Arch Toxicol DOI 10.1007/s00204-014-1300-0

Molecular Toxicology

Quercetin induces mitochondrial‑derived apoptosis via reactive oxygen species‑mediated ERK activation in HL‑60 leukemia cells and xenograft Wei‑Jiunn Lee · Michael Hsiao · Junn‑Liang Chang · Shun‑Fa Yang · Tsui‑Hwa Tseng · Chao‑Wen Cheng · Jyh‑Ming Chow · Ke‑Hsun Lin · Yung‑Wei Lin · Chung‑Chi Liu · Liang‑Ming Lee · Ming‑Hsien Chien 

Received: 24 December 2013 / Accepted: 17 June 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Quercetin is a plant-derived bioflavonoid that was recently shown to have multiple anticancer activities in various solid tumors. Here, novel molecular mechanisms through which quercetin exerts its anticancer effects in acute myeloid leukemia (AML) cells were investigated. Results from Western blot and flow cytometric assays revealed that quercetin significantly induced caspase-8, caspase-9, and caspase-3 activation, poly ADP-ribose polymerase (PARP) cleavage, and mitochondrial membrane depolarization in HL-60 AML cells. The induction of PARP cleavage by quercetin was also observed in other AML cell lines: THP-1, MV4-11, and U937. Moreover, treatment of HL-60 cells with quercetin induced sustained activation of extracellular signal-regulated kinase (ERK), and inhibition of ERK by an ERK inhibitor significantly

abolished quercetin-induced cell apoptosis. MitoSOX red and 2′,7′-dichlorofluorescin fluorescence, respectively, showed that mitochondrial superoxide and intracellular peroxide levels were higher in quercetin-treated HL-60 cells compared with the control group. Moreover, both N-acetylcysteine and the superoxide dismutase mimetic, MnTBAP, reversed quercetin-induced intracellular reactive oxygen species production, ERK activation, and subsequent cell death. The in vivo xenograft mice experiments revealed that quercetin significantly reduced tumor growth through inducing intratumoral oxidative stress while activating the ERK pathway and subsequent cell apoptosis in mice with HL-60 tumor xenografts. In conclusions, our results indicated that quercetin induced cell death of HL-60 cells in vitro and in vivo through induction of intracellular

W.-J. Lee · K.-H. Lin · Y.-W. Lin · C.-C. Liu · L.-M. Lee (*)  Department of Urology, Wan Fang Hospital, Taipei Medical University, 111 Hsing Long Road, Section 3, Taipei 116, Taiwan e-mail: [email protected]

T.-H. Tseng  School of Applied Chemistry, Chung Shan Medical University, Taichung, Taiwan

M. Hsiao  Genomics Research Center, Academia Sinica, Taipei, Taiwan J.-L. Chang  Department of Medical Management, Taoyuan Armed Forces General Hospital, Taoyuan County, Taiwan J.-L. Chang  Biomedical Engineering Department, Ming Chuan University, Taoyuan County, Taiwan S.-F. Yang  Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan

C.-W. Cheng · M.-H. Chien (*)  College of Medicine, Graduate Institute of Clinical Medicine, Taipei Medical University, 250 Wu‑Hsing Street, Taipei 110, Taiwan e-mail: [email protected] J.-M. Chow  Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan M.-H. Chien  Department of Medical Education and Research, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan

S.-F. Yang  Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan

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oxidative stress following activation of an ERK-mediated apoptosis pathway. Keywords  Acute myeloid leukemia · Apoptosis · Extracellular signal-regulated kinase · Quercetin · Reactive oxygen species

