Human acute promyelocytic leukemia NB4 cells are sensitive to esculetin through induction of an apoptotic mechanism

Chemico-Biological Interactions 220 (2014) 129–139

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Human acute promyelocytic leukemia NB4 cells are sensitive to esculetin through induction of an apoptotic mechanism Virginia Rubio, Eva Calviño, Ana García-Pérez, Angel Herráez, José C. Diez ⇑ Unidad de Bioquímica y Biología Molecular, Departamento de Biología de Sistemas, Facultad de Medicina y Ciencias de la Salud, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares (Madrid), Spain

a r t i c l e

i n f o

Article history: Received 6 September 2013 Received in revised form 8 May 2014 Accepted 19 June 2014 Available online 1 July 2014 Keywords: Apoptosis Caspases Intracellular kinases Leukemia Peroxides Superoxides

a b s t r a c t Acute promyelocytic leukemia (APL) is a type of cancer, in which immature cells called promyelocytes proliferate abnormally. Human NB4 cell line appears to be a suitable in vitro model to express the characteristics of APL. In this work, we have investigated the effects of esculetin, a coumarin derivative with antioxidant properties, on the viability, the induction of apoptosis and the expression of apoptotic factors in NB4 cells. Cells treated with esculetin at several concentrations (20–500 lM) and for different times (5–24 h) showed a concentration- and time-dependent viability decrease with increased subdiploid DNA production. Esculetin inhibited cell cycle progression and induced DNA fragmentation. Moreover, annexin-V-FITC cytometry assays suggested that increased toxicity is due to both early and late apoptosis. This apoptosis process is be mediated by activation of caspase-3 and caspase-9. Treatments with progressively increasing concentrations (from 100 lM to 500 lM) of esculetin produced a reduction of Bcl2/Bax ratio in NB4 cells at 19 h, without affecting p53 levels. Proapoptotic action of esculetin involves the ERK MAP kinase cascade since increased levels of phosphorylated ERK were observed after those treatments. Increments in the levels of phosphorylated-Akt were also observed. Additionally, esculetin induced the loss of mitochondrial membrane potential with a release of cytochrome c into the cytosol which starts at 6 h of treatment with esculetin and increases up to 24 h. Esculetin induced an increase in superoxide anion at long times of treatment and a reduction of peroxides at short times (1 h) with an observed increase at 2–4 h of treatment. No significant changes in NO production was observed. Esculetin reduced the GSH levels in a time-dependent manner. In summary, the present work shows the cytotoxic action of esculetin as an efficient tool to study apoptosis mechanism induction on NB4 cell line used as a relevant model of APL disease. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Acute promyelocytic leukemia (APL) is a malignant disorder of the white blood cells. In this disease, a rapid growth of immature cells (promyelocytes) is observed with their accumulation in peripheral blood and bone marrow. APL is considered as a subtype of acute myeloid leukemia (AML) characterized by chromosomal translocation t (15; 17) [1] that occurs between the promyelocytic leukemia (PML) gene and the retinoic acid receptor-a (RARa) gene, resulting in the generation of a PML–RARa fusion protein [2]. NB4 cell line appears to be the most suitable in vitro model to express the characteristics of APL. This cell line shows the unique property of carrying this chromosomal translocation, expressing different levels of some enzymatic markers and of different differentiation ⇑ Corresponding author. Tel.: +34 91 8854582/8854579; fax: +34 91 8854585. E-mail address: [email protected] (J.C. Diez). http://dx.doi.org/10.1016/j.cbi.2014.06.021 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

behavior as compared to other APL-derived cell lines, such as HL-60 [3,4]. NB4 cell line is known to show resistance to several chemotherapeutic drugs [5] such as all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) [6–8]. The efficiency of the antitumor action depends on the cell death induction to eliminate malignant cells. To this respect, we have recently shown the induction of apoptosis in NB4 cells by novel reagents such as dequalinium [9,10], active compounds present in Ganoderma lucidum [11,12] and antimitotic compounds such as paclitaxel or vinblastine that induce cytotoxicity in the NB4 cell line by regulation of signal transduction kinases and apoptotic factors [13]. Esculetin (6,7-dihydroxycoumarin) is a coumarin, produced by many plants traditionally used as natural medicines. Coumarins have a diversity of biological and pharmacological activities, such as antioxidative, anti-inflammatory and antiviral effects [14]. Esculetin inhibits lipoxygenase and tyrosinase [15,16] and displays cytoprotective properties against oxidative stress-induced cell

