Quercetin Induces Tumor-Selective Apoptosis through ...clincancerres.aacrjournals.org/content/clincanres/16/23/5679.full.pdf · Quercetin Induces Tumor-Selective Apoptosis through

Cancer Therapy: Preclinical

Quercetin Induces Tumor-Selective Apoptosis through Downregulationof Mcl-1 and Activation of Bax

Senping Cheng1, Ning Gao1, Zhuo Zhang1, Gang Chen2, Amit Budhraja1, Zunji Ke2, Young-ok Son1,Xin Wang1, Jia Luo2, and Xianglin Shi1

AbstractPurpose: To investigate the in vivo antitumor efficacy of quercetin in U937 xenografts and the functional

roles of Mcl-1 and Bax in quercetin-induced apoptosis in human leukemia.

Experimental Design: Leukemia cells were treated with quercetin, after which apoptosis, Mcl-1

expression, and Bax activation and translocation were evaluated. The efficacy of quercetin as well as

Mcl-1 expression and Bax activation were investigated in xenografts of U937 cells.

Results: Administration of quercetin caused pronounced apoptosis in both transformed and primary

leukemia cells but not in normal blood peripheral mononuclear cells. Quercetin-induced apoptosis was

accompanied by Mcl-1 downregulation and Bax conformational change and mitochondrial translocation

that triggered cytochrome c release. Knockdown of Bax by siRNA reversed quercetin-induced apoptosis and

abrogated the activation of caspase and apoptosis. Ectopic expression of Mcl-1 attenuated quercetin-

mediated Bax activation, translocation, and cell death. Conversely, interruption of Mcl-1 by siRNA

enhanced Bax activation and translocation, as well as lethality induced by quercetin. However, the absence

of Bax had no effect on quercetin-mediated Mcl-1 downregulation. Furthermore, in vivo administration of

quercetin attenuated tumor growth in U937 xenografts. The TUNEL-positive apoptotic cells in tumor

sections increased in quercetin-treated mice as compared with controls. Mcl-1 downregulation and Bax

activation were also observed in xenografts.

Conclusions: These data suggest that quercetin may be useful for the treatment of leukemia by

preferentially inducing apoptosis in leukemia versus normal hematopoietic cells through a process

involving Mcl-1 downregulation, which, in turn, potentiates Bax activation and mitochondrial transloca-

tion, culminating in apoptosis. Clin Cancer Res; 16(23); 5679–91. �2010 AACR.

The natural product quercetin (3,5,7,30,40-pentahy-droxyflavone) is a widely distributed flavonoid that ispresent in many fruits and vegetables (e.g., onions andapples). Previous research has shown that quercetininduces apoptosis in a variety of tumors (1–3), includingleukemia (4). However, the molecular mechanisms ofquercetin-induced apoptosis are unclear. There is noavailable information concerning the in vivo efficacy ofquercetin against leukemia.Apoptosis involves 2 distinct pathways, one engaging

death receptor-initiated extrinsic pathway and the otherinvolving mitochondria-mediated intrinsic pathway (5).

The intrinsic pathway involves the release of proapoptoticproteins (e.g., cytochrome c) into the cytosol, formationof the apoptosome, and activation of caspase-9 (6, 7),which subsequently cleaves and activates procaspase-3(8). The mitochondrial pathway is mainly regulated byBcl-2 family proteins (9–11). Antiapoptotic Bcl-2 familyproteins (e.g., Bcl-2, Bcl-xL, and Mcl-1) antagonize mem-brane permeabilization and prevent the release of cyto-chrome c from mitochondria (12). Proapoptotic Bcl-2family proteins can be further divided into 2 subgroups.The multidomain proapoptotic proteins (e.g., Bax andBak) participate in the formation of mitochondrial porethrough which cytochrome c releases (13–16). The BH3-only proteins (e.g., Bim and Bid) are required for theactivation of multidomain proapoptotic proteins throughassociation of antiapoptotic Bcl-2 proteins (17, 18). It hasbeen reported that quercetin-mediated cell apoptosisinvolves mitochondria-mediated caspase activation (1,4, 19–22).

Notably, Mcl-1 is a highly expressed antiapoptoticprotein (23) implicated in malignant hematopoietic sur-vival (23, 24). It has been shown that depletion of Mcl-1,using antisense oligonucleotides, rapidly triggers apopto-sis in U937 cells (25). In contrast, selective expression of

Authors' Affiliations: 1Graduate Center for Toxicology and 2Departmentof Internal Medicine, College of Medicine, University of Kentucky,Lexington, Kentucky

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Xianglin Shi, Graduate Center for Toxicology,College of Medicine, University of Kentucky, Lexington, KY 40536. Phone:859-257-4054; Fax: 859-323-1059; E-mail: [email protected].

doi: 10.1158/1078-0432.CCR-10-1565

�2010 American Association for Cancer Research.

ClinicalCancer

Research

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Mcl-1 in hematopoietic tissues of transgenic mice pro-motes the survival of hematopoietic cells and enhancesthe outgrowth of myeloid cell lines (26). Furthermore,overexpression of Mcl-1 protects cells from apoptosisinduced by a variety of agents, including UV, etoposide,staurosporine, actinomycin D, and others (27–30). Twogroups (4, 31) have indicated a decrease of Mcl-1 level inquercetin-treated cells.

It has been proposed that alteration of Bax conformationand its redistribution tomitochondria play a key role in theinduction of cell death (32, 33). In healthy cells, Bax ispredominantly located in the cytoplasm. Upon apoptoticsignals, Bax undergoes a conformational change thatexposes the N-terminus and the hydrophobic C-terminusthat targets mitochondria (34, 35). The membrane inser-tion of Bax is essential for the release of cytochrome c andapoptosis (36, 37). It has been shown that quercetin caninduce apoptosis in multiple cancer cells through upregu-lation of Bax expression (19, 20, 22, 38). It has also beenreported that apoptotic process caused by quercetin aremediated by the dissociation of Bax from Bcl-xL in humanprostate cancer cells (39). Granado-Serrano et al. haveprovided evidences indicating that quercetin promotestranslocation of Bax to mitochondria membrane in humanhepatoma cells (1).