Introduction Acute myeloid leukemia (AML) is the most common type of leukemia, while has the lowest survival rate of all leukemias. A better understanding of the molecular biology of AML will be helpful in developing new therapeutic strategies to specifically target molecular abnormalities. Treatment options for leukemia include radiotherapy, chemotherapy, hormonal therapy, immune therapy, and symptomatic and supportive therapy. Nowadays, there is interest in therapy with drugs of plant origins because conventional medicines may be inefficient and result in side effects (Rates 2001). Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a bioflavonoid, is widely distributed in plants and fruits, and the chief dietary sources of quercetin are apples, onions, and tea. Quercetin has been used as a dietary supplement for many years, and thus, its use does not raise concerns regarding its toxicity. Previous research showed that quercetin is among the most mutagenic of the flavonoids, and subsequent studies showed it to be a powerful natural anticancer agent (Hirpara et al. 2009). The majority of studies indicated that quercetin appears to have anti-inflammatory and antioxidant properties, which may be responsible for its beneficial effects (Wang et al. 2006). Recently, quercetin has garnered much attention in relation to its anticancer activities in many cancer cell models including leukemia (Duo et al. 2012; Granado-Serrano et al. 2006; Lee et al. 2011), whereas the complicated mechanisms behind the cancer preventive effects of quercetin have not been fully investigated. Apoptosis is an active process of endogenous programmed cell death, which is a key pathway for regulating homeostasis and morphogenesis and is connected to several diseases, especially cancers (Goldsworthy et al. 1996). One of the hallmarks of cancer is the deregulation of apoptosis; thus, increasing apoptosis in tumors is one of the best ways for anticancer agents to treat all types of cancer. Apoptosis is controlled by two diverse pathways, either the death receptor-mediated (extrinsic) pathway or the mitochondria-dependent (intrinsic) pathway. The receptor-mediated pathway is dependent upon upstream activation of caspase-8, which results in activation of caspase-3 that then cleaves various substrates and ultimately leads to apoptosis (Ashkenazi and Dixit 1999). On the other hand,

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the mitochondrial-mediated pathway begins with disruption of the mitochondrial membrane potential (MMP, ΔΨm), which is regulated by B cell leukemia/lymphoma (Bcl)-2 family proteins, thereby promoting the release of cytochrome c, that activates caspase-9, which in turn activates caspase-3, thus resulting in apoptosis (Cory and Adams 2002). Recently, it was reported that the redox status of a cell plays a key role in the cell’s fate. Indeed, deregulation of the balance between the rates of production and breakdown of reactive oxygen species (ROS) can lead to excessive production of ROS in cells followed by induction of cell apoptosis (Martindale and Holbrook 2002; Sastre et al. 2000). In addition, ROS appear to mainly function at the initiation/activation step of apoptosis, since most apoptosis inducers produce ROS (Jacobson 1996). A previous study indicated that extracellular signal-regulated kinase (ERK) is an important downstream target of the ROS-mediated apoptotic process (Kong et al. 2008). Although the ERK pathway is normally associated with enhanced cell proliferation, conversely, many studies on bioactive food components showed that ERK activation upregulates expressions of apoptotic genes which contribute to cell death (Cagnol and Chambard 2010). Moreover, mitochondria are known to be an important target of cellular stressors, and mitochondrial abnormalities associated with altered oxidative metabolism appear to contribute to intracellular oxidative stress (Hsu and Sabatini 2008). Indeed, mitochondria are the major source of superoxide production and are directly attacked by ROS. Interestingly, recent studies revealed that activation of ERK by quercetin resulted in antiproliferative effects such as apoptosis, senescence, or autophagy in cancer cells (Kim et al. 2008). On the other hand, quercetin was shown to induce the production of superoxide anions, hydrogen peroxide, and other ROS (De Marchi et al. 2009). Until now, whether and how ROS signals impact quercetin-induced cell apoptosis and what the underlying mechanisms of quercetin are which cause the antitumor activity remain largely unknown. In the present study, we investigated the cytotoxic effects of quercetin on four AML cell lines (THP1, U937, HL-60, and MV4-11) and its underlying mechanisms in vitro and in an AML xenograft model.

Materials and methods Chemicals Quercetin, dimethyl sulfoxide (DMSO), 2′,7′-dichlorofluorescein diacetate (DCFDA), dihydroethidium (DHE), 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), N-acetylcysteine (NAC), z-IETD-fmk