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damage [17–19]. It shows anti-inflammatory activities [20] and induces apoptosis in some cell lines such as adipocyte 3T3-L1 cells [21], human leukemia U937 cells [22,23], HL-60 cells [24] and in human oral cancer SAS cells [25]. Some intracellular kinases are key proteins in the regulation of cellular functions. MAPKs and PI3K/Akt are both activated in response to various stimuli leading to activation of genes involved in cell progression, differentiation, proliferation and apoptosis. At least three MAPK families have been characterized: ERK, JNK and p38MAPK. ERK is directly downstream of the ERK pathway and some reports indicate that ERK is constitutively activated in several human leukemias [26] while JNK activation is associated with induction of apoptosis [27]. Apoptosis may proceed through either the intrinsic (or mitochondrial) pathway or the extrinsic (death receptor) pathway. Stimulation of these pathways leads to the activation of caspases which begins the process of apoptosis [28,29]. Activation of the intrinsic pathway can produce mitochondrial damage with release of cytochrome c. The intrinsic pathway is regulated through proteins of the Bcl-2 family: the subfamily of antiapoptotic proteins like Bcl-2 that inhibit apoptosis and the subfamily of proapoptotic proteins like Bax that promotes apoptosis. Some of them can be activated by tumor suppressor protein (p53) when there is DNA damage [30,31]. The role of these signaling proteins may be particularly relevant in order to know the mechanism by which esculetin could eliminate leukemia human cells. From different studies, it appears that the involvement of such proteins is variable and depends on the cellular model. Esculetin-induced apoptosis may proceed, without alteration in the expression of Bcl2 [25] or may require down-regulation of Bcl2 [24]. Similarly, an increase in the levels of Bax [32] may be needed in esculetin induced apoptosis. In contrast, a translocation of this protein from the cytosol to mitochondria could be observed in some cellular models [22]. Thus, the mechanism of induction of apoptosis by esculetin is dependent on the cell type and lineage. Differences may even exist among different lineages of the same leukemia disease model. The activation of ERK in esculetin-induced apoptosis seems also controversial [22,23,32]. Some authors claim the inhibition of ERK for induction of apoptosis by esculetin in hepatoma HepG2 cells [32], while ERK activation seems to be relevant in U937 leukemia cells [22,23]. The use of new unstudied cell lines or the discovery of new relationships between pathways may explain some differences on the activation in this kinase [32]. Esculetin could produce changes in ROS species and imbalance the redox equilibrium producing changes in the levels of reduced glutathione what might play a role in the cytotoxic effect on leukemia cells. Esculetin could act as a scavenger compound changing the redox balance in NB4 leukemia cell line [10]. Thus, the study of superoxide anion and peroxide levels could be relevant to know their role in the apoptosis induced by esculetin in this leukemia cell line that shows a high level of oxidative stress [10]. Additionally, intracellular GSH depletion is an early hallmark in the progression of cell death in response to different apoptotic stimuli including cytotoxic chemotherapeutic agents inducing oxidative stress. Since leukemia cells, as other cancer cells, are under an increased oxidative stress, ROS overproduction has been proposed as a useful mechanism for killing these malignant cells [33]. The effect of esculetin in NB4 leukemia cell model has not yet been reported, so the purpose of our study was to investigate the effect of esculetin on cell viability, induction of apoptosis and expression of apoptotic factors as well as the relationship between cell death and intracellular kinases in this cell model. Furthermore, we aimed to know if redox imbalance in NB4 leukemia cells could be relevant in the cytotoxicity of esculetin in these cells. We also intended to elucidate the roles of superoxide anion, of peroxides

and reduced glutathione in this process. Further investigation can establish whether the changes in ROS levels are the result of the induced apoptosis process or they are important effector of the observed cell death. 2. Material and methods 2.1. Reagents and antibodies Esculetin (6,7-dihydroxycoumarin, 98% purity) was obtained from Sigma–Aldrich (Steinheim, Germany) and prepared as 196 mM stock solution in dimethyl sulfoxide (DMSO) and stored at 20 °C. BSO (DL-Buthionine-[S,R]-Sulfoximine) was purchased from Sigma–Aldrich (Steinheim, Germany) and prepared as 1M stock solution in distilled water at the time of use. Primary antibodies to Bcl-2, Bax, ERK, P-ERK1/2, Akt, P-Akt, Caspase-3, Caspase-9 and anti-a-tubulin were purchased from Santa Cruz Biotechnology, p53 from Calbiochem (Darmstadt, Germany) and anti-cytochrome c was purchased from BD Biosciences. The HRPconjugated secondary antibodies from Promega (Madison, USA) were detected using an ECL detection system. For fluorometry analyses, IRDye680 conjugated goat anti-rabbit IgG and IRDye800CW conjugated goat anti-mousse IgG secondary antibodies were purchased from Li-cor (Nebraska, USA). Rhodamine 123 probe was purchased from Sigma Chemical Co. (St. Louis, MO); fluorescent probes hydroethidine, 20 ,70 -dichlorodihydrofluorescein diacetate and monochlorobimane (mBCl) were obtained from Molecular Probes (Eugene, Oregon, USA). 2.2. Cell culture The human NB4 leukemia cell line was maintained in culture at a density of 3  105 cells/ml in RPMI medium (Gibco-Life Technologies) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 0.02 mg/ml gentamicin at 37 °C in a humidified 5% CO2 atmosphere. 2.3. Treatments for flow cytometric analysis Kinetic assay: NB4 cells (5  105 cells/ml) were seeded in 12-well plates and treated with esculetin (100 lM and 250 lM) for different times (5, 14, 19 and 24 h). Cells without treatment were used as negative control and cells treated with 5 ll DMSO as control for the action of this solvent. Concentration assay: NB4 cells (5  105 cells/ml) were treated with esculetin at various concentrations (20, 50, 100, 250 and 500 lM) for 19 h. We used the same controls as in the kinetics assay. 2.4. Cell viability study Cell viability of NB4 cells treated with esculetin was determined by measuring the level of impermeability to propidium iodide (PI) by flow cytometry. After treatments, the cells (2.5  105 cells) were washed with 500 ll phosphate buffered saline (PBS) and resuspended in 300 ll PBS, then 15 ll propidium iodide (Calbiochem) were added and the fluorescence of each well was measured using a Becton Dickinson FACScalibur flow Cytometer (San José, CA, USA). 2.5. Cell cycle study: analysis of the DNA content After treatments with esculetin, cells pellets (2.5  105 cells) were washed with 500 ll PBS and then 300 ll PBS, with 0.1% NP-40 and 0.5 mg/ml ribonuclease A (Sigma Chemical) were added and 0.05 mg/ml PI was added immediately before measuring the

