Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Dec 1.
Published in final edited form as: Clin Cancer Res. 2010 Dec 1;16(23):5679–5691. doi: 10.1158/1078-0432.CCR-10-1565

Quercetin induces tumor-selective apoptosis through down-regulation of Mcl-1 and activation of Bax

Senping Cheng 1, Ning Gao 1, Zhuo Zhang 1, Gang Chen 2, Amit Budhraja 1, Zunji Ke 2, Young-ok Son 1, Xin Wang 1, Jia Luo 2, Xianglin Shi 1
PMCID: PMC3069720  NIHMSID: NIHMS246540  PMID: 21138867

Abstract

Purpose

To investigate the in vivo antitumor efficacy of querctin in U937 xenografts and the functional role of Mcl-1 and Bax in quercetin-induced apoptosis in human leukemia cells.

Experimental Design

Leukemia cells were treated with quercetin, after which apoptosis, Mcl-1 expression, and Bax activation and translocation were evaluated. The efficacy of quercein, as well as Mcl-1 expression and Bax activation were investigated in xenografts of leukemia 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 down-regulation and Bax conformational change and mitochondrial translocation which triggered cytochrome c release. Knockdown of Bax by siRNA reversed querctin-induced apoptosis. Knockout of Bax 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 down-regulation. 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 down-regulation and Bax activation were 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 down-regulation, which in turn potentiates Bax activation and mitochondrial translocation, culminating in apoptosis.

Keywords: Apoptosis, Leukemia, Quercetin, Mcl-1, Bax

Introduction

The natural product quercetin (3,5,7,3’,4’-pentahydroxy-flavone) is a widely distributed flavonoid that is present in many fruits and vegetables (e.g., onions and apples). Previous research has shown that quercetin induces apoptosis in a variety of tumors (1–3), including leukemia (4). However, the molecular mechanisms of quercetin-induced apoptosis are unclear. There is no available information concerning quercetin’s in vivo efficacy against leukemia.

Apoptosis involves two distinct pathways, one engaging death receptor-initiated extrinsic pathway and the other involving mitochondria-mediated intrinsic pathway (5). The intrinsic pathway involves the release of pro-apoptotic proteins (e.g., cytochrome c) into the cytosol, formation of the apoptosome, and activation of caspase-9 (6–7), which subsequently cleaves and activates pro-caspase-3 (8). The mitochondrial pathway is mainly regulated by Bcl-2 family proteins (9–11). Anti-apoptotic Bcl-2 family proteins (e.g., Bcl-2, Bcl-xL, and Mcl-1) antagonize membrane permeabilization and prevent the release of cytochrome c from mitochondria (12). Pro-apoptotic Bcl-2 family proteins can be further divided into two subgroups. The multi-domain pro-apoptotic proteins (e.g., Bax and Bak) participate in the formation of mitochondrial pore through which cytochrome c releases (13–16). The BH3-only proteins (e.g., Bim and Bid) are required for activation of multi-domain pro-apoptotic proteins, through association of anti-apoptotic Bcl-2 proteins (17–18). It is well known that quercetin-mediated cell apoptosis involves mitochondria-mediated caspase activation (1, 4, 19–22).

Notably, Mcl-1 is a highly expressed anti-apoptotic protein (23) implicated in malignant hematopoietic survival (23–24). It has been shown that depletion of Mcl-1 using antisense oligonucleotides rapidly triggers apoptosis in U937 cells (25). In contrast, selective expression of Mcl-1 in hematopoietic tissues of transgenic mice promotes the survival of hematopoietic cells and enhances the outgrowth of myeloid cell lines (26). Furthermore, over-expression of Mcl-1 protects cells from apoptosis induced by a variety of agents, including UV, etoposide, staurosporine, actinomycin D, and others (27–30). Two groups (4, 31) have indicated a decrease of Mcl-1 level in quercetin-treated cells.