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(a caspase-8 inhibitor), zDEVD-fmk (a caspase-3 inhibitor), and a general inhibitor of caspases (zVAD-fmk) were purchased from Sigma–Aldrich (St. Louis, MO, USA). MitoSOX red, propidium iodide (PI), and annexin V were purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), antibiotics, molecular weight standards, trypsin– EDTA, trypan blue stain, and all medium additives were obtained from Life Technologies (Gaithersburg, MD, USA). N,N-Methylenebisacrylamide, acrylamide, sodium dodecylsulfate (SDS), ammonium persulfate, Temed, and Tween 20 were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Anti-phospho-ERK1/2 (Thr202/Tyr204), poly (ADP-ribose) polymerase (PARP), and cleaved PARP antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-ERK1/2, cleaved caspase-3, Ki67, caspase-3, caspase-8, and caspase-9, LC-3, and β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The MEK inhibitor (U0126) and SOD mimetic compound, manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), were obtained from Calbiochem (La Jolla, CA, USA). Cell culture Human leukemic HL-60, THP-1, MV4-11, and U937 cell lines, breast carcinoma MDA-MB-231 cell line, lung carcinoma A549 cell line, hepatoma SK-Hep1 cell line, normal prostatic epithelial cells PNT2, and human lung fibroblast cells WI38 were obtained from the American Type Culture Collection (Manassas, VA, USA). HL-60, THP-1, MV411, U937, and PNT2 cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA), and the other cell lines were maintained in DMEM (Gibco BRL, Grand Island, NY, USA) supplemented with 10 % FBS, 2 mM glutamine, 200 µg/ml penicillin, and 100 µg/ml streptomycin sulfate. Cells were maintained at 37 °C in a humidified atmosphere of 95 % air and 5 % CO2 unless specifically described in some experiments. Preparation of total cell extracts and Western blot analysis Cells were washed with phosphate-buffered saline (PBS) plus zinc ions (1 mM) and lysed in radio immunoprecipitation assay (RIPA) buffer (50 mM Tris buffer, 5 mM EDTA, 150 mM NaCl, 1 % NP40, 0.5 % deoxycholic acid, 1 mM sodium orthovanadate, 81 μg/ml aprotinin, 170 μg/ml leupeptin, and 100 μg/ml PMSF; pH 7.5). After mixing for 30 min at 4 °C, the mixtures were centrifuged (104×g) for 25 min, and the supernatants were collected as whole-cell extracts. The protein content was determined with the BioRad protein assay reagent using bovine serum albumin as a standard. Samples containing 10–50 μg of proteins were

boiled in Laemmli sample buffer, separated on SDS polyacrylamide gels, electrophoretically transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL, USA), and blotted with the indicated primary antibodies. Proteins were visualized with horseradish peroxidase-conjugated secondary antibodies (Zymed Laboratory, South San Francisco, CA, USA) followed by chemiluminescence detection (ECL-Plus; Santa Cruz Biotechnology). Cell viability assay Cells were seeded in 24-well plates (5 × 104 cells/well) and cultured in complete growth medium for 24 h. Cells were pretreated with or without various antioxidants, NAC, or the MnSOD mimetic, MnTBAP; then, various concentrations of quercetin were added to the wells. After the required time point, the medium was changed and incubated with 0.5 mg/ml of MTT for 4 h. The number of viable cells was directly proportional to the production of formazan, which was dissolved by DMSO, and measured spectrophotometrically at 570 nm on an Emax Microplate reader (Molecular Devices, Sunnyvale, CA, USA). The comparative cell viability of quercetin treatment in various types of human cell lines was evaluated, and the 50 % of effective dose (IC50) was further determined. DAPI staining Morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with the DNA-binding fluorochrome, DAPI. After quercetin treatment, cells were fixed with methanol, stained with the DAPI solution, and examined under a fluorescence microscope. Apoptotic cells exhibited morphological features of apoptosis including chromatin condensation and nuclear fragmentation. Cell cycle analysis Briefly, 5 × 105 cells per well were cultured in 6-well plates and incubated overnight. Thereafter, cells were treated with 100  μM of quercetin for the indicated time. After treatment, cells were fixed in chilled 75 % methanol and stained with a PI solution (100 μg/ml RNase and 10 μg/ml PI in PBS). Data acquisition and analysis were performed using a flow cytometric system, and the percentage of cells in each phase was determined. Apoptosis assays Apoptosis was further quantitated by flow cytometry using a PI/Annexin V-FITC kit following the manufacturer’s guidelines. Annexin V fluorescence was quantitated with a