The present study shows that quercetin has an anti-leukemia ability by inhibition of xenografts growth ofU937 cells. Our study also shows an increase in apop-tosis in human leukemia cells and tumor sections uponquercetin treatment. In addition, our results indicate thatthis phenomenon stems from a novel mechanism invol-ving cooperation between Bcl-2 family proteins: 1) quer-cetin mediates Mcl-1 downregulation and activates Bax;

and 2) Mcl-1 regulates quercetin-mediated Bax activa-tion.

Materials and Methods

CellsHuman leukemia U937, Jurkat, and HL-60 cells were

obtained from American Type Culture Collection andcultured in RPMI 1640 supplemented with 10% fetalbovine serum (FBS), L-glutamine, and antibiotics. U937cells stably overexpressing Mcl-1, and their empty vectorcounterpart (pCEP) were kindly provided by Dr. RuthCraig (Dartmouth Medical School). HL-60 cells stablyoverexpressing Bcl-2 (HL-60/Bcl-2) and Bcl-xL (HL-60/Bcl-xL) were kindly provided by Dr. Ming Ding (TheNational Institute for Occupational Safety and Health).Mononuclear cells were isolated from peripheral bloodor bone marrow of leukemia patients or healthy donorswere purchased from AllCells, LLC.Mononuclear cells weresuspended in RPMI 1640 medium containing 10% fetalcalf serum at 8 � 105/mL for treatment. Baxþ/� and Bax�/�

human colon cancer HCT116 cells were kindly providedby Dr. Bert Vogelstein (Johns Hopkins University) andKenneth W. Kinzler (Howard Hughes Medical Institute)and sustained in MyCoy’s 5A medium containing 10% FBSand antibiotics.

Chemicals and reagentsQuercetin (>99% pure) was purchased from Sigma Che-

mical Co., dissolved in DMSO, aliquoted, and stored at�20�C. The pan-caspase inhibitor Z-VAD-FMK was pur-chased from EMD Biosciences.

Assessment of apoptosisThe extent of apoptosis in leukemia cells was evaluated

by flow cytometric analysis using FITC-conjugated AnnexinV/propidium iodide (PI; BD PharMingen) staining as perthe manufacturer’s instructions as previously described(40). Both early apoptotic (Annexin V–positive, PI-nega-tive) and late apoptotic (Annexin V–positive and PI-posi-tive) cells were included in cell death determinations.

Western blot assayCells were lysed and sonicated in 1� NuPAGE LDS

sample buffer (Invitrogen). The protein concentrationwas measured using Coomassie Protein Assay Reagent(Pierce), and 30 mg of sample proteins were separated bySDS-PAGE and incubated with antibodies. The blots werethen reprobed with an antibody against b-actin (SantaCruz Biotechnology) or Cox IV (Cell Signaling) to ensureequal loading of proteins. Primary antibodies used wereas follows: cytochrome c (Santa Cruz Biotechnology);cleaved caspase-3, cleaved capase-9, and Bcl-xL (CellSignaling); Mcl-1 and Bax (N-20) (BD PharMingen);PARP (Biomol Research Laboratories); and Bcl-2(DAKO). Secondary antibodies conjugated to horseradishperoxidase were obtained from Kirkegaard and PerryLaboratories, Inc.

Translational Relevance

The hematologic malignancies are the types of cancerthat arise frommalignant transformation of cells derivedfrom peripheral blood, lymphatic system, and bonemarrow. Because of the increase in the morbidity andmortality of human leukemia in recent years, chemo-prevention or intervention toward the control of humanleukemia is highly desirable. The present study hasprovided evidences that the natural product quercetininduces human leukemia cell death through downregu-lation of Mcl-1 and activation of Bax. The data presentedhere suggest that the 2 Bcl-2 family proteins Mcl-1 andBax may represent attractive targets for quercetin-induced apoptosis in human leukemia cells. In vivo dataconfirmed the antitumor efficacy of quercetin via induc-tion of apoptosis by targeting Mcl-1 and Bax in xeno-grafts of leukemia cells. The results of this study couldhave implications for the incorporation of agents such asquercetin into the chemopreventionor therapeutic inter-vention against hematologic malignancies.

Cheng et al.

Clin Cancer Res; 16(23) December 1, 2010 Clinical Cancer Research5680



Subcellular fractionationFor cytosol isolation, cells were lysed in lysis buffer

(75 mmol/L of NaCl, 8 mmol/M of Na2HPO4, 1 mmol/M of NaH2PO4, 1 mmol/L of EDTA, and 350 mg/mL ofdigitonin) and needle strokes. After sequential centrifuga-tion (1,000 � g to pellet nuclei, 10,000 � g to pelletmembrane fraction, and 100,000 � g), the supernatant(S-100, cytosolic fraction) were collected and subjected toimmunoblot. For mitochondrial and cytosol fractiona-tions, cells (50 � 106) were fractionated by mitochondrialfractionation kit (Active Motif) as per manufacturer’sinstructions.

Analysis of Bax conformational changeTo analyze the conformational change of Bax by flow

cytometry, cells were fixed and permeabilized using FIX &PERM cell permeabilization reagents (Caltag Lab) as permanufacturer’s instructions. Cells were incubated withFITC-conjugated anti-Bax (clone 6A7; Santa Cruz Biotech-nology) and then analyzed by flow cytometry. The resultsfor each condition were calibrated by values for cellsstained with mouse IgG (Santa Cruz Biotechnology) asthe primary antibody. Values for untreated controls werearbitrarily set to 100%. For analysis of Bax conformationalchange by immunoprecipitation (IP), cells were lysed inCHAPS lysis buffer and 500 mg of total lysates were immu-noprecipitated using anti-Bax 6A7 antibody. Resultingimmune complexes were analyzed by immunoblottingwith anti-Bax antiserum (N-20).