It has been proposed that alteration of Bax conformation and its redistribution to mitochondria play a key role in the induction of cell death (32–33). In healthy cells, Bax is predominantly located in the cytoplasm. Upon apoptotic signals, Bax undergoes a conformational change that exposes the N-terminus and the hydrophobic C-terminus that targets mitochondria (34–35). The membrane insertion of Bax is essential for the release of cytochrome c and apoptosis (36–37). It has been demonstrated that quercetin is able to induce apoptosis in multiple cancer cells through up-regulation of Bax expression (19–20, 22, 38). It has also been reported that apoptotic process caused by quercetin are mediated by the dissociation of Bax from Bcl-xL in human prostate cancer cells (39). Granado-Serrano et al. have provided evidences indicating that quercetin promotes translocation of Bax to mitochondria membrane in human hepatoma cells (1).

The present study shows that quercetin has an anti-cancer ability by inhibition of xenografts growth of U937 cells. Our study also demonstrates an increase of apoptosis in human leukemia cells and tumor sections upon quercetin treatment. In addition, our results indicate that this phenomenon stems from a novel mechanism involving two levels of cooperation between Bcl-2 family proteins: (1) quercetin mediates Mcl-1 down-regulation and activates Bax; and (2) Mcl-1 regulates quercetin-mediated Bax activation.

Materials and methods

Cells

Human leukemia U937, Jurkat, and HL-60 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS), L-glutamine and antibiotics. U937 cells stably over-expressing Mcl-1 and their empty vector counterpart (pCEP) were kindly provided by Dr. Ruth Craig (Dartmouth Medical School, Hanover, NH). HL-60 cells stably over-expressing Bcl-2 (HL-60/Bcl-2) and Bcl-xL (HL-60/Bcl-xL) were kindly provided by Dr. Ming Ding (The National Institute for Occupational Safety and Health, Morgantown, WV). Mononuclear cells were isolated from peripheral blood or bone marrow of leukemia patients or healthy donors were purchased from AllCells, LLC. (Emeryville, CA). Mononuclear cells were suspended in RPMI1640 medium containing 10% fetal calf serum at 8 × 105 /mL for treatment. Bax+/− and Bax−/− human colon cancer HCT116 cells were kindly provided by Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD) & Kenneth W. Kinzler (Howard Hughes Medical Institute, Chevy Chase, MD) and sustained in MyCoy’s 5A medium containing 10% FBS and antibiotics.

Chemicals and reagents

Quercetin (>99% pure) was purchased from Sigma Chemical Co. (St. Louis, MO), dissolved in DMSO, aliquoted, and stored at −20°C. The pan-caspase inhibitor Z-VAD-FMK was purchased from EMD Biosciences (La Jolla, CA).

Assessment of apoptosis

The extent of apoptosis in leukemia cells was evaluated by flow cytometric analysis using FITC conjugated Annexin V/ propidium iodide (PI) (BD PharMingen, San Diego, CA) staining as per the manufacturer’s instructions as previously described (40). Both early apoptotic (Annexin V-positive, PI-negative) and late apoptotic (Annexin V-positive and PI-positive) cells were included in cell death determinations.

Western blot assay

Cells were lysed and sonicated in 1 × NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA). The protein concentration was measured using Coomassie Protein Assay Reagent (Pierce, Rockford, IL) and 30 µg of sample proteins were separated by SDS-PAGE, and incubated with antibodies. The blots were then reprobed with an antibody against β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) or Cox IV (Cell signaling, Beverly, MA) to ensure equal loading of proteins. Primary antibodies used were as follows: cytochrome c (Santa Cruz Biotechnology); Cleaved-caspase-3, Cleaved-capase-9, Bcl-xL (Cell Signaling, Beverly, MA); Mcl-1 and Bax (N-20) (BD PharMingen); PARP (Biomol Research Laboratories, Plymouth Meeting, PA); Bcl-2 (DAKO, Carpinteria, CA). Secondary antibodies conjugated to horseradish peroxidase were obtained from Kirkegaard and Perry Laboratories, Inc (Gaithersburg, MD).

Subcellular fractionation

For cytosol isolation, cells were lysed in lysis buffer (75mM NaCl, 8 mM Na2HPO4, 1 mM NaH2PO4, 1 mM EDTA, and 350 µg/mL digitonin) and needle strokes. After sequential centrifugation (1,000 × g to pellet nuclei, 10,000 × g to pellet membrane fraction, and 100,000 × g), the supernatant (S-100, cytosolic fraction) were collected and subjected to immunoblot. For mitochondrial and cytosol fractionations, cells (50 × 106) were fractionated by Mitochondrial Fractionation Kit (Active Motif, Carlsbad, CA) as per manufacturer’s instructions.