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Becton–Dickinson FACSCalibur or LSRII flow cytometer (BD Biosciences, San Jose, CA, USA). HL-60 cells were pre-incubated with or without 50 μM of inhibitors specific to caspase-3 (zDEVD-fmk) or caspase-8 (zIETD-fmk), or a general inhibitor of caspases (zVAD-fmk) for 1 h. Cells were then exposed to quercetin at the indicated concentration for 12 h. Subsequently, cells were washed twice with PBS, and cell pellets were suspended in 150 µl binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2; pH 7.4), and stained with 5 µl annexin V-FITC in the dark at room temperature for 15 min. Prior to a fluorescenceactivated cell sorting (FACS) analysis, 250 µl binding buffer and 5 µl PI (250 µg/ml) were added. Cells stained with annexin V plus PI was considered apoptotic. Isolation of mitochondrial and cytosolic proteins Cells were harvested and pelleted by centrifugation at 1,500×g for 3 min at 4 °C. The pellet was resuspended in 500  μl of fractionation buffer (250 mM sucrose, 10 mM KCl, 0.15 mM MgCl2, 2 mM EGTA, and 1 mM DTT) containing a cocktail of protease inhibitors and incubated 7 min on ice. After incubation, cells were homogenized with a manual homogenizer for seven passages and then centrifuged at 1,500×g for 15 min at 4 °C. The supernatant obtained was centrifuged at 104×g for 15 min at 4 °C. The mitochondrial pellets were lysed in standard RIPA lysis buffer, and the supernatants were used as the cytosolic fractions.

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harvested, washed with PBS, and stained with DCFDA, MitoSOX red, and 2.5 μg/ml of a DAPI solution for 30 min at room temperature. Cells were washed twice with PBS, captured with a fluorescence microscope (Nikon Eclipse TE 300, Germany), and processed with Adobe Photoshop. Animals Male athymic nude mice (SCID), 6–8 weeks of age, were purchased from BioLASCO Taiwan Co., Ltd., Taipei, Taiwan, and maintained in caged housing in a specifically designed pathogen-free isolation facility with a 12/12 h light/dark cycle. The mice were provided with rodent chow and water ad libitum. All experiments were conducted in accordance with the guidelines of the Taipei Medical University Animal Ethics Research Board. In vivo antitumor activity by quercetin treatment

HL-60 cells (106) were resuspended in RMPI 1640 supplemented with 10 % FBS and stained with 2.5 μg/ml JC-1, followed by incubation for 10 min at room temperature. Cells were then washed, and the pellet was resuspended in PBS for fluorescence microscopic and flow cytometric analyses.

HL-60 cells (5 × 105) were injected subcutaneously (s.c.) into the right dorsal flank of SCID mice (6–8 weeks old). Ten days after the injection, mice were randomized into experimental and control groups according to the tumor size, such that treatment began with similar mean tumor sizes in each group. Ten days after tumor transplantation, treated animals received daily intraperitoneal injections of 500 mg/ kg quercetin dissolved in DMSO. The injection volume was 200  μl (containing 25 μl of a stock solution and 175 μl PBS) each time. The control group was given 200 μl of vehicle (25 ml DMSO and 175 μl PBS) only. Antioxidant-treated mice received drinking water supplemented with 40 mM NAC to yield an average dose of 1 g NAC/kg of body weight/day, and NAC-treated water was replaced every third day. The length (L) and width (W) of the tumor were measured with calipers every third day, and the tumor volume (TV) was calculated as TV (mm3) = (L × W2)/2. Results are expressed as the mean of the tumor volume ± the standard error of the mean (SEM; n = 5 mice in each group).

Measurement of ROS production

Tumor immunohistochemistry

HL-60 cells were treated with quercetin for the indicated time in the presence or absence of 5 mM NAC or 100 μM MnTBAP; then, ROS production was measured by staining with ROS probes (5 μM DCFDA or 5 μM MitoSOX red) in RPMI 1640 medium for 30 min. After being washed with PBS or medium, the ROS production of DCFDA- or MitoSOX red-preloaded cells was measured using an RF5301PC spectrofluorophotometer (Shimadzu, Japan). The following settings were used: 488 nm excitation and 522 nm emission for DCF-DA and 510 nm excitation and 580 nm emission for MitoSOX red. For ROS live imaging, after treatment with 100 μM quercetin for 6 h, cells were

All tumor tissue samples were fixed in a 10 % buffered formaldehyde solution. Specimens were embedded in paraffin blocks and 4-μm sections were cut. All specimens were deparaffinized and immersed in 10 mM sodium citrate buffer (pH 6.0) in a microwave oven twice for 5 min to enhance antigen retrieval. After washing, slides were incubated with 0.3 % H2O2 in methanol to quench endogenous peroxidase activity. Slides were washed with PBS and incubated with anti-Ki67, anti-cleaved PARP, anti-cleaved caspase-3, anti-mouse IgG, and anti-rabbit IgG antibodies for 2 h at room temperature. After washing in PBS, slides were developed with a VECTASTAIN ABC (avidin–biotin