ImmunofluorescenceTo analyze cellular distribution of Bax cells were fixed,

permeabilized and probed with FITC-conjugated anti-Bax(Santa Cruz Biotechnology). Mitochondria were stainedwith 300 nmol/L of MitoTracker (Molecular Probes). Cellswere plated on 35-mm collagen-coated, glass-bottomeddishes (MatTek Corportation) and visualized using aninverted Leica TCS SP5 laser scanning confocal microscopeunder a 60� oil immersion objective.

RNA interferenceU937 cells (1.5 � 106) were transiently transfected with

10 nmol/L of siRNA specific for Mcl-1 or Bax (Santa CruzBiotechnology), using Amaxa Nucleofector device (pro-gram V-001) with Kit V (Amaxa GmbH), as per man-ufacturer’s instructions. After 24 hours of transfection,cells were treated and analyzed for protein expression,apoptosis, or viability.

Therapeutic evaluation of quercetin in xenograftmodelThe in vivo evaluation of quercetin was carried out using

xenograft model of human U937 cells. Athymic nude mice(NU/NU, 6–8 weeks old; Charles River) were housed in aspecific pathogen-free room within the animal facilities atthe University of Kentucky, Chandler Medical Center.Animals were allowed to acclimatize to their new environ-ment for 1 week prior to use. All animals were handled

according to the Institutional Animal Care and Use(IACUC), University of Kentucky. U937 cells (6 � 106)were resuspended in serum-free RPMI 1640 medium withMatrigel basement membrane matrix (BD Biosciences) at a1:1 ratio (total volume ¼ 100 mL) and then were subcu-taneously injected into the flanks of nude mice. From thesecond day of injection, mice were randomly assigned to3 treatment groups (n ¼ 6 for each group) and admini-strated intraperitoneally with quercetin (0, 20, and 40 mg/kg of body weight) in 150 mL of DMSO/0.9% physiologicsaline (1:0.5) daily for consecutive 15 days. Body weightand tumor mass were measured every 5 days. Tumorvolumes was determined by a caliper and calculatedaccording to the following formula: (width2 � length)/2.All animals were sacrificed immediately if tumor volumereached an approximate volume of 1,500 mm3 at day 16.

Detection of apoptosis by TUNEL, detection of Mcl-1expression and Bax activation in tumor tissue sections

Tumor tissue sections of formalin-fixed, paraffin-embedded specimens were dewaxed in xylene and rehy-drated in a graded series of ethanol. Apoptosis was detectedusing the TUNEL in situ apoptosis detection kit (DeadEndFluorometric TUNEL System; Promega). Briefly, tumorsamples were incubated with proteinase K (2 mg/mL)and the TUNEL staining was done according to the man-ufacturer’s instructions. The percentage of TUNEL-positivecells relative to total cells was calculated for each sampleunder a fluorescent microscope, counting at least 200 cells.Mcl-1 expression in tumor samples was analyzed by immu-nofluorescence, using a primary anti-Mcl-1 antibody(Abcam) following FITC-conjugated anti-mouse secondaryantibody (Invitrogen). Tumor samples were homogenizedand lysed in CHAPS lysis buffer and then buffered andimmunoprecipitated using anti-Bax 6A7 antibody. Result-ing immune complexes were analyzed by immunoblottingwith an anti-Bax antiserum (N-20).

Statistical analysisThe values were presented as means � SD. Two-way

analysis of variance (ANOVA) and Student’s t test were usedfor statistical analysis. P < 0.05 was considered significantlydifferent.

Results

Exposure to quercetin led to pronounced apoptosis inU937 cells, associated with a decrease in Mcl-1expression

A dose–response analysis of U937 cells revealed a mod-erate increase in apoptosis 9 hours after exposure to quer-cetin at concentrations of 10 to 20 mmol/L and extensiveapoptosis at concentrations 30 mmol/L or greater (Figs. 1Aand 3A). A time-course study of cells exposed to 40 mmol/Lof quercetin showed amarked increase in apoptosis as earlyas 2 hours after drug exposure and reached near-maximallevels after 9 hours (Fig. 1B).

Expression of antiapoptotic Bcl-2 family proteins inU937 cells was monitored following treatment with

Quercetin-Induced Apoptosis in Human Leukemia Cells

www.aacrjournals.org Clin Cancer Res; 16(23) December 1, 2010 5681



quercetin. As shown in Figure 1C, a marked dose-depen-dent decrease of Mcl-1 expression was observed in quer-cetin-treated cells. Exposure of U937 cells to 40 mmol/L ofquercetin at varying intervals resulted in a rapid down-regulation of Mcl-1 that was detectable as early as 2 hours,and by 4 hours of treatment expression was almost absent(Fig. 1D). In contrast, the other 2 important antiapoptoticBcl-2 family proteins Bcl-2 or Bcl-xL expression remainedunaffected. Together, these findings show that quercetininduces rapid and dose-dependent downregulation of Mcl-1 in U937 cells.

Quercetin induced Bax conformational change andmitochondrial translocation in U937 cells

Following death stimuli, Bax undergoes an N-terminalconformational change, which can be detected by means

of an antibody specifically recognizing the active proteinconformer (6A7). Although no change in the overallprotein levels of Bax was noted upon quercetin treatment(Fig. 2A), an increase in the expression of the active,conformationally changed form of Bax was observed(Fig. 2B, immunoblot data) in quercetin (40 mmol/L)exposed cells. The Bax conformational change appearedas early as 3 hours after addition of quercetin andincreased progressively over the ensuing 9 hours. Flowcytometric analysis (Fig. 2B, flow data) also showed a40% increase in the number of Bax-positive cells (controlwas set to 100%; quercetin treatment group was 140%)following incubation with quercetin (40 mmol/L) for 9hours.

