-kB p105 in the cytosol as time and the esculetin concentration increased. The levels of NF-kB p50 increased in

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the nucleus and became lower in the cytosol at longer times of treatment. The levels of NF-kB p65 slightly decreased in the cytosol and did also so significantly in the nucleus. In all cases, similar results were obtained independently of the esculetin concentration used 100 µM or 500 µM (Figure 5).

Treatment with 25 µM quercetin for 48 h highly decreased NB4 metabolic activity

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(Fig.1) therefore we only analyzed NF-kB intracellular location at 24 h incubation time. Levels of NFkB p50 in the cytosol and in the nucleus were similar (Figure 5). However, NFkB p65 levels decreased in the cytosol but do not significantly increase in the nucleus after treatment with quercetin (Figure 5).

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The response related to transcription factor NF-kB was studied as induction of LOX-5. Levels of LOX-5, an enzyme which generates ROS, were studied in presence of

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esculetin or quercetin. After treatment with either quercetin or esculetin, levels of LOX5 were significantly lower than in control untreated NB4 human leukemia cells (Figure 6). It correlates with the citosolic location of NF-Kb p65 subunit. The antioxidant response related to transcription factor Nrf2 was studied

analyzing induction of SOD. Treatment with quercetin for 24 h showed a significant decrease of SOD levels (Figure 6). Treatment with 25 µM quercetin for 48 h highly decreased NB4 metabolic activity (Fig.1) therefore we only analyzed Nrf2 intracellular location at an incubation time of 24 h. Figure 6B also shows that treatment with 100 µM esculetin for different time periods restored the expressed levels of SOD after 19 h treatment. NB4 cells SOD

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ACCEPTED MANUSCRIPT activity was determined by a colorimetric method using the Kit SOD Assay (Sigma). 100 µM esculetin increased SOD activity when applied for 19 h (Figure 6D). This correlated with the higher SOD expression (Figure 6B). This also agrees with the Nrf2 nuclear location at 19 h (Fig. 4B). We have studied SOD activity after esculetin treatment because of the increase in SOD levels produced by this antioxidant compound.

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These increased levels of SOD could be correlated with an increased activity of the enzyme (Figure 6).

The involvement of the phosphorylated forms of some intracellular kinases (pJnk and p-p38) on the induction of antioxidant response was studied. Figure 7 shows a

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progressive increase of the levels of both phosphorylated forms when NB4 cells were treated with 100 µM or 500 µM esculetin for 0.5, 3, 6 and 19 hours. This increase was mainly significant at long times of treatment for both concentrations of esculetin (100

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and 500 µM).

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ACCEPTED MANUSCRIPT 4. Discussion.

We have studied the biological effects of two antioxidant compounds on NB4 leukemia cells. We focused our studies on the relevance of changes in NFkB and Nrf2 as factors involved in inflammatory processes and proliferation.

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First, we checked the action of quercetin or esculetin on metabolic activity of NB4 cells. We used a quercetin concentration that has been extensively used in toxicity studies on malignant cells [21-23]. 25 µM quercetin produced a 50% reduction of metabolic activity at 24 h (Fig. 1A). In the case of esculetin, after 19 h. of treatment at

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concentrations of 100 µM or higher metabolic activity was reduced to 40% of that present in untreated cells (Fig. 1B) [35]. Thus, we fixed the conditions used in this study on the basis of these results.

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Since we have previously shown the apoptotic effects of esculetin and its action on changes in the cell cycle of NB4 cells [35], we analysed the toxicity of quercetin in this leukemia cell line (Fig. 2). The effects of quercetin in the conditions used are very similar to that shown by esculetin [35, 21-23] at 100 µM either with respect to apoptosis induction or cell cycle changes (Figures 2A, B and 2C). Quercetin as antioxidant alteres

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the cellular ROS levels [39] and when applied on NB4 leukemia cells (Fig. 3), it produces an increase of superoxides mainly after 48 h. with a significant reduction of peroxides at 24 h. Esculetin increased the levels of superoxides and also reduced the levels of peroxides at treatments for long time periods (19-24 h) [35-37].