Analysis of the MMP

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complex) peroxidase kit (Vector Laboratories, Burlingame, CA, USA) and a 3,3,9-diaminobenzidine (DAB) peroxidase substrate kit (Vector Laboratories) according to the manufacturer’s instructions. All specimens were deparaffinized and stained with hematoxylin and eosin that were used as a light counterstain. Tumor fluorescence imaging and data analysis The fluorescent oxidation products of DCFDA and DHE were used to demonstrate ROS production ex vivo. DCFDA and DHE were dissolved in 10 % DMSO in PBS at a final concentration of 200 μM and warmed to 37 °C. After tumor resection, to monitor ROS generation, DCFDA and DHE were added to the tumor for 30–45 min, and the fluorescence was imaged ex vivo and quantified using a noninvasive bioluminescence system (IVIS spectrum, Caliper Life Sciences) by photon excitation at 465 nm and emission at 520 nm for DCF and excitation at 500 nm and emission at 620 nm for DHE.

Fig. 1  Quercetin markedly induces apoptosis in human leukemia cells. Cells were incubated with 100 μM of quercetin for 6 and 12 h, and then, cell lysates were assayed by Western blotting for cleavage of PARP in different types of cancer cells (a) and various AML cells (b). β-Actin was used as a loading control Table 1  Evaluation of quercetin on cell viability

Statistical analysis Data points represent the mean ± standard deviation (SD). Data were analyzed using Student’s t test when two groups were compared. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to analyze three or more groups. Differences were considered significant at the 95 % confidence interval (p 400 237.4 ± 50.4 >400 216.9 ± 9.6

HL-60 THP-1 MV4-11 U937 MDA-MB-231 A549 SKHep-1 PNT2

Leukemia Leukemia Leukemia Leukemia Breast cancer Lung cancer Hepatoma Normal prostatic epithelial cells

57.1 ± 1.1 67.2 ± 1.1 69.4 ± 0.4 73.5 ± 0.8 >400 333.5 ± 42.4 >400 >400

WI38

Lung fibroblast cells

306.9 ± 25.4 255.3 ± 10.9

Cell viability was determined by MIT assay of three independent experiments as described in “Materials and methods” section. The comparative effect with control was evaluated, and IC50 of quercetin was expressed. Values are mean ± SD

MTT assay. The results showed that quercetin exhibited a more potent cytotoxicity against leukemia cell lines such as HL-60, THP-1, MV4-11, and U937 than other cancer cell types and normal cells. The IC50 values of all different cell lines are presented in Table 1. These findings suggest that quercetin would likely be useful as a therapeutic agent in managing leukemia. Quercetin‑induced caspase‑dependent apoptosis in HL‑60 cells We next detected condensed and fragmented nuclei, another marker of apoptosis, after treatment of HL-60 cells with 100 μM of quercetin for 12 h (Fig. 2a). To further

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Fig. 2  Quercetin induces caspase-dependent apoptosis in HL-60 cells. a HL-60 cells were treated with 100 μM quercetin for 12 h, and apoptotic body formation (arrows) as an indicator of apoptosis was analyzed by fluorescence microscopy after DAPI staining (×100). b HL-60 cells were incubated with 100 μM quercetin for the indicated time points. The cell cycle distribution was analyzed by FACS after PI staining. The data are shown as the cell cycle distribution profile by FACS and the percentage distribution of cells in the G0/G1, S, and G2/M phases. Data are presented as the mean ± SD of n = 3 experi-

ments. Results were analyzed using one-way ANOVA with Tukey’s post hoc tests at 95 % confidence intervals. Different letters represent different levels of significance. c Analysis of cleaved PARP, and caspase-3, caspase-8, and caspase-9 from lysates of HL-60 cells treated with 100 μM quercetin for the indicated time. d HL-60 cells were incubated with 50 and 100 µM quercetin for 12 h, in the absence or presence of various caspase inhibitors (100 µM). Apoptotic cells were determined and quantified by FACS after staining with annexin V-FITC/PI