We further examined the intracellular Bax localizationby immunoblot in mitochondrial and cytosolic protein

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Fig. 1. Quercetin (Quer) markedly induces apoptosis in U937 human leukemia cells in dose- and time-dependent manners and is associated withdownregulation of Mcl-1. A, U937 cells were treated with 0, 10, 20, 30, and 40 mmol/L of quercetin for 9 hours. B, the cells were treated with40 mmol/L of quercetin for 0, 1, 2, 4, 6, 9, 12, and 24 hours. For A and B, after treatment the cells were stained with Annexin V/PI, and the percentage ofapoptotic cells was determined using flow cytometry. C, the cells were treated with 0, 10, 20, 30, and 40 mmol/L of quercetin for 6 and 12 hours. D,the cells were treated with 40 mmol/L of quercetin for 0, 1, 2, 4, 6, 9, 12, and 24 hours. After treatment in C and D, immunoblot analysis was done to monitorexpression of Mcl-1, Bcl-2, and Bcl-xL. Blots were subsequently stripped and reprobed with antibody against b-actin to ensure equivalent loading.

Cheng et al.




fractions. As shown in Figure 2C, Bax was found predo-minantly in cytosolic fraction in untreated cells. Incubationwith quercetin induced a redistribution of Bax from cyto-solic to themitochondrial compartment. These results werecorroborated by visualization of immunostaining withFITC-conjugated Bax and confocal imaging (Fig. 2D). Inuntreated cells, Bax was sparsely distributed in the cytosol,whereas upon quercetin treatment, Bax exhibited a morepunctuate pattern and had an overlap with mitochondriathat was stained with MitoTracker Red CMXRos, indicating

a clear shift of the cellular localization of Bax from cytosolto mitochondria. These data suggest that quercetin treat-ment leads to a significant Bax conformational change,accompanied by its translocation to mitochondria. BesidesBax, we also investigated the overall expression of otherproapoptotic Bcl-2 proteins, Bak and Bim, which did notchange following quercetin treatment (data not shown).These findings suggest that activation of Bax may contri-bute to the induction of apoptosis in cells exposed toquercetin.

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Fig. 2.Quercetin (Quer) induces Bax conformational change andmitochondrial translocation in U937 cells. A, U937 cells were treated with 0, 10, 20, 30, and 40mmol/L of quercetin for 6 and 12 hours, after which immunoblot analysis was done to monitor the total levels of Bax. B, U937 cells were treated with40 mmol/L of quercetin for 0, 3, 6, and 12 hours, after which cells were lysed in CHAPS buffer and subjected to IP by using anti-Bax (6A7) and thenimmunoblotted with anti-Bax antibody (N-20). For comparison, the lysate (middle) was loaded with whole-cell lysate. Alternatively, cells were treated with 40mmol/L of quercetin for 9 hours, after which cells were stained with FITC-conjugated anticonfirmationally changed Bax (6A7) and subjected to flowcytometry. This is the representative histogram (solid, control; dotted, quercetin treatment) of flow data. Untreated control was set up as 100%. C, U937 cellswere treated with 0, 10, 20, 30, and 40 mmol/L of quercetin for 9 hours, after which cytosolic and mitochondrial fractions were isolated and subjected toimmunoblot analysis by using an anti-Bax antibody. For Western blot analysis, blots were subsequently stripped and reprobed with an antibody againstb-actin (cytosolic fraction) or Cox IV (mitochondrial fraction) to ensure equivalent loading. D, U937 cells were untreated (a–c) or treated (d–f) with40 mmol/L of quercetin for 9 hours. Bax was stained green with FITC-conjugated anti-Bax antibody (a and d). Mitochondria were stained red with MitoTracker(b and e). c, overlay of (a) and (b). f, overlay of (d) and (e). Colocalization of Bax and mitochondria is shown in yellow.





Quercetin induced lethality in association with Mcl-1downregulation and Bax activation in multipleleukemia cell lines as well as primary human leukemiacells but not in normal human peripheral bloodmononuclear cell

To determine whether quercetin-mediated lethalityobserved in U937 cells also occur in other cell lines, parallelstudies were done in Jurkat and HL-60 cells. As shownin Figure 3A, 9-hour exposure to quercetin resulted in a

dose-dependent cell death in both cell lines. Markedly, arapid decline in Mcl-1 protein levels (Fig. 3B, immunoblotdata) and an obvious increase in active Bax (Fig. 3B, flowdata) were observed in Jurkat and HL-60 cells. Moreover,these leukemia cells exhibited different susceptibilities toquercetin-mediated lethality (Fig. 3A). U937 cells, whichwere the most sensitive of the 3 cell lines, exhibited rela-tively high basic Mcl-1 expression (Fig. 3A, immunoblotdata), most rapid degradation of Mcl-1 (Figs. 1D and 3B),

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Fig. 3.Quercetin (Quer) downregulates Mcl-1 and promotes Bax activation in multiple human leukemia cell lines and primary leukemia blasts but not in normalhuman peripheral blood mononuclear cells. A, U937, Jurkat, and HL-60 cells were exposed to 0, 20, 40, and 60 mmol/L of quercetin for 9 hours, after which thepercentage of apoptotic cells (Annexin V/PI staining) was determined by flow cytometry. Untreated U937, HL-60, and Jurkat cells were lysed andsubjected to immunoblot analysis to detect basic protein levels of Mcl-1 (inset). B, Jurkat and HL-60 cells were treated with 40 mmol/L of quercetin for 0, 2, 4, 8,and 12 hours, after which immunoblot analysis was done to monitor Mcl-1 expression. For Western blot analysis, blots were subsequently stripped andreprobed with an antibody against b-actin to ensure equivalent loading. Alternatively, cells were treated with 40 mmol/L of quercetin for 9 hours,and flow cytometry analysis was done to detect the percentage of cells with active Bax (6A7 positive). Untreated control was set up as 100%. C, mononuclearcells were isolated from the BM (bone marrow) or PM (peripheral blood) of 5 patients with leukemia (designated as 1–5), including 2 AML, 1 MM, and 2 CLLpatients. Cells were then incubated with 0, 20, 40, and 60 mmol/L of quercetin for 9 hours. At the end of this period, the percentage of apoptotic cells(Annexin V/PI staining) was determined by flow cytometry. The blasts were incubated with 40 mmol/L of quercetin and then lysed for immunoblot using Mcl-1primary antibody (patients 2 and 5; inset, left) or analyzed by flow cytometry by using FITC-Bax 6A7 antibody (patient 5; inset, right). For Western blotanalysis, blots were subsequently stripped and reprobed with an antibody against b-actin to ensure equivalent loading. For flow cytometry analysis, untreatedcontrol was set up as 100%. D, mononuclear cells were isolated from the PM of 2 healthy donors. Cells were then incubated with 0, 20, 40, 60, and80 mmol/L of quercetin for 9 hours. At the end of this period, the percentage of apoptotic cells (Annexin V/PI staining) was determined by flow cytometry. Theblasts were incubated with 40 and 80 mmol/L of quercetin and then lysed for immunoblot by using Mcl-1 primary antibody (donor 1; inset, left) oranalyzed by flow cytometry by using FITC-Bax 6A7 antibody (donor 1; inset, right). For Western blot analysis, blots were subsequently stripped and reprobedwith an antibody against b-actin to ensure equivalent loading. For flow cytometry analysis, untreated control was set up as 100%.