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NFkB is a crucial survival factor in lymphopoiesis [10, 11]. In several malignant hematological processes, constitutive activation of NFkB contributes to an abnormal proliferation and survival of transformed cells [40, 17]. NF-kB is sensitive to

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intracellular redox state and is modulated by ROS and iNOS [41]. It has been described that many antioxidants such as quercetin at low doses can exert a benefitial effect through activation of Nrf2/ARE, promoting the expression of antioxidant enzymes such as superoxide dismutase and the activation of cell survival pathways inhibiting proapoptotic and proinflammatory genes regulated by NFkB [17,39]. Thus, the expression and translocation to the nucleus of these two factors after antioxidant treatment should be relevant. In Figure 4 we demonstrate that esculetin but not quercetin increase the Nrf2 presence in the nucleus of treated NB4 cells. On the contrary, treatments with quercetin has an effect antagonic increasing the presence of Nrf2 in the cytoplasm. 13

ACCEPTED MANUSCRIPT Quercetin and esculetin show a different control of NFkB factors localization in the cytoplasm and nucleus of NB4 cells (Figure 5). Esculetin increased the levels of NFkB p50 in the nucleus reducing significantly the presence of this factor in the cytoplasm (Figure 5B). Also, the antioxidant produced a significant reduction of the factor NFkB p65 in the nucleus (Figure 5B). An increase of

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p50 in the nucleus has been related with antiinflammatory properties whereas an increase of p65 in the related to pro-inflammatory processes. Thus, esculetin shows an antiinflammatory effect on NB4 cells.

Quercetin did not significantly change the levels of NFkB p50 in cytosol and

pro-inflammatory effects on NB4 cells.

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nucleus but increased NFkB p65 in nucleus reducing it in the cytosol. Thus, it shows

Quercetin shows significant inhibitory effects on the growth of malignant cells in

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leukaemia, ovary, bladder, liver, colorectal, etc. [21,22]. Quercetin produced apoptosis in P39 leukemia cells, with reduction of Bcl-2, Bcl-xL and Mcl-1, increase of Bax and mitochondrial translocation, releasing cytochrome c and activating caspases with expression of FasL showing antioxidant activity in myeloid leukemia P39 cells [23]. Quercetin also attenuated neuroinflammatory processes through control of oxidative

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stress by regulation of apoptosis through Nrf2 and NFkB [42]. Thus, leukaemia cells can be sensitive to alteration of redox equilibrium produced by antioxidants compounds what could be useful for a possible cytotoxic action as antitumor compounds [43-46]. We have also previously shown the antitumor effects of esculetin on acute

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promyelocytic leukemia (APL) cells [35]. Furthermore, we have correlated ROS imbalance produced by esculetin to apoptosis processes in NB4 cells [36-37]. It is plausible that Nrf2 and NFkB could be involved in these antitumor activities.

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Cu-Zn superoxide dismutase-1 (SOD-1) is a well characterized cytosolic

scavenger of oxygen free radicals that requires copper and zinc binding to potentiate its enzymatic activity. Enzymatically, SOD-1 facilitates the dismutation of oxygen radicals to hydrogen peroxide, and it also catalyzes prooxidant reactions, which include the peroxidase activity and hydroxyl radical generating activity. SOD-1 is ubiquitously expressed in somatic cells and functions as a homodimer. Defects in the gene encoding SOD-1 have been implicated in the progression of neurological diseases, including amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by the loss of spinal motor neurons, Down syndrome and Alzheimer’s disease. In familial ALS, several mutations in SOD-1 predominate, and they result in the loss of zinc binding and 14

ACCEPTED MANUSCRIPT the loss of scavenging activity of SOD-1 and correlate with an increase in neurotoxicity and motor neuron death [47]. Furthermore a correlation between SOD activity and GSH levels were significantly observed with the relapse of acute lymphoblastic leucemia (ALL) patients [48]. On the basis of this previous knowledge, we studied SOD-1 in NB4 leukemia

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cells treated with antioxidants. Quercetin reduced significantly SOD-1 levels in treatments for 24 h. Esculetin seemed to reduce SOD-1 levels at short times. At longer times (19 h.) the amount of SOD-1 was higher than that observed in control cells what correlated with high activity.