confirm activation of apoptosis by quercetin, a flow cytometric analysis was used. Quercetin induced time-dependent increases in the sub-G1 population (Fig. 2b) compared with the control group. Because PARP is a substrate for certain activated caspases during apoptosis, we delineated more details about the spectrum of caspases which were activated in quercetin-induced apoptosis, including initiator (caspase-9 and

caspase-8) and executioner (caspase-3) caspases. The results clearly showed that quercetin induced PARP cleavage and hydrolysis of procaspase-8, procaspase-9, and procaspase-3 in time-dependent manners (Fig. 2c). Next, we further determined whether activation of caspases was required for quercetin-triggered apoptosis in HL-60 cells by a flow cytometric analysis using specific inhibitors that, respectively, inhibit caspase-3 (zDEVD-fmk), caspase-8

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(zIETD)-fmk, or a broad-spectrum caspase (zVAD-fmk). As shown in Fig. 2d, all caspase inhibitors significantly attenuated 50 or 100 μM quercetin-induced increases in annexin V and PI double-positive cells. Taken together, the results indicate that quercetin induced rapid and time- or dose-dependent apoptosis in a caspase-dependent pathway. Quercetin‑induced extensive apoptosis was accompanied by altered expressions of Bcl‑2 family proteins and depolarization of mitochondrial membranes In order to gain insights into the molecular mechanisms by which quercetin induces apoptosis, we first investigated the effects of quercetin on levels of Bcl-2 family proteins in HL-60 cells. Figure 3a shows that exposure of HL-60 cells to quercetin (0–100 μM for 12 h) caused significant concentration-dependent increases in the proapoptotic proteins, Bax and Bak, and a decrease in the antiapoptotic protein, Bcl-XL, but not Bcl-2. Because Bcl-2 family proteins were reported to regulate cell apoptosis via controlling mitochondrial membrane permeability and inducing cytochrome c release (Cory et al. 2003), we determined whether quercetin-induced apoptotic cell death involved the mitochondrial pathway. Translocation of proapoptotic Bcl-2 family members to the outer mitochondrial membrane is an important event associated with the loss of MMP (Cory et al. 2003). Under the same treatment conditions of quercetin which induced apoptosis, we found that quercetin concentrationdependently enhanced distributions of Bax, Bak, and t-Bid on mitochondria and elevated the release of cytochrome c from mitochondria to the cytoplasm (Fig. 3b). To further investigate the effect of quercetin on the MMP, the fluorescent cationic dye, JC-1, was used to detect the mitochondrial permeability transition. In healthy, non-apoptotic cells, the dye accumulated and aggregated within mitochondria, resulting in bright-red staining. In apoptotic cells, due to collapse of the membrane potential, JC-1 did not accumulate within the mitochondria and remained in the cytoplasm in its green fluorescent monomeric form. In Fig. 3c, d, immunofluorescence and flow cytometric analyses of JC1-stained HL60 cells treated with 100 μM quercetin for 9 h are shown. Quercetin induced concentration-dependent collapse of the MMP, and 100 μM quercetin led to mitochondrial damage in nearly 30 % of HL-60 cells (Fig. 3d). These data suggest that disruption of mitochondrial membranes by quercetin coincides with the presence of apoptotic death. Increased intracellular oxidative stress is linked to quercetin‑induced apoptosis Mitochondria rapidly lose their transmembrane potential and generate ROS, which are related to the induction of mitochondrial damage and apoptosis (Schonfeld and