Analysis of Bax conformational change

To analyze the conformational change of Bax by flow cytometry, cells were fixed and permeabilized using FIX & PERM Cell Permeabilization Reagents (Caltag Lab, Burlingame, CA) as per manufacturer’s instructions. Cells were then incubated with FITC-conjugated anti-Bax (clone 6A7) (Santa Cruz Biotechnology), and then analyzed by flow cytometry. The results for each condition were calibrated by values for cells stained with mouse IgG (Santa Cruz Biotechnology) as the primary antibody. Values for untreated controls were arbitrarily set to 100%. For analysis of Bax conformational change by immunoprecipitation, cells were lysed in CHAPS lysis buffer and 500 µg of total lysates were immunoprecipitated using anti-Bax 6A7 antibody. Resulting immune complexes were analyzed by immunoblotting with anti-Bax antiserum (N-20).

Immunofluorescence

To analyze cellular distribution of Bax and Cytochrome c, cells were fixed, permeabilized and probed with FITC-conjugated anti-Bax or Alexa Fluor 594 conjugated anti-Cytochrome c (both from Santa Crua Biotechnology). Mitochondria were stained with 300 nM MitoTracker (Molecular Probes, Eugene, OR). Cells were plated on 35 mm collagen coated glass-bottomed dishes (MatTek Corportation, Ashland, MA) and visualized using an inverted Leica TCS SP5 laser scanning confocal microscope under an 60× oil immersion objective.

RNA interference

U937 cells (1.5 × 106) were transiently transfected with 10 nM siRNA specific for Mcl-1 or Bax (Santa Cruz Biotechnology) using Amaxa Nucleofector™ device (program V-001) with Kit V (Amaxa GmbH, Cologne, Germany) as per manufacturer’s instructions. After 24 h of transfection, cells were treated and analyzed for protein expression, apoptosis or viability.

Therapeutic effect evaluation of quercetin in xenograft model

The 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, Wilmington, MA) were housed in a specific pathogen-free room within the animal facilities at the University of Kentucky, Chandler Medical Center. Animals were allowed to acclimatize to their new environment 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 re-suspended in serum-free RPMI 1640 medium with Matrigel basement membrane matrix (BD Biosciences, Bedford, MA) at a 1:1 ratio (total volume 100 µL), then were subcutaneously injected into the flanks of nude mice. From the second day of injection, mice were randomly assigned to three treatment groups (n=6 for each group) and administrated intraperitoneally (i.p.) with quercetin (0, 20 and 40 mg/kg body weight) in 150 µL of DMSO/0.9% physiological saline (1:0.5) daily for consecutive 15 days. Body weight and tumor mass were measured every 5 days. Tumor volumes was determined by a caliper and calculated according to the formula (width2×length)/2. All animals were sacrificed immediately if tumor volume reached an approximate volume of 1500 mm3 (Day 16).

Apoptosis detection by TUNEL, Mcl-1 expression and Bax activation in tumor tissue sections

Tumor tissue sections of formalin-fixed, paraffin-embedded specimens were dewaxed in xylene and rehydrated in a graded series of ethanol. Apoptosis was detected using the TUNEL in situ apoptosis detection kit (DeadEnd™ Fluorometric TUNEL System, Promega, Madison, WI). Briefly, tumor samples were incubated with proteinase K (2 mg/ml), and the TUNEL staining was performed according to the manufacturer’s instructions. The percentage of TUNEL positive cells relative to total cells was calculated for each sample under a fluorescent microscope, counting at least 200 cells. Mcl-1 expression in tumor samples was analyzed by immunofluorescence using primary anti-Mcl-1 antibody (Abcam, Cambridge, MA) following FITC-conjugated anti-mouse secondary antibody (Invitrogen). Tumor samples were homogenized and lysed in CHAPS lysis buffered and immunoprecipitated using anti-Bax 6A7 antiboy. Resulting immune complexes were analyzed by immunoblotting with anti-Bax antiserum (N-20).