Cheng et al.




and most active Bax cells (Figs. 2B and Fig. 3B, flow data),suggesting that levels and turnover rate of Mcl-1 as well asextent of Bax activation may be related to the sensitivity ofleukemia cells to quercetin.Further attempts were made to determine whether quer-

cetin could also trigger cell death in primary human leu-kemia blasts. Parallel experiments were done on primarymononuclear cells isolated from blood or bone marrow of5 leukemia patients [2 acute myeloid leukemia (AML), 1multiple myeloma (MM), and 2 chronic lymphocytic leu-kemia (CLL)]. Treatment with quercetin (0–60 mmol/L) for9 hours resulted in a dose-dependent increase in celldeath in mononuclear cells of all human leukemia types(Fig. 3C). A marked decrease in Mcl-1 expression (Fig. 3C,immunoblot data) and increase in active Bax cells (Fig. 3C,flow data) were also observed in leukemia blasts. Thesefindings indicate that quercetin induces cell lethality inprimary leukemia blasts in association with Mcl-1 down-regulation and Bax activation, analogous to findings incontinuously cultured human leukemia cell lines.In contrast, quercetin exerted little toxicity toward nor-

mal human peripheral blood mononuclear cells (Fig. 3D).Neither Mcl-1 expression or Bax activation was changedupon quercetin treatment.

Treatment with quercetin resulted in a markedinduction of mitochondrial injury and caspaseactivation in human leukemia cells but not in normalhuman peripheral blood mononuclear cellsIt has been reported that quercetin-induced apoptosis is

related to mitochondria-mediated caspase activation (21).We investigated mitochondria alterations as well as caspaseactivation in response to quercetin treatment. For JC-1staining, if mitochondria membrane potential (DYm)decreases, the fluorescence will change from red to green.As shown in Supplementary Figure 1A, there was anobvious shift of fluorescence from red to green after quer-cetin exposure, which indicates the occurrence of themitochondrial membrane depolarization following quer-cetin exposure. Moreover, administration with quercetintriggered a pronounced increase in the release of cyto-chrome c and Smac/Diablo into the cytosolic fraction (S-100), which was noted after 4-hour treatment and becamemore apparent at later exposure intervals (8 and 12 hours;Supplementary Fig. 1B). Furthermore, the caspase cascadewas activated, as determined by the detection of the activeform of caspase-9, -3, and -7, and the proteolytic cleavageof PARP, an endogenous substrate of caspases (Supple-mentary Fig. 1C). These events were readily apparent after8 hours of treatment. The findings show that mitochon-drial injury and caspase activation were detected later thanMcl-1 degradation and Bax activation, indicating the pos-sibility of a primary role for Mcl-1 downregulation and Baxactivation in quercetin-mediated cell death.To confirm the role of caspase cascade, U937 cells were

treated with 40 mmol/L of quercetin in the presence ofpan-caspase inhibitor Z-VAD-FMK at 20 mmol/L. Addi-tion of Z-VAD-FMK significantly diminished the extent of

cell death (data now shown) and abrogated quercetin-induced caspase activation and PARP cleavage (Supple-mentary Fig. 1D), showing the caspase dependence of thecell death phenomena. However, Z-VAD-FMK was inef-fective in preventing downregulation of Mcl-1 protein,suggesting that Mcl-1 reduction is not a consequence ofcaspase process.

In contrast, quercetin had little effects on the mitochon-dria membrane depolarization in normal human periph-eral blood mononuclear cells (Supplementary Fig. 1E), nordid it induce PARP cleavage (Supplementary Fig. 1F).

Overexpression of Mcl-1 substantially attenuatedquercetin-mediated mitochondrial injury, caspaseactivation, and apoptosis

If downregulation of Mcl-1 is responsible for the sub-sequent induction of apoptosis, thenmaintenance of Mcl-1levels is expected to prevent apoptosis. To test this hypoth-esis, studies were conducted by employing U937 cellsstably overexpressing Mcl-1 (30). Enforced Mcl-1 expres-sion decreased lethal effects of quercetin (Fig. 4A), whereasempty vector control U937/pCEP cells were approximatelyas sensitive as parental cells. As shown in Figure 4B, treat-ment with quercetin diminishedMcl-1 expression in U937/pCEP cells but just had a little inhibition on Mcl-1 expres-sion in their U937/Mcl-1 counterparts. Bcl-2 and Bcl-xLexpression in U937/Mcl-1 was roughly equivalent to thosein U937/pCEP cells. Moreover, enforced expression of Mcl-1 blocked quercetin-mediated mitochondrial membranedepolarization (Supplementary Fig. 2A) and cytochrome crelease (Supplementary Fig. 2B). In addition, exposure toquercetin failed to promote caspase-9, -3, and -7 cleavagesand PARP degradation (Supplementary Fig. 2C) in Mcl-1overexpressed U937 cells. To determine whether overex-pression of Bcl-2 and Bcl-xL could compensate the reduc-tion of Mcl-1 at the mitochondrial outer membrane andprevent apoptosis (41), we compared the sensitivity toquercetin of the HL-60/Bcl-2 and HL-60/Bcl-xL cell lineswith the parental HL-60 cell line. Overexpressing Bcl-2 orBcl-xL did not either protect cells from quercetin-mediatedlethality (Supplementary Fig. 2D) or protect Mcl-1 andPARP from degradation (data not shown). These dataindicate that it is the downregulation of Mcl-1 expressionthat may play a specific role in quercetin-induced selectiveapoptosis in leukemia.