Wojtczak 2007). To determine whether ROS are involved in quercetin-induced apoptosis, cellular ROS were monitored with the fluorescent, redox-sensitive dyes, H2DCFDA and MitoSOX red, after exposure of HL-60 cells to quercetin. H2DCFDA is widely used to measure cellular H2O2 and other ROS, and MitoSOX red is a selective indicator of mitochondrial superoxide. Our results showed that compared with those of the control group, treatment of cells with 100 μM quercetin initially decreased DCF fluorescence until 3 h after treatment and subsequently increased DCF fluorescence at 6–9 h after treatment. In contrast to H2DCFDA, a more rapid increase in MitoSOX red fluorescence at 15 min after quercetin treatment was seen, which was maintained for at least 12 h (Fig. 4a). The following fluorescence microscopic analysis revealed that almost all apoptotic cells caused by 6 h of quercetin treatment showed increased DCF and MitoSOX red fluorescence compared with those of the control group (Fig. 4b). To more definitively study whether the increased intracellular H2O2 and mitochondrial superoxide were implicated in quercetin-induced apoptosis in HL-60 cells, we pretreated HL-60 cells with two antioxidants, NAC and the MnSOD mimetic, MnTBAP, followed by exposure of cells to 100 μM quercetin for 1 or 6 h. Results showed that the increase in mitochondrial superoxide after 1 h of quercetin treatment was significantly attenuated by pretreating cells with NAC or MnTBAP (Fig. 4c, left panel). Moreover, increases in DCF and MitoSOX red fluorescence caused by 6 h of quercetin treatment were also inhibited by NAC and MnTBAP treatment (Fig. 4c, right panel). In order to further explore the significance of ROS production in quercetin-induced apoptosis, antioxidants were used to suppress the apoptosis-inducing effect of quercetin. Pretreatment with NAC or MnTBAP dramatically attenuated quercetin-induced PARP cleavage (Fig. 4d). Taken together, these results suggest that quercetin exposure is directly involved in the production of ROS, which led to apoptosis of HL-60 cells. Quercetin‑induced intracellular oxidative stress as an initial signal for ERK‑mediated apoptosis in HL‑60 cells A recent report suggested that ERK activation is associated with ROS-induced cell apoptosis (Luchetti et al. 2009). To ascertain the correlation between ERK activation and quercetin-induced apoptosis, we examined activation of ERK in quercetin-treated HL-60 cells. Our results showed that ERK was time-dependently phosphorylated after 100 μM quercetin treatment, but the protein level of ERK did not change. This activation had begun by 15 min, reached a maximum effect at 1 h, and remained in an active status for 12 h after quercetin treatment (Fig. 5a). Moreover, pretreatment of HL-60 cells with the ERK-specific inhibitor, U0126, effectively attenuated quercetin-induced

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Fig. 3  Extensive quercetin-induced apoptosis is accompanied by altered expressions of Bcl-2 family proteins and depolarized mitochondrial membranes. a HL-60 cells were incubated with the indicated concentration of quercetin for 12 h, and cell lysates were assayed by Western blotting for Bcl-2 family members; β-actin was used as an equal loading control. b HL-60 cells were treated with 50 and 100 μM quercetin for 6 h, while mitochondrial and cytoplasmic extracts were prepared as described in “Materials and methods” section. The mitochondrial and cytoplasmic levels of Bax, Bak, and truncated (t)-Bid were assayed by Western blotting. α-Tubulin and Tom20 antibodies were, respectively, used as cytoplasmic and mito-

chondrial markers. c, d Cells were treated with 100 μM quercetin for 9 h, then stained with JC-1, and analyzed by fluorescence microscopy and FACS. c An immunofluorescence analysis showed that the green fluorescent monomeric form increased in HL-60 cells after treatment with quercetin (×100). d The red-to-green fluorescence ratio indicates functional mitochondria with membrane potential. A significant lower red-to-green fluorescence ratio was observed in quercetin-treated cells compared with untreated cells. Values represent the mean ± SD (n = 3). Results were analyzed using one-way ANOVA with Tukey’s post hoc tests at 95 % confidence intervals, and different letters represent different levels of significance

cleavage of PARP (Fig. 5b), indicating that activation of ERK plays an important role in quercetin-induced apoptosis of HL-60 cells. To further investigate the correlation between ROS production and ERK activation in quercetin-induced cell death, we found that pretreating HL-60 cells with NAC or MnTBAP significantly blocked quercetin-induced ERK activation (Fig. 5c) and subsequently reversed quercetin-induced cell death (Fig. 5d). Overall, these data demonstrated that the increase in intracellular

oxidative stress acts upstream of the ERK activation pathway in quercetin-induced apoptosis.

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Quercetin induces tumor cell apoptosis via enhancing intratumoral oxidative stress in HL‑60 xenograft mice Given the above findings of ROS playing critical roles in quercetin-induced cell apoptosis in AML in vitro, we next analyzed the in vivo antitumor effect of quercetin and

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Fig. 4  Increased mitochondrial superoxide production is linked to cell apoptosis induced by quercetin. a HL-60 cells were treated with 100 μM quercetin for the indicated time and stained with H2DCF-DA or MitoSOX red; then, total ROS or mitochondrial superoxide levels were analyzed by spectrofluorophotometry, and data are presented as the mean multiples of increase in fluorescence compared with the control ± SD. #p