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5-lipoxygenase is expressed primarily in polymorphonuclear leukocytes, macrophages, and mast cells. 5-LO performs the first two catalytic reactions in the biosynthesis of leukotrienes, lipid metabolites that induce contractions of airway smooth

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muscle and increase vascular permeability during anaphylaxis. In activated blood polymorphonuclear leukocytes 5-LO undergoes calcium-dependent translocation from the cytosol to the nuclear envelope. This intracellular shuttling of 5-LO is dependent on the association with various signaling molecules, phosphorylation and the presence of a distinct nuclear localization signal, which is encoded at the amino terminus of 5-LO

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[49,50].

Both quercetin and esculetin induced reductions of 5-LO levels in NB4 leukemia cells as it can be seen in Figure 7 B. Quercetin at 25 µM produces an inhibition of 5-LO. Thus, it may be acting as an anti-inflammatory compound. Treatment with 100 µM

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esculetin for different times gave rise to inhibition 5-LO as an inflammatory enzyme. Esculetin produced an increase of transcription factor Nrf2 in the nucleus at 19 h of NB4 human leukemia cells what can be related with activation of cellular antioxidant

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response with increase of SOD. Esculetin increased the levels of NF-kB p50 in the nucleus with a possible formation of inhibitory homodimers p-50 and reduction of 5-LO levels which may correlate with anti-inflammatory properties of esculetin. On the contrary, quercetin decreased the levels of Nrf2 in the nucleus. These

changes match with reduction of SOD and 5-LO levels and suggest antiinflamatory properties for quercetin and a different antioxidant mechanism and response than esculetin in human leukaemia cells. It has been previously shown that quercetin induced an increase in p38 phosphorylation, but not JNK1,2 phosphorylation in the human leukemia cell line, MOLT-4

[51].

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ACCEPTED MANUSCRIPT the phosphorylation of members of the mitogen activated protein kinases including p38MAPK and JNK/SAPK in several leukemia cells (HL-60, U-937, MOLT-3, K-562, NALM-6, Raji) [52]. Esculetin produced an increase of the levels of the phosphorylated forms (p-Jnk and p-p38) of intracellular kinases in NB4 human leukemia cells as other cytotoxic and antitumor compounds [52].

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Some questions are opened to be solved in further investigations. Apoptosis process promoted by esculetin or quercetin could share some similarities in relation to the involved pathways.

The possibility of a direct interaction of Keap1 with Nrf2 related to esculetin

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antitumor activity could be tested in the future. It has been described, in three pancreatic tumor cell lines, that the action of esculetin is mediated by binding with Keap1

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promoting Nrf2 nuclear accumulation producing apoptosis by reducing NFkB [53].

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ACCEPTED MANUSCRIPT Conflict of interest The authors declare that there is no conflict of interest with the work entitled “Different roles of Nrf2 and NFKB in the antioxidant imbalance produced by esculetin or quercetin on NB4 leukemia cells” including any financial, personal or other relationships with other people or organizations within three years of beginning the

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submitted work that could inappropriately influence, or be perceived to influence, the

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work.

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ACCEPTED MANUSCRIPT Acknowledgements

This work was supported in part by Grants from F.I.S. PI060119, CCG10UAH/SAL-5966 and UAH2011/BIO-006. We also want to thank Isabel Trabado for her

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technical assistance in cytometric analyses (C.A.I. Medicina-Biología. Unidad de

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Cultivos. Universidad de Alcalá).

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Figure 1. Metabolic activity analyses of cells treated with quercetin or esculetin. NB4 cells (5 × 105 cells/ml) were incubated with either 25 µM quercetin or 20 - 500 µM esculetin for up to 48 hours, and MTT activity was measured by spectrophotometric

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analysis. Data are expressed as means ± SEM of 3 (quercetin) or 7 (esculetin) independent experiments.