Knockdown of Mcl-1 via RNA interference enhancedquercetin-mediated cell death

To further confirm the functional role of Mcl-1 in quer-cetin-mediated apoptosis, RNA interference of Mcl-1 wasemployed to U937 cells. A large reduction in Mcl-1 proteinlevels was detected by immunoblot analysis after U937cells were transfected withMcl-1 siRNA (Fig. 4D). Althoughresidual Mcl-1 expression was very low after Mcl-1 siRNAtransfection, a further decline could be discerned afterquercetin exposure (Fig. 4D), which sensitized U937 cellsto quercetin lethality (P < 0.01, compared with thoseuntreated cells tranfected with Mcl-1 siRNA; see Fig. 4C).





The siRNA transfection had no effect on Bcl-xL proteinlevels, but it decreased Bcl-2 expression in the presence ofquercetin. Taken together, these findings suggest that thereduction in Mcl-1 levels following quercetin exposuremay be a critical factor contributing to the induction ofapoptosis.

Knockdown of Bax substantially diminished thelethality of quercetin and knockout of Bax completelyabrogated quercetin-induced cell death

Our study showed that Bax activation occurred in quer-cetin-treated cells. We therefore tested the role of Bax in thelethality by quercetin. RNA interference was used to knock-down Bax in U937 cells prior to treatment with quercetin.Transfection with Bax siRNA yielded a sharp reduction in

Bax protein level (Fig. 5B). Apoptosis induced by quercetinwas significantly reduced from 59% to 21% by knockdownof Bax (Fig. 5A). The Mcl-1 protein level was unperturbedby Bax siRNA transfection compared with control siRNAtreated cells, and exposure to quercetin resulted in equiva-lent decrease in Mcl-1 expression (Fig. 5B), arguing againstthe possibility that quercetin downregulates Mcl-1 throughBax. We further used HCT116 Baxþ/� and HCT 116 Bax�/�

cells to investigate the role of Bax in quercetin-induced celldeath. Firstly, we compared the cell viability upon querce-tin exposure and found that administration of quercetincould still induce cell death dramatically in Baxþ/� cells butfailed to induce the lethality in Bax�/� cells (Supplemen-tary Fig. 3A). Also, we observed that PARP cleavageoccurred in Baxþ/� cells but not in Bax�/� cells during

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Fig. 4. Ectopic expression of Mcl-1 markedly protects cells from quercetin (Quer)-induced apoptosis, whereas Mcl-1 siRNA transfection renders cells moresusceptible to quercetin-induced apoptosis. U937 cells stably transfected with an empty vector (pCEP) and Mcl-1 construct were obtained.A, cells were treated with 0, 20, 40, and 60 mmol/L of quercetin for 9 hours, after which apoptosis was analyzed using Annexin V/PI assay. *, values for Mcl-1cells were significantly decreased compared with those for pCEP cells after quercetin treatment at concentrations of 40 and 60 mmol/L by ANOVA;P < 0.05. B, cells were treated with 0, 10, 20, 30, and 40 mmol/L of quercetin for 9 hours, after which total cellular extracts were prepared and subjected toWestern blot analysis by using antibodies against Mcl-1, Bcl-2, and Bcl-xL. Blots were subsequently stripped and reprobed with an antibody against b-actin toensure equivalent loading. C and D, U937 cells were transfected with Mcl-1 siRNA or control siRNA and incubated for 24 hours at 37�C, after whichcells were treated with 20 mmol/L of quercetin for additional 9 hours, after which (C) the percentage of apoptotic cells was determined using the Annexin V/PIassay by flow cytometry. *, values for quercetin-treated cells were significantly increased compared with those for untreated cells after transfected with Mcl-1siRNA by ANOVA; P < 0.05. D, cells were lysed and subjected to immunoblot analysis by using antibodies against Mcl-1, Bcl-2, and Bcl-xL.For Western blot analysis, blots were subsequently stripped and reprobed with an antibody against b-actin to ensure equivalent loading.

Cheng et al.




treatment with quercetin (Supplementary Fig. 3B). Thelevel of Mcl-1 and changes of Mcl-1 expression were similarbetween Baxþ/� and Bax�/� cell types. Bcl-2 and Bcl-xLlevels were roughly equivalent in these 2 cell types in theabsence or presence of quercetin (Supplementary Fig. 3B).As Supplementary Fig. 3C showed, in the absence ofquercetin, Mcl-1 siRNA decreased cell survival in Baxþ/�

cells but had no effect on cells lacking Bax, suggesting thatthe presence of Bax is required for Mcl-1 to exert itsantiapoptotic activity. Collectively, these results indicate

that apoptosis induced by quercetin is dependent upon thepresence and activation of Bax.