Statistical analysis

The values were presented as means ± SD. Two-way analysis of variance (ANOVA) and Student’s t-test were used for statistical analysis. P < 0.05 was considered significantly different.

Results

Exposure to quercetin led to pronounced apoptosis in U937 cells, associated with decrease in Mcl-1 expression

A dose-response analysis of U937 cells revealed a moderate increase in apoptosis 9 h after exposure to quercetin at concentration of 10–20 µM and extensive apoptosis at concentrations ≥ 30 µM (Figs. 1A and 3A). A time-course study of cells exposed to 40 µM of quercetin demonstrated a marked increase in apoptosis as early as 2 h after drug exposure and reached near-maximal levels after 9 h (Fig. 1B).

Figure 1.

Figure 1

Open in a new tab

Quercetin markedly induces apoptosis in U937 human leukemia cells in dose-and time-dependent manners, which is associated with down-regulation of Mcl-1. (A) U937 cells were treated with 0, 10, 20, 30, and 40 µM of quercetin for 9 h. (B) The cells were treated with 40 µM of quercetin for 0, 1, 2, 4, 6, 9, 12, and 24 h. For A and B, after treatment the cells were stained with annexin V/propidium iodide (PI), and the percentage of apoptotic cells was determined using flow cytometry. (C) The cells were treated with 0, 10, 20, 30, and 40 µM of quercetin for 6 h and 12 h. (D) The cells were treated with 40 µM quercetin for 0, 1, 2, 4, 6, 9, 12, and 24 h. After treatment in C and D, immunoblot analysis was done to monitor expression of Mcl-1, Bcl-2, and Bcl-xL. Blots were subsequently stripped and reprobed with antibody against β-actin to ensure equivalent loading.

Figure 3.

Figure 3

Open in a new tab

Quercetin down-regulates Mcl-1 and promotes Bax activation in multiple human leukemia cell lines and primary leukemia blasts, but not in normal human peripheral blood mononuclear cells. (A) U937, Jurkat, and HL-60 cells were exposed to 0, 20, 40, and 60 µM of quercetin for 9 h, after which the percentage of apoptotic cells (Annexin V/PI staining) was determined by flow cytometry. Untreated U937, HL-60, and Jurkat cells were lysed and subjected to immunoblot analysis to detect basic protein levels of Mcl-1 (inset). (B) Jurkat and HL-60 cells were treated with 40 µM of quercetin for 0, 2, 4, 8, and 12 h, after which immunoblot analysis was done to monitor Mcl-1 expression. For Western blot analysis blots were subsequently stripped and reprobed with an antibody against β-actin to ensure equivalent loading. Alternatively, cells were treated with 40 µM of quercetin for 9 h, and flow cytometry analysis was performed to detect the percentage of cells with active Bax (6A7 positive). Untreated control was set up as 100%. (C) Mononuclear cells were isolated from the BM (bone marrow) or PM (peripheral blood) of five leukemia patients (designated as # 1–5), including two AML (acute myeloid leukemia), one MM (multiple myeloma), and two CLL (chronic lymphocytic leukemia) patients. Cells then incubated with 0, 20, 40, and 60 µM of quercetin for 9 h. 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 µM of quercetin and then lysed for immunoblot using Mcl-1 primary antibody (Patient #2 and Patient #5) (inset, left) or analyzed by flow cytometry using FITC-Bax 6A7 antibody (Patient #5) (inset, right). For Western blot analysis blots were subsequently stripped and reprobed with an antibody against β-actin to ensure equivalent loading. For flow cytometry analysis, untreated control was set up as 100%. (D) Mononuclear cells were isolated from the PM of two healthy donors. Cells then incubated with 0, 20, 40, 60 and 80 µM of quercetin for 9 h. 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 and 80 µM of quercetin and then lysed for immunoblot using Mcl-1 primary antibody (Donor #1) (inset, left) or analyzed by flow cytometry using FITC-Bax 6A7 antibody (Donor #1) (inset, right). For Western blot analysis blots were subsequently stripped and reprobed with an antibody against β-actin to ensure equivalent loading. For flow cytometry analysis, untreated control was set up as 100%.