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Figure 2. A) Cell viability of NB4 cells (5 x 105 cells/ml) treated with 25 µM quercetin for 24 hours. Cell viability was determined measuring the impermeability to PI by flow cytometry. The results show one representative experiment of three previous. B)

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Apoptosis analyses of NB4 cells (5 x 105 cells/ml) treated with 25 µM quercetin for 24 hours. Cells were stained with FITC-conjugated Annexin V-FITC and PI and measured by flow cytometry. The results show one representative experiment of three previous. C) Cell cycle analyses of NB4 cells (5 x 105 cells/ml) treated with 25 µM for 24 hours. DNA content was analized by a cytometry assay. The results show one representative

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experiment of three previous and the mean ± SEM of three independent experiments. Figure 3. ROS levels in NB4 cells (5 x 105 cells/ml) treated with 25 µM quercetin for 24 or 48 hours. ROS levels were detected by flow cytometric assays using a specific probe.

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A) Cells were incubated with 2 µM DHE during the last 15 min of esculetin treatment and intracellular superoxide levels were measured by flow cytometry. The results show one representative experiment of three previous experiments and mean ± SEM of three

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independent experiments. B) Cells were incubated with 10 µM of H2DCFDA for 30 minutes at 37ºC and intracellular peroxides levels were analized by flow cytometry. The results show one representative experiment of three previous experiments and the mean ± SEM of three independent experiments.

Figure 4. Expression levels of Nrf2 protein in NB4 cells treated with esculetin or quercetin. Cells (1 x 107 cells/ml) were incubated with 25 µM of quercetin for 24 hours or 100 µM of esculetin for 0.5, 3, 6 or 19 hours. A) The Nrf2 expression levels in cytosol or nucleus were examined by Western blot. α-tubulin or nucleoporin, respectively, were used as internal controls. Nucleoporin was undetectable in the 26

ACCEPTED MANUSCRIPT cytosolic fraction and α-tubulin was undetectable in the nuclear fraction. The results also represent the mean ± SEM of three independent experiments.

Figure 5. Expresion levels of NF-kB protein in NB4 cells treated with esculetin or quercetin. Cells (1 x 107 cells/ml) were incubated with 25 µM of quercetin for 24 hours

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or 100 µM and 500 µM of esculetin for 0.5, 3, 6 or 19 hours. The NF-kB p105, NF-kB p50 and NF-kB p65 expression levels in cytosol or nucleus were examined by Western blot. α- tubulin or nucleoporin proteins were used as internal controls. Nucleoporin was undetectable in the cytosolic fraction and α-tubulin was undetectable in the nuclear

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fraction. The results represent the mean ± SEM of three independent experiments.

Figure 6. Determination of superoxide dismutase and lipoxygenase. A) Expression of

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superoxide dismutase and lipoxygenase in cells treated with esculetin or quercetin. NB4 cells (1 × 107 cells/ml) were incubated with either 100 µM esculetin for up to 19 hours or 25 µM quercetin for 24 hours. The protein expression was examined by Western blot. β-tubulin was used as internal control. B) The results represent the mean ± SEM of three independent experiments. C) SOD activity after treatment with esculetin. NB4 cells (1 ×

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106 cells/ml) were treated with 100 µM esculetin for up to 19 hours and the SOD activity was determined by colorimetry using the SOD Assay Kit from Sigma.

Figure 7. Expression levels of intracellular proteins related to transduction signalling and

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oxidative stress in NB4 cells treated with esculetin. Cells (3 x 106 cells/ml) were incubated with 100 µM of esculetin for 0.5, 3, 6 or 19 hours or 500 µM of esculetin for

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19 hours and the p-Jnk and p-p38 expression levels proteins were examined by Western blot. In all experiments, α-tubulin was used as internal control. The results also represent the mean ± SEM of three independent experiments.

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Esculetin or quercetin differently affected NFkB levels in NB4 leukemia cells. Nrf2 levels in nucleus and cytosol were also regulated by esculetin or quercetin. Lipoxygenase expression was reduced either by esculetin or quercetin.

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Superoxide dismutase was differently affected by esculetin or quercetin.

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In NB4 cells, a distinct role for NFkB and Nrf2 in redox response can be stated.

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