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fluorescence. We used a Becton Dickinson FACScalibur flow cytometer and the results were analyzed using WinMDI 2.8 software. 2.6. 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 using the Apoptosis Detection Kit (Calbiochem). After treatments with esculetin, the cells (2.5  105 cells) were centrifuged at 1200 rpm for 5 min and incubated with 300 ll PBS, 10 ll Media Binding Reagent and 1 ll annexin V-FITC for 15 min at room temperature in the dark. Then, the mixture was centrifuged at 1500 rpm for 5 min and the pellets were resuspended in 500 ll binding buffer 1 diluted in PBS. 10 ll of PI were added and the apoptotic cells were counted by fluorescence using a Becton Dickinson FACScalibur flow cytometer. The results were analyzed using WinMDI 2.8 software. 2.7. Treatments for Western blot analysis 3  106 NB4 cells were seeded at 5  105 cells/ml and treated with esculetin (between 100 lM and 500 lM) for 14 h and 19 h. 2.8. Protein extraction and Western blot analyses After treatments, cells (5  105 cells/ml) were harvested and centrifuged at 2000 rpm for 10 min at 4 °C, then the pellets were resuspended in 100 ll lysis buffer (50 mM Tris–HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM PMSF, 1 lg/ml leupeptin, 1 lg/ml bestatin, 10 lg/ml aprotinin and 10 lg/ml antipain) for 30 min at 4 °C., then cells were sonicated for 20 s (duty cycle 100%, output control 50%). After centrifugation for 5 min at 14,000 rpm at 4 °C supernatants were collected and protein concentration was determined by the Bradford method using the Bio-Rad protein assay and bovine serum albumin as a standard. An equal amount of total proteins (20–40 lg/well) plus 5% sample buffer (300 lM Tris–HCl, pH 6.8, 50% glycerol, 50% sodium dodecylsulfate (SDS) and 10% bromophenol blue) were heated at 100 °C for 5 min and electrophoresed in 10% or 15% polyacrylamide gels with SDS, with protein standard (Precision Plus from Bio-Rad), then the proteins were transferred to nitrocellulose membranes (Pure Nitrocellulose Membrane; 0,45 lm; 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 4 °C. We used the following antibodies at the indicated dilutions: Anti-p53 mouse monoclonal IgG from Calbiochem (Ab-1, OP03), 1:50; anti-Bcl-2 mouse monoclonal IgG1 (C-2; sc7382), 1:200; anti-Bax mouse monoclonal IgG2b (B-9; sc-7480), 1:100; anti ERK (sc-154), 1:3000; anti p-ERK1/2 (Tyr204, sc-7383), 1:200; anti-Akt1/2/3 (H-136, sc-8312), 1:400; antipAkt1/2/3 (Ser437, sc-7985-R), 1:400; anti-caspase-3 (H-277, sc-7148), 1:200 and anti-caspase-9 (9CSP03, sc-73548) from Santa Cruz Biotechnology. Subsequently, membranes were washed four times for 10 min each in TTBS buffer and then incubated with secondary antibodies (1:10,000 dilution). After washing for 10 min in TTBS, the chemiluminescence was visualized using ECL Western blotting kit (Amersham) or the fluorescence by infrared detection (Li-cor Odyssey). The blots were washed with Re-Blot PlusMild Solution (Millipore, California, USA) at room temperature for 30 min, blocked and reprobed with anti human a-tubulin mouse monoclonal antibodies purchased from Santa Cruz Biotechnology and diluted at 1:10,000. For detection of cytosolic cytochrome c, NB4 cells (15  106 cells) were seeded and treated with esculetin (final concentration

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of 100 lM) for 3, 6, 19 and 24 h and cells without treatment were used as a negative control. After treatments, cells were harvested and washed with PBS, then resuspended in 75 ll lysis buffer (250 mM sucrose, 1 mM EDTA, 0.05% digitonin, 25 mM Tris–HCl pH 6.8, 1 lg/ml leupeptin, 1 lg/ml aprotinin, 0.1 mM PMSF) for 30 s at 4 °C. After centrifugation for 2 min at 15,000g at 4 °C, the supernatants were collected and protein concentration was determined as above. We used anti-cytochrome c mouse monoclonal IgG at a dilution 1:1000. The intensities of the bands were always corrected with respect to the intensity of a constitutively expressed protein (a-tubulin) in the same blot, and then quantified relative to control untreated cells. All the experiments were repeated at least three times. For graphical representation of the statistical treatment of the results, control (in black in the graphs of Fig. 5) corresponds to the intensity of the studied band in any of the cases with respect to the intensity of a-tubulin band at the beginning of the treatment (time: 0 h). This ratio was considered in each case as 100. The relationship of the intensity of each band corrected with respect to a-tubulin band intensity at a defined time was compared to the ratio obtained at time zero. 2.9. Determination of mitochondrial membrane potential (wm) Mitochondrial membrane potential was assessed using the fluorescent probe rhodamine 123 (Sigma Chemical Co., St. Louis, MO), which accumulates in mitochondria and its fluorescence correlates with membrane potential. NB4 cells (5  105 cells/ml) were treated with 100 lM esculetin for different times (0.5, 1, 2, 3, 4, 6, 14, 19 and 24 h). During the last 15 min of esculetin treatment the cells received 1 lg/ml of rhodamine 123 at 37 °C, then the cells were washed twice with PBS and their fluorescence was measured using a Becton Dickinson FACScalibur flow Cytometer (San José, CA, USA). 2.10. Measurement of intracellular ROS levels Intracellular ROS levels were detected using fluorescent probes, H2DCFDA (20 ,70 -dichlorodihydrofluorescein diacetate) and DHE (hydroethidine). H2DCFDA is a non-fluorescent molecule which accumulates intracellularly and reacts with reactive oxygen species, especially hydrogen peroxide, becoming green fluorescent 20 ,70 -dichlorofluorescein (DCF). NB4 cells (5  105 cells/ml) were treated with 100 lM esculetin for different times (0.5, 1, 2, 3, 4, 6, 14, 19 and 24 h). After treatment, cells were incubated with 10 lM H2DCFDA for 30 min at 37 °C, then cells were washed with PBS and the fluorescence intensity was measured using flow cytometer FACScalibur. To measure intracellular superoxide, we treated NB4 cells (5  105 cells/ml) as above and then, cells were incubated with 2 lM DHE during the last 15 min of the esculetin treatment. The fluorescence intensity was determined by flow cytometry. 2.11. Determination of NO production To determine the presence or absence of NO we used the Griess method that determines the production of NO by the accumulation of nitrites in the culture medium. Thus, NB4 cells (5  105 cell/ml) were treated with 20, 50, 100, 250 and 500 lM esculetin for 15, 30 min, 1, 2 and 19 h. After treatment, 400 ll of culture supernatant was removed and combined with 800 ll of Griess reagent containing 0.15% N-naphthyl ethylenediamine dihydrochloride and 1.5% sulfanilamide at room temperature for 10 min and the absorbance was measured using a spectrophotometer Spectronic Genesis5 at 540 nm. The nitrite concentrations in the supernatants