Expression of anti-apoptotic Bcl-2 family proteins in U937 cells was monitored following treatment with quercetin. As shown in Fig. 1C, a marked dose-dependent decrease of Mcl-1 expression was observed in quercetin-treated cells. Exposure of U937 cells to 40 µM of quercetin at varying intervals resulted in a rapid down-regulation of Mcl-1 that was detectable as early as 2 h, and by 4 h of treatment expression was almost absent (Fig. 1D). In contrast, the other two important anti-apoptotic Bcl-2 family proteins Bcl-2 or Bcl-xL expression remained unaffected. Together, these findings demonstrate that quercetin induces rapid and dose-dependent down-regulation of Mcl-1 in U937 cells.

Quercetin induced Bax conformational change and mitochondrial translocation in U937 cells

Following death stimuli, Bax undergoes an N-terminal conformational change which can be detected by means of an antibody specifically recognizing the active protein conformer (6A7). Although no change in the overall protein levels of Bax was noted upon quercetin treatment (Fig. 2A), a dramatic increase in the expression of the active, conformationally changed form of Bax was observed (Fig. 2B, immunoblot data) in quercetin (40 µM) exposed cells. The Bax conformational change appeared as early as 3 h after addition of quercetin, and increased progressively over the ensuing 9 h. Flow cytometry analysis (Fig 2B, flow data) also showed a 40% increase in the number of Bax-positive cells (control was set to 100%, quercetin treatment group was 140%) following incubation with quercetin (40 µM) for 9 h.

Figure 2.

Figure 2

Open in a new tab

Quercetin induces Bax conformational change and mitochondrial translocation in U937 cells. (A) U937 cells were treated with 0, 10, 20, 30, and 40 µM of quercetin for 6 h and 12 h, after which immunoblot analysis was done to monitor the total levels of Bax. (B) U937 cells were treated with 40 µM of quercetin for 0, 3, 6, and 12 h, after which cells were lysed in CHAPS buffer and subjected to immunoprecipitation (IP) using anti-Bax (6A7) and then immunoblotted with anti-Bax antibody (N-20). For comparison, the lower panel (designated as Lysate) was loaded with whole-cell lysate. Alternatively, cells were treated with 40 µM of quercetin for 9 h, after which cells were stained with FITC conjugated anti-confirmationally changed Bax (6A7) and subjected to flow cytometry. This is the representative histogram (solid, control; dotted, quercetin treatment) of flow data. Untreated control was set up as 100%. (C) U937 cells were treated with 0, 10, 20, 30, and 40 µM of quercetin for 9 h, after which cytosolic and mitochondrial fractions were isolated and subjected to immunoblot analysis using an anti-Bax antibody. For Western blot analysis blots were subsequently stripped and reprobed with an antibody against β-actin (cytosolic fraction) or Cox IV (mitochondrial fraction) to ensure equivalent loading. (D) U937 cells were untreated (panels a–c) or treated (panels d–f) with 40 µM of quercetin for 9 hours. Bax was stained green with FITC conjugated anti-Bax antibody (panel a and d). Mitochondria were stained red with MitoTracker (panel b and e). Panel (c) is the overlay of panels (a) and (b). Panel (f) is the overlay of panels (d) and (e). Co-localization of Bax and mitochondria is shown as yellow.

We further examined the intracellular Bax localization by immunoblot in mitochondrial and cytosolic protein fractions. As shown in Fig. 2C, Bax was found predominantly in cytosolic fraction in untreated cells. Incubation with quercetin induced a redistribution of Bax from cytosolic to the mitochondrial compartment. These results were corroborated by visualization of immunostaining with FITC conjugated Bax and confocal imaging (Fig. 2D). In untreated cells, Bax was sparsely distributed in the cytosol, whereas upon quercetin treatment Bax exhibited a more punctuate pattern and had an overlap with mitochondria which was stained with Mito Tracker Red CMXRos, indicating a clear shift of the cellular localization of Bax from cytosol to mitochondria. The above data suggests that quercetin treatment leads to a significant Bax conformational change, accompanied by its translocation to mitochondria. Besides Bax, we also investigated the overall expression of other pro-apoptotic Bcl-2 proteins, Bak and Bim. The expression of Bak and Bim did not change following quercetin treatment (data not shown). These findings suggest that activation of Bax may contribute to the induction of apoptosis in cells exposed to quercetin.