Mcl-1 inhibited conformational change andtranslocation of Bax without direct interaction

Recent studies suggest that Mcl-1 antiapoptotic activitymay be related to its inactivation of Bax (42), whichprompted our investigation of the regulation of Bax byMcl-1. The effects of Bax by Mcl-1 were addressed usingcells that are either overexpressing or knocking down

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Fig. 5. A and B, downregulation of Bax successfully protects cells from cell death induced by quercetin (Quer). U937 cells were transfected with either BaxsiRNA or control siRNA for 24 hours, after which cells were treated with 40 mmol/L of quercetin for additional 9 hours, after which (A) apoptosis wasdetermined using the Annexin V/PI assay by flow cytometry. *, values for Bax siRNA-treated cells were significantly decreased compared with those for controlsiRNA-treated cells after treatment with quercetin by ANOVA; P < 0.05, (B) total cellular extracts were prepared and subjected to immunoblot analysis usingantibodies against Bax, Mcl-1, and PARP. CF ¼ cleaved fragment. Blots were subsequently stripped and reprobed with an antibody against b-actinto ensure equivalent loading . C–E, Mcl-1 inhibits Bax conformational change and translocation. C, wild-type (WT) U937, U937 stably overexpressing Mcl-1,and U937 transfected with Mcl-1 siRNA cells were treated with 40 mmol/L of quercetin for 9 hours, after which the percentage of 6A7 Bax-positive cells (WTU937 –untreated cells were set up as 100%) was determined by flow cytometry. *, values for U937/Mcl-1 cells were significantly decreased compared withthose for WT U937 cells after treatment with quercetin by ANOVA; P < 0.05. *, values for Mcl-1 siRNA-treated cells were significantly increasedcompared with those for WT U937 cells after treatment with quercetin by ANOVA; P < 0.05. U937/Mcl-1 and its empty vector control (U937/pCEP) cells (D) andU937 transfected with Mcl-1 siRNA or control siRNA cells (E) were treated with 0 and 40 mmol/L of quercetin, after which cytosolic (designated as C) andmitochondrial (designated as M) fractions were prepared and subjected to immunoblot analysis by using an anti-Bax antibody. For Western blotanalysis, blots were subsequently stripped and reprobed with an antibody against b-actin (cytosolic fraction) or Cox IV (mitochondrial fraction) to ensureequivalent loading.





Mcl-1. Wild-type U937 cells treated with quercetin dis-played a rapid increase in conformationally changed Bax(Fig. 5C). In contrast, overexpression of Mcl-1 partiallyreduced Bax conformational change after exposure to quer-cetin (Fig. 5C). Knockdown of Mcl-1 expression by siRNAexhibited an increase in Bax conformational change uponquercetin treatment compared with wild-type cells. Takentogether, these results provide clear evidences that Mcl-1 isa critical determinant of quercetin-mediated Bax confor-mational change.

To test whether Mcl-1 affects the translocation of Bax tomitochondria, U937 cells stably overexpressing Mcl-1 orvector alone were treated with quercetin and the mitochon-dria and cytosol were isolated. As shown in Figure 5D, Bax

underwent a shift from cytosol to mitochondria in U937/pCEP cells whereas enforced expression of Mcl-1 preventedBax translocation from cytosol to mitochondria followingquercetin treatment.On theotherhand, knockdownofMcl-1 by siRNA enhances quercetin-induced Bax translocationto mitochondria (Fig. 5E). These data indicate that the Baxredistribution following quercetin treatment is regulated byMcl-1.

One possible explanation for the inhibition of Bax byMcl-1 is that Bax is sequestered in a Bax/Mcl-1 complex. Totest this possibility, co-immunoprecipitation (co-IP) wascarried out to determine the interaction between these 2proteins. As shown in Supplementary Figure 3D, no directinteraction between Bax and Mcl-1 was detected in either

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Fig. 6. Quercetin (Quer) inhibits tumor growth and induces apoptosis in the xenograft animal model. Six- to 8-week-old nude mice received subcutaneoustransplants of 6 � 106 U937 cells. From the second day, mice were randomized into a control group (6 mice per group) and 2 treated groups(6 mice per group, quercetin 20 and 40 mg/kg). Intraperitoneal administration of quercetin and tumor volume assessment were conducted as described in``Materials and Methods.'' Representative animals with solid tumor volume are shown in (A), tumor volume measured on day 11 and day 16 isshown in (B), body weight is shown in (C), and representative immunohistochemical images for TUNEL staining and percentage of apoptotic cells(TNUNEL-positive cells) in tumor tissue are shown in (D), representative IF images for Mcl-1 expression are shown in (E), and Bax activation using IP (anti-Bax6A7) following immunoblotting (anti-Bax N-20) is shown in (F) in tumor samples. *, values of tumor volume for the quercetin treatment groups weresignificantly decreased compared with those for the nontreatment group by Student's t test; P < 0.05. *, values of TUNEL-positive cells for thequercetin treatment groups were significantly increased compared with those for the nontreatment group by Student's t test; P < 0.05.

Cheng et al.




control or quercetin-treated conditions. It may therefore beconcluded that Mcl-1 inhibits Bax activation through amechanism(s) that does not require a direct interactionbetween these two proteins.

Quercetin exhibited antitumor activity in xenograftsof human leukemia U937 cellsThe in vitro data described earlier prompted us to further

test the anticancer efficacy of quercetin in an in vivomodel,that is, inmice xenografted subcutaneously withU937 cells.Treatment of mice with 20 and 40 mg/kg of quercetinresulted in 53% and 90% inhibition of tumor growthcompared with controls in day 16 (Fig. 6A and B). AsFig. 6C showed, the body weights of the xenograft micewere not significantly variable between different treatmentgroups after 16 days. We further determined apoptotic cellsin tumor tissue by TUNEL assay. Our data showed thatTUNEL-positive apoptotic cells of tumor sections signifi-cantly increased in quercetin (20 and 40 mg/kg)-treatedU937 xenograft mice as compared with the control group[TUNEL-positive cells: control 11.5 � 1.03%; quercetin(20 mg/kg) treatment: 26.7 � 2.03%; quercetin (40 mg/kg) treatment: 56.3 � 4.02%; Fig. 6D). Moreover, Mcl-1expression in tumor samples decreased upon quercetintreatment, as shown by immunofluorescence results(Fig. 6E). Bax activation was observed in quercetin-exposedxenografts (Fig. 6F). Thesedata implicate a therapeutic valueof quercetin in preventing or eradicating tumor growth inxenograft models of human hematologic malignancies.