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were determined by comparison with a sodium nitrite standard curve (range 0–100 lM). 2.12. Measurement of intracellular GSH levels We used monochlorobimane fluorometric method to analyze of intracellular glutathione content. Monochlorobimane (mBCL) forms a fluorescent adduct with GSH in a reaction catalyzed by glutathione S-transferase. NB4 cells (3  106 cells/ml) were treated with BSO for 24 h or/and 100 lM of esculetin for different times (0.5, 1, 2, 3, 4, 6, 14, 19 and 24 h). After treatments, the cells were washed twice with phosphate buffered saline (PBS) and resuspended in 500 ll PBS containing 2 mM monochlorobimane, after incubated for 20 min at 37° in the dark, the fluorescence was measured at an excitation wavelength of 390 nm and emission wavelength of 490 nm using a Perkin Elmer LS 50 B Fluorimeter. 2.13. Statistical analysis Data are expressed as the mean ± standard error of the mean from at least three independent experiments. The differences between the control group and treatments groups were determined using the Student’s t-test and p < 0.05 was considered statistically significant. The  indicates p < 0.05,  indicates p < 0.01 and  indicates p < 0.001. The results in Figs. 1–6 were treated by Student’s t analysis. In the results of Fig. 7, significance of differences between treated groups was determined by one-way analysis of variance (ANOVA) followed by the Bonferroni post-hoc test. ,  and  represent significant differences of p < 0.05, p < 0.01 and p < 0.001, respectively, in the comparison between treated cells and controls. #, ## and ### or +, ++ represent significant differences of p < 0.05, p < 0.01 and p < 0.001, respectively in the comparison between cells treated with different compounds. 3. Results 3.1. Esculetin causes a decrease in cell viability To determine the effect produced by esculetin on NB4 cell viability, we incubated cells with 100 and 250 lM esculetin for 5, 14, 19 and 24 h. Fig. 1A shows that, at either of these concentrations, esculetin reduced viability at all concentrations tested. Decrease in the number of living cells was observed in the first for 5–14 h to decrease (accounting for 75% and 65% of the initial cell viability, respectively) after 19 and 24 h treatment with different concentrations of esculetin. Since treatments with esculetin for 19 h induced significant reduction in cell viability, we studied the dependence on the concentration of esculetin (20, 50, 100, 250 and 500 lM). Fig. 1B and C show that esculetin inhibited the viability in a concentration-dependent manner. 3.2. Induction of apoptosis by esculetin We analyzed cellular DNA content by flow cytometry to know whether esculetin induced DNA fragmentation. Fig. 2A shows that the number of cells with sub-G1 DNA content increased in a timedependent manner after treatment with 100 or 250 lM esculetin. Sub-G1-DNA content also increased when human leukemia cells were treated with esculetin at different concentrations (20, 50, 100, 250 and 500 lM) for 19 h (Fig. 2B). As shown in Fig. 2B, 20 and 50 lM esculetin produced levels of DNA subdiploid similar to that seen in control cells whereas higher concentrations (100, 250 and 500 lM) of esculetin induced a gradual increase of DNA fragmentation.

Fig. 1. Cell viability of NB4 cells treated with esculetin. (A) Cells (5  105 cells/mL) were treated with esculetin at a concentration of 100 and 250 lM for 5, 14, 19 and 24 h. Cell viability was determined measuring the impermeability to PI by flow cytometry. Data are expressed as means ± SEM of seven independent experiments. (B) Cells (5  105 cells/ml) were treated with 20, 50, 100, 250 and 500 lM of esculetin for 19 h. Cell viability was determined measuring the impermeability to PI by flow cytometry. Data are expressed as means ± SEM of five independent experiments. (C) The results shown one representative experiment of five previous experiments. The differences between the control group and treatments groups were determined using the Student’s t-test and p < 0.05 was considered statistically significant. The  indicates p < 0.05,  indicates p < 0.01 and  indicates p < 0.001. The results in Figs. 1–6 were treated by Student’s t analysis.