Quercetin induced lethality in association with Mcl-1 down-regulation and Bax activation in multiple leukemia cell lines as well as primary human leukemia cells, but not in normal human peripheral blood mononuclear cells

To determine whether quercetin-mediated lethality observed in U937 cells also occur in other cell lines, parallel studies were performed in Jurkat and HL-60 cells. As shown in Fig. 3A, 9 h exposure to quercetin resulted in a dose-dependent cell death in both cell lines. Markedly, a rapid decline in Mcl-1 protein levels (Fig. 3B, immunoblot data) and an obvious increase in active Bax (Fig. 3B, flow data) were observed in Jurkat and HL-60 cells. Moreover, these leukemia cells exhibited different susceptibilities to quercetin-mediated lethality (Fig. 3A). U937 cells, which were the most sensitive of the three cell lines, exhibited relatively high basic Mcl-1 expression (Fig. 3A immunoblot data), most rapid degradation of Mcl-1 (Figs. 1D and 3B), and most active Bax cells (Figs. 2B and Fig. 3B flow data), suggesting that levels and turnover rate of Mcl-1 as well as extent of Bax activation may be related to the sensitivity of leukemia cells to quercetin.

Further attempts were made to determine whether quercetin is also able to trigger cell death in primary human leukemia blasts. Parallel experiments were performed on primary mononuclear cells isolated from blood or bone marrow of five leukemia patients (two acute myeloid leukemia (AML), one multiple myeloma (MM), and two chronic lymphocytic leukemia (CLL)). Treatment with quercetin (0–60 µM) for 9 h resulted in a dose-dependent increase in cell death 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. These findings indicate that quercetin induces cell lethality in primary leukemia blasts in association with Mcl-1 down-regulation and Bax activation, analogous to findings in continuously cultured human leukemia cell lines.

In contrast, quercetin exerted little toxicity toward normal human peripheral blood mononuclear cells (Fig. 3D). Neither the expression of Mcl-1 or Bax activation was changed upon quercetin treatment.

Treatment with quercetin resulted in a marked induction of mitochondrial injury and caspase activation in human leukemia cells, but not in normal human peripheral blood mononuclear cells

It has been reported that quercetin induced apoptosis is related to mitochondria-mediated caspase activation (21). We investigated mitochondria alterations as well as caspase activation in response to quercetin treatment. For JC-1 staining, if mitochondria membrane potential (ΔΨm) decreases, the fluorescence will change from red to green. As shown in Supplementary Fig. 1A, there was an obvious shift of fluorescence from red to green after quercetin exposure, which indicates the occurrence of the mitochondrial membrane depolarization following quercetin exposure. Moreover, administration with quercetin triggered a pronounced increase in release of cytochrome c and Smac/Diablo into the cytosolic fraction (S-100), which was noted after 4 h treatment and became more apparent at later exposure intervals (8 and 12 h; Supplementary Fig. 1B). Furthermore, the caspase cascade was activated, as determined by the detection of the active form of caspase-9, -3 and -7, and the proteolytic cleavage of PARP, an endogenous substrate of caspases (Supplementary Fig 1C). These events were readily apparent after 8 h of treatment. The findings show that mitochondrial injury and caspase activation were detected later than Mcl-1 degradation and Bax activation, indicating the possibility of a primary role for Mcl-1 down-regulation and Bax activation in quercetin-mediated cell death.

To confirm the role of caspase cascade, U937 cells were treated with 40 µM of quercetin in the presence or absence of pan-caspase inhibitor Z-VAD-FMK at 20 µM. Addition of Z-VAD-FMK significantly diminished the extent of cell death (data now shown) and abrogated quercetin-induced caspase activation, and PARP cleavage (Supplementary Fig. 1D), demonstrating the caspase dependence of the cell death phenomena. However, Z-VAD-FMK was ineffective in preventing down-regulation of Mcl-1 protein, suggesting that Mcl-1 reduction is not a consequence of caspase process.

In contrast, quercetin had little effects on the mitochondria membrane