Discussion

The present study is focused on the tumor-selectiveapoptosis induced by quercetin, a prospective anticancerdrug that has been used in preclinical and small phase Iclinical evaluation (43).In view of the extensive evidence that Mcl-1 plays an

important role in the survival of malignant hematopoieticcells (25, 44, 45), the development of anticancer com-pounds that can diminish Mcl-1 protein levels has been thefocus of intense interest. Because Mcl-1 protein has a shorthalf-life [(30minutes (46, 47)], it is particularly susceptibleto downregulation by agents. Here, we discovered thatquercetin is efficient in killing tumor cells exhibiting rela-tively high levels of Mcl-1. The findings that overexpressionof Mcl-1 diminished quercetin lethality highlight the cen-tral role of Mcl-1 downregulation. This interpretation isfurther supported by the results showing that knockdownof Mcl-1 could enhance quercetin-mediated apoptosis. It isnoteworthy that overexpression of Bcl-2 or Bcl-xL failed toprotect leukemia cells from quercetin-induced cell death,reflecting the important contribution of Mcl-1 downregu-lation to the lethality of this drug. Our unpublished dataindicate that quercetin-induced manganese superoxide dis-mutase downregulation, Akt inactivation as well as atranslational initiation factor eIF4E-mediated translationalmechanism are responsible for this Mcl-1 reduction. There-fore, interventions disabling Mcl-1 may be an optimal way

to kill leukemia cells. It should be noted that Mcl-1 over-expression did not completely block quercetin-induced celldeath, suggesting that elimination of Mcl-1 may be neces-sary but not sufficient to trigger apoptosis, and otherapoptosis-inducing actions might be required for achievingthe full therapeutic effects.

In our study, although the overall Bax protein levels arenot altered during quercetin-induced apoptosis, our dataclearly show that Bax conformational change is one of theearly steps in drug-induced apoptosis. Our data also showthat an increase in Bax translocation leads tomitochondria-mediated caspase activation: mitochondria dysfunctionpromoted by Bax translocation leads to leakage of cyto-chrome c frommitochondria, which subsequently activatescaspase-9 and -3. The importance of Bax in quercetin-treated lethality is confirmed by the observation that knockdown of Bax effectively protects cells from quercetin-mediated cell death and that cells lacking Bax displayedcomplete resistance to quercetin. These results suggest thatthe presence of Bax is essential for quercetin-treated leth-ality and that activation of Bax represents a highly potentapoptotic stimulus in the presence of quercetin.

Mcl-1 has previously been shown to inhibit Bax activa-tion when overexpressed at high levels (36). In accord withthis observation, our findings that enforced expression ofMcl-1 essentially diminishes quercetin-induced Bax activa-tion strongly support that Mcl-1 plays an important role inregulating the function of Bax. Conversely, low levels ofMcl-1 enhance the formation of active Bax following expo-sure to quercetin. These findings are consistent with theresults described by Nijhawan et al. (27) that Mcl-1 oper-ates upstream of Bax and Bcl-xL translocation to the mito-chondria in UV-treated HeLa cells. The inhibition of Bax byMcl-1 in the absence of direct interactions between the 2proteins could occur through several possible mechanisms.One would be that Mcl-1 acts through other multidomainproapoptotic Bcl-2 family proteins, such as active Bak, in anas yet to be defined way. However, in our case, Bakactivation has not been observed. Therefore, this possibilitycan be excluded. A second possibility is that Mcl-1 inhibitsBax through a process involving the Bax ‘‘activator.’’ BH3-only proteins, such as Bid or Bim, could be possible targetsfor Mcl-1 in this regard (48). Very recent studies from ourgroup have uncovered a novel mechanism of quercetin-mediated Bax activation that involves Bid cleavage (unpub-lished data).

It has been reported that administration of quercetineliminates colorectal cancer xenografts (49) as well ashuman breast cancer MDA-MB-435 cells xenografts (50).In our in vivo studies using a nude mice U937 xenograftmodel, tumor volumes were reduced compared with con-trols after quercetin treatment, indicating an antileukemiaactivity of this compound. To further validate the apoptoticmechanism found in vitro, we next examined the TUNELstaining, Mcl-1 expression, and Bax activation in tumorspecimens obtained from control and quercetin-treatedanimals. The increase in TUNEL-positive cells and Baxactivation as well as the decrease in Mcl-1 expression were





detected in the quercetin-treated xenografts compared withthe control group. To the best of our knowledge, this is thefirst report that describes an effective extrapolation of the invitro apoptosis-inducing effects of quercetin on the leuke-mia cells to the in vivo situation.

In summary, the present findings indicate that quercetineffectively induces cell death in human leukemia cells,including primary leukemia blasts, as well as in leukemiaxenografts. This effect occurs in association with the rapiddownregulation of Mcl-1 and is accompanied by the con-formational change and translocation of Bax, which aredependent on Mcl-1 reduction. The potent antileukemiaactivity of quercetin found both in vitro and in vivo in thisstudy along with the novel mode of action make thiscompound an attractive antitumor agent for hematologicmalignancies. In addition, this work also identifies Mcl-1and Bax as potential biomarkers of quercetin activity thatdirectly relate to its mechanism of action. Such biomarkersmay be useful to show the mode of action of quercetin invivo, using patient biopsy samples. Further efforts are

warranted to elucidate the mechanism(s) by which quer-cetin inhibits Mcl-1, as well as to identify other possiblefactors that contribute to Bax activation during quercetintreatment. This study could provide a better understandingof how this compound exerts its antitumor activity in vivoand aid in developing this compound either aloneor in combination with established chemotherapeuticagents to treat leukemia and potentially other hematologicmalignancies.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

NIH grant RO1 ES015375 (X.S.).

Received 06/16/2010; revised 09/22/2010; accepted 10/08/2010; pub-lished online 12/07/2010.

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2010;16:5679-5691. Clin Cancer Res Senping Cheng, Ning Gao, Zhuo Zhang, et al. Downregulation of Mcl-1 and Activation of BaxQuercetin Induces Tumor-Selective Apoptosis through

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