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The analysis of DNA content also provides information on cell cycle progression. As shown in Fig. 2C, cells in S and G2/M phases decreased after treatment with 100, 250 and 500 lM esculetin for 19 h while cells in sub-G1 increased. Treatments for 5, 14 and 24 h were also carried out in order to analyze cell cycle progression (data not included) showing reduction in the percentage of cells in S and G2/M phases with an increase in sub-G1. To further confirm the apoptotic induction by esculetin, we used annexin-V-FITC cytometry assay. Data shown in Figs. 3 and 4 indicate that treatments with esculetin for different times (5, 14, 19 and 24 h) and at different concentrations (20 and 250 lM) lead to an increase in early and late apoptosis in a time- and concentration-dependent manner. According to these results, treatment with 100 lM esculetin for 14 and 19 h, which causes 20–30% apoptosis was selected for analyses of caspase 3 and caspase 9 protein expression. 3.3. Activation of caspases We evaluated whether the apoptotic cell death was induced by activation of caspase-3 and caspase-9. As shown in Fig. 5A, Western Blot analyses revealed that treatment even with 100 lM of esculetin for 14 or 19 h increased cleaved caspase-3 bands (tenfold increase in caspase-3 bands intensity was observed at 19 h) which in turn induced significantly caspase-9 cleavage (a twofold increase in caspase-9 band at 19 h). A concomitant decrease of procaspase-9 levels compared with the untreated control cells was also seen. These results seem to indicate that esculetin induced apoptosis occurs through activation of caspase 3 and caspase 9. 3.4. Effect of esculetin on pro- and anti- apoptotic proteins (p53, Bcl-2 and Bax) We analyzed the levels of p53 protein, which is activated by damage of DNA resulting in triggering cell death process (Fig. 5B). We used progressive (increasing) concentrations of esculetin from 100 and 500 lM applied to the cell culture for 19 h. We observed no significant changes in p53 levels even at the highest esculetin concentration used (500 lM). Western blot analyses showed changes in Bcl-2 and Bax levels after treatment with concentrations of esculetin from 100 to 500 lM for 19 h (Fig. 5B). The Bcl2/Bax ratio decreased (0.91, 0.80 and 0.66 as the esculetin concentration used increased from 100 to 250 and 500 lM). This is a rationally understandable result that can be correlated with the observed increased apoptosis obtained at high concentrations of esculetin. 3.5. Effect of esculetin on signaling pathways

Fig. 2. Cell cycle analyses of NB4 cells treated with esculetin. (A) Cell cycle analyses of NB4 cells treated with esculetin for different times. Cells (5  105 cells/ml) were exposed to 100 and 250 lM of esculetin for 5, 14, 19 and 24 h. DNA content was analyzed by cytometry assay. The results represent the mean ± SEM of seven independent experiments. (B) Effect of different concentrations of esculetin on the sub-G1 DNA content of NB4 cells. Cells (5  105 cells/ml) were exposed to 20, 50, 100, 250 and 500 lM of esculetin for 19 h. DNA content was analyzed by cytometry assay. Data are expressed as mean ± SEM of five independent experiments. (C) The results shown one representative experiment of five previous experiments. The differences between the control group and treatments groups were determined using the Student’s t-test and p < 0.05 was considered statistically significant. The  indicates p < 0.05,  indicates p < 0.01 and  indicates p < 0.001. The results in Figs. 1–6 were treated by Student’s t analysis.

In order to examine the involvement of MAPKs (ERK pathway) and Akt in apoptosis induced by esculetin, we measured the levels of ERK, p-ERK, Akt and p-Akt in NB4 cells treated with different esculetin concentrations for 19 h (Fig. 5C and D). As shown in Fig. 5C, the levels of ERK increased after esculetin treatment. In NB4 cells treated with 500 lM the proportion of ERK reached 2.5-fold the amount of ERK present in control cell. Activation of ERK phosphorylation was observed (Fig. 5C). p-ERK levels increased 10-folds after treatment with esculetin 500 lM for 19 h in relation to untreated cells. The levels of p-ERK increased significantly in relation to ERK. Thus, an increase in p-ERK/ERK ratio was observed as a result of the increase of esculetin concentration. We obtained a p-ERK/ERK ratio increase of 7.3-fold. We found that levels of Akt increased slightly after treatments with different esculetin concentrations in comparison to untreated cells. Phosphorylated-Akt levels increased with increasing esculetin concentration (Fig. 5D). This increase in p-Akt was significantly

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Fig. 3. Apoptosis and necrosis analyses of NB4 cells treated with esculetin. Cells (5  105 cells/ml) were treated with 20, 50, 100, 250 and 500 lM of esculetin for 19 h. Cells were stained with FITC-conjugated annexin and PI and measuring by flow cytometry. The results show one representative experiment of five previous experiments.

Fig. 4. Apoptosis analyses of NB4 cells treated with esculetin for different times. Cells (5  105 cells/ml) were treated with esculetin at a concentration of 100 and 250 lM for 5, 14, 19 and 24 h. Cells were stained with FITC-conjugated annexin V-FITC and PI and measuring by flow cytometry. Data are expressed as means ± SEM of seven independent experiments.

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Fig. 5. Expression levels of intracellular proteins related to apoptosis and transduction signaling in NB4 cells treated with esculetin. (A) Cells (5  105 cells/ml) were incubated with 100 lM of esculetin for 14 and 19 h, the caspase-3 and caspase-9 expression levels were examined. Cells (5  105 cells/ml) were incubated with 0, 100, 250 and 500 lM of esculetin for 19 h and the protein expression levels were examined by Western blot. (B) Bcl-2, Bax and p-53 expression. (C) ERK and P-ERK expression. (D) Akt and P-Akt expression. In D the arrows show the positions of the phosphorylated forms for Akt that were quantitated in the graph. In all experiments, a-tubulin was used as internal control for relative densitometric quantitation of changes in expression levels of the studied proteins. The differences between the control group and treatments groups were determined using the Student’s t-test and p < 0.05 was considered statistically significant. The  indicates p < 0.05,  indicates p < 0.01 and  indicates p < 0.001.

higher than found for Akt. The proportion of the phosphorylated forms of Akt after treatment at 500 lM was 4 times higher than in untreated cells. Increasing esculetin concentration produced increments in the p-Akt/Akt ratio.

that treatments with 100 lM of esculetin for different times (0.5– 24 h) lead to loss of mitochondrial membrane potential in NB4 cells from 3 to 24 h preserving, 70% cells with intact mitochondrial potential at 24 h.

3.6. Esculetin induces the loss of mitochondrial membrane potential

3.7. Esculetin induces the release of cytochrome c into the cytosol

We analyzed mitochondrial membrane potential (Dwm) by flow cytometry using Rhodamine 123 to know whether esculetin induced mitochondrial dysfunction. Data shown in Fig. 6A indicate

A reduction in mitochondrial membrane potential was clearly detected 3 h after exposure of cells to esculetin. According to these results, to determine whether the loss of Dwm by esculetin could

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Fig. 6. Effects of esculetin on mitochondrial membrane and ROS levels. (A) Effects of 100 lM of esculetin for different times (0.5–24 h) on mitochondrial membrane potential. During the last 15 min of treatment the cells were incubated with 1 lg/ml of rhodamine 123 at 37 °C and mitochondrial membrane potential was measured by flow cytometry. (B) Analysis of mitochondrial cytochrome c release. Cells (5  105 cells/ml) were incubated with 100 lM of esculetin for 3, 6, 19 and 24 h and the expression levels of mitochondrial cytochrome c were examined by Western blot. a-Tubulin was used as internal control for relative densitometric quantitation of changes in expression levels. Statistical analyses correspond to the mean value of three different experiments. NB4 cells (5  105 cells/ml) were treated with 100 lM of esculetin for different times (0.5– 24 h) and intracellular ROS levels was detected by flow cytometric assay using a specific probe. (C) Cells were incubated with 2 lM DHE during the last 15 min the esculetin treatment and intracellular superoxide levels were measured. (D) Cells were incubated with 10 lM of H2DCFDA for 30 min at 37 °C and intracellular peroxides levels were analyzed. Statistical analyses correspond to the mean value of six different experiments. The differences between the control group and treatments groups were determined using the Student’s t-test and p < 0.05 was considered statistically significant. The  indicates p < 0.05,  indicates p < 0.01 and  indicates p < 0.001.

lead to the release of cytochrome c from mitochondria to cytosol, we treated NB4 cells with 100 lM esculetin for 3, 6, 19 and 24 h and determined cytochrome c protein in the cytosol by Western blotting. As shown in Fig. 6B, the levels of cytosolic cytochrome c were similar to control (untreated) cells in cells treated for 3 h and increased progressively after esculetin treatment for 6, 19 and 24 h (400%, 500% and 800% over the control, respectively). Thus, the release of cytochrome c into the cytosol starts at 6 h of treatment with esculetin and increasing up to 24 h.

and a later increase from 15 to 19 h of treatment with esculetin (23% over the control). This increase was maintained after 24 h treatment. In contrast, esculetin at either of these times of treatment reduced the levels of intracellular peroxides from 0.5 h (45%) to 24 h (20%) in comparison to control (untreated) cells (Fig. 6). Additionally, we measured NO production to assess the involvement of NOS in esculetin-induced apoptosis on NB4 cells. Our results showed that 20, 50, 100, 250 and 500 mM esculetin treatments for 15, 30 min, 1, 2 and 19 h do not induce significant changes in NO production in NB4 cells (data not shown).

3.8. Effect of esculetin on intracellular ROS and NOS levels 3.9. Esculetin causes a decrease of intracellular GSH levels After treatment with 100 lM esculetin, we evaluated ROS levels by using the probes DHE for superoxide anion and H2DCFDA for peroxides. Fig. 6C indicates that NB4 cells treated with esculetin showed a significant decrease for superoxide ions from 2 to 6 h

To determine the effect produced by esculetin on intracellular GSH levels in NB4 cells, we treated cells with 100 lM of esculetin for 0.5–24 h. Fig. 7A shows that esculetin diminished GSH in a

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time-dependent manner from 4 to 19 h. When cells were treated with BSO, we observed a significant reduction of GSH. Pretreatments with BSO previous to esculetin incubation clearly decreased GSH levels (Fig. 7B). The relationship between GSH depletion and apoptosis was then analyzed (Fig. 7C). Increased apoptosis was clearly observable after long term treatments with either esculetin or BSO plus esculetin while BSO alone did not produce observable apoptosis in NB4 after 24 h (Fig. 7C). Moreover, apoptosis was similar in the absence or presence of BSO. These results might indicate that GSH depletion is not related to apoptosis induced by esculetin in NB4 cells. 4. Discussion

Fig. 7. Effects of esculetin on the levels of GSH in NB4 leukemia cells. (A) Effects of 100 lM of esculetin for different times (0.5–24 h) on GSH levels. (B) NB4 cells (5  105 cells/ml) were pretreated with or without 1 mM BSO for 24 h and afterwards with 100 lM of esculetin for 0.5, 2 and 19 h and then intracellular levels of GSH were measured by fluorimetry analysis. (C) Apoptosis analyses of NB4 cells (5  105 cells/ml) pretreated with or without 1 mM BSO for 24 h and afterwards with 100 lM esculetin for 0.5, 2 and 19 h. Cells were stained with FITC-conjugated annexin V-FITC and PI and measuring by flow cytometry. Total apoptosis was calculated considering the percentage of annexin-FITC positive cells in treated samples in relation to the percentage of annexin-FITC positive cells in control samples. Values are expressed as the mean ± standard error of the mean (SEM) of at least three independent experiments. The differences between the control group and treated groups were determined. Significance of differences between treatments was determined by one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. ,  and  represent significant differences of p < 0.05, p < 0.01 and p < 0.001, respectively, in the Anova-Bonferroni comparison between treated cells and controls. #, ## and ### represent significant differences of p < 0.05, p < 0.01 and p < 0.001, respectively in the comparison between cells treated with BSO only and the cells treated with BSO and esculetin. +, ++ and +++ represent significant differences of p < 0.05, p < 0.01 and p < 0.001, respectively in the comparison between cells treated with esculetin only and the cells treated with BSO and esculetin.

Oxidative balance is a determinant fact that should be controlled for cells to be maintained functionally active. Promotion of cell oxidation by chemical compounds, pathological disease processes or environmental changes gives rise to lose of the physiological equilibrium of the cell. Eventually altered cells could die by different death mechanism. Compounds that are known to show antioxidant properties could prevent cell oxidation. Intriguingly, some antioxidant compounds when applied on growing cells can produce some extent of cytotoxicity. These phenomena might be useful for treating tumor cells whose oxidative equilibrium could be deregulated. Esculetin is an antioxidant that may be an effective agent to inhibit cell proliferation [34]. Acute promyelocytic leukemia cell line (NB4) has been used as a model to study the action of esculetin in this work. This cell line shows unique features with respect to cell progression, differentiation and resistance to antitumor drugs [3] and it can be used to study the toxicity of antitumor agents [11,13]. Esculetin showed toxic effects on NB4 leukemia cells with reduction of cell viability in a time- and concentration-dependent manner (Figs. 1 and 2) becoming evident from 14 h of treatment. Interestingly, this decrease in cell viability seems to be caused by induction of apoptosis as a function of the duration and concentration of esculetin treatment (Figs. 3 and 4). Our results indicate a cytotoxic effect that is clearly visible starting at a concentration of 100 lM. Furthermore, treatments for 19 h and longer also gave rise to NB4 cell death. Thus, these can be considered adequate conditions for studying apoptosis and cytotoxicity induced by esculetin in NB4 leukemia cells. Moreover, the range of concentrations of esculetin used in this study is consistent with other in vitro studies that used similar esculetin concentrations [23,31,35] in other cell types. In recent years, some authors have found toxic responses to esculetin in different human leukemia cell lines. These effects have been explained by different mechanisms exerted by esculetin. For example, it has been reported that esculetin exhibits an antiproliferative effect by inducing apoptosis in HL-60 cells [23,35] and U937 cells [21,22,36]. NB4 leukemia cell line shows a differential response to several compounds such as ATRA, DMSO, vitamin D3 or TPA [3] in comparison to other leukemia cell models such as HL60 or U937 lines. Differential resistance to differentiation induction shown by NB4 cell line might be correlated with different cytotoxicity resistance or apoptosis mechanisms. In non-leukemia cells such as 3T3-L1 adipocytes [20,37] and hepatoma HepG2 cells [31], esculetin could also induce cell death presumably by an apoptosis mechanism. In acute promyelocytic leukemia NB4 cells, esculetin induced significant apoptosis (Figs. 5 and 6) as observed by propidium iodide and annexin V-FITC cytometry. In the conditions used, no relevant necrosis can be seen. In order to know more precisely the way how cell toxicity and apoptosis develop in NB4 cells as a result of esculetin treatment,

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we studied changes in relevant apoptosis factors that correlate with the observed toxicity. Apoptosis may proceed through the intrinsic or mitochondrial pathway and the extrinsic or death receptor pathway. Stimulation of these pathways leads to the activation of caspases that begins the process of apoptosis. Thus, to further confirm an apoptosismediated cytotoxic effect of esculetin on NB4 cells, we analyzed activation of caspases. Indeed, esculetin-mediated apoptosis in NB4 cells showed to proceed through caspase-3 and caspase 9 activation (Fig. 7A). These results clearly confirm an apoptotic process through intrinsic pathway although it is not possible to determine whether activation of caspase 9 could be previous to the action of caspase 3 as an executer of the apoptosis process. Our results give support to the hypothesis of caspase activation proposed from results previously obtained in human leukemia U937 [21], 3T3-L1 adipocytes [37] and human cervical cancer cells [34]. We investigated the expression levels of p53, Bcl-2 and Bax, finding that p53 levels seem not to change upon treatment with esculetin. Some authors have also observed that p53 was not altered after esculetin exposure in human oral cancer SAS cells [24]. This suggests that p53 might not be related directly to the activation of apoptosis by esculetin although this possibility cannot be disregarded. In other cell types such as 3T3-L1 adipocytes, other authors found that esculetin induced apoptosis by changes in mitocondria with reduction of Bcl-2 and Bax increase. In contrast, these proteins were not altered in SAS cells. Our results show that treatment of esculetin on NB4 cells produced a reduction of the value of Bcl2/Bax ratio. These results support the induction of an apoptosis process by esculetin in NB4 cells. Thus, the expression changes in Bcl2 and Bax could be significant in NB4 leukemia cells apoptosis induction by esculetin in this model. In some tumor cell lines, it has been seen that apoptosis induction renders alteration of the levels and activity of intracellular kinases such as ERK and Akt. In NB4 cells treated with esculetin we observed a slight increase in ERK levels as esculetin concentration increased. These results show the relevant role of ERK phosphorylation in esculetin-induced apoptosis of NB4 cells. This stimulation of ERK phosphorylation has also been observed in other leukemia cell lines [21] although might not be a general feature of the action of esculetin in different leukemia cell types [38]. The role played by p-ERK induction in leukemia cells might not be the same than that played in other kinds of cells such as 3T3-L1 adipocytes [37] or human hepatoma HepG2 cells [32]. Our results are also in accordance with recently published results that claim in favor of a correlation of increased ERK expression and phosphorylation and the induction of apoptosis in those cases where reactive oxygen species are implicated [39]. Anyhow, the involvement of ERK should be considered while studying response mechanisms to this compound which involves oxidative changes [40]. Also, ERK phosphorylation as a secondary effect of the toxic action of esculetin cannot be disregarded since we observed these changes 19 h after the beginning of esculetin treatment. Thus, a direct correlation between the phosphorylation of ERK and apoptosis could be considered if inhibition of ERK activation showed reduction of apoptosis at short times after the beginning of esculetin treatment. We have observed that levels of Akt are slightly altered with respect to control cells in treatments with different concentrations of esculetin for 19 h. p-Akt levels increased with increasing concentrations of esculetin (from 100 lM to 500 lM for 19 h). From these results, a role for Akt phosphorylation might be considered at least in this NB4 human leukemia cell model in contrast to other human leukemia cells [21]. Akt activity has been related to redox balance. ROS induce Akt dephosphorylation [41]. However, Akt is activated by esculetin in a time-dependent way what could be due to the claimed antioxidant activity of esculetin.

As in the case of other factors involved in apoptosis, some changes in expression and/or phosphorylation of Akt can be raised as a response to the agent which promotes cell toxicity. Thus, an adaptative response cannot be disregarded for avoiding apoptosis cascade. This could also be the reason for the observed increment in phosphorylated Akt. The expression of NFkB and Nrf2 as a result of the antioxidant response to esculetin is under current investigation in order to study possible inflammation processes [42]. We have also carried out experiments in order to determine whether changes in oxidative equilibrium involving mitochondria can eventually explain or not the apoptosis produced by esculetin in NB4 cells. We have shown a decrease of mitochondrial membrane potential and release of cytochrome c to cytosol. Thus, mitochondrial damage correlated with apoptosis induction by esculetin in NB4 cells. Mitochondrial alteration could be consequence of induction of apoptosis by esculetin or the result of redox imbalance on mitochondrial membrane. We favor the first hypothesis since we have clear results demonstrating DNA fragmentation, membrane phospholipids and variations in Bcl2/Bax ratio. Anyhow, the correlation of mitochondrial membrane damage and redox imbalance should be deeply studied. Thus, we studied the production of ROS species such as superoxide anion and peroxides. Our results indicate a decrease in superoxide anion at short times after esculetin incubation (6 h) together with a concomitant reduction of peroxides. This means that esculetin behaved as a ROS scavenger. These results are hence in accordance with the effects of antioxidant compounds. However, after 6 h of treatment, we observed an increase of superoxide anions that could be a consequence of mitochondrial alterations (release of cytochrome c) and the beginning of an apoptosis process. Although, some role for ROS in the apoptosis could be postulated (Fig. 6), our results seem to suggest that the increment of superoxide levels and the great decrease of peroxides observed at 24 h might be the result of the mitochondrial alteration in apoptosis and not the cause. The antioxidant action of esculetin could be producing this decrease of ROS. A redox imbalance might be implicated in the described effects of esculetin on NB4 cells [10,33]. As we have also shown, esculetin reduced the levels of intracellular glutathione. Since esculetin is considered an antioxidant compound we wanted to know whether increments of reduced glutathione were obtained after treatment of NB4 cells. The decrease in GSH which is remarkable after treatment for 24 h could be the result of the apoptosis process induced in these cells by esculetin. Furthermore, successive treatments with BSO and esculetin produced an even more important decrease in GSH than that produced by separate treatments with BSO or esculetin (Fig. 7). Thus, it might be considered that depletion of GSH could be a final effect of the apoptosis process which is clearly observable after 6 h of the esculetin treatment. It has been described that NB4 cells are under a high level of oxidative stress [10,43]. Thus, a decrease of ROS caused by esculetin might be contributing to apoptosis induction. Since from these results, a role of ROS species on the apoptosis process could not be definitively stated, the implication of antioxidant effect on the apoptosis process of NB4 cells will be more extensively studied. In conclusion, we have demonstrated that esculetin induced apoptosis in acute promyelocytic leukemia NB4 human cells. This apoptosis process correlated with activation of caspases cascade by the intrinsic pathway, changes in Bcl2/Bax ratio and alterations in the levels of expression and phosphorylation of intracellular kinases such as ERK and Akt. These results can benefit the understanding of the cytotoxic action of esculetin and even the study of its possible effect on tumor processes in comparison to healthy cells. Even more, both the studied cytotoxic effects of esculetin and the observed changes in peroxide and superoxide species could be

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a key to understand the role of redox balance on leukemia cells proliferation and treatment. Conflict of Interest The authors declare that there is no conflict of interest. Transparency Document The Transparency document associated with this article can be found in the online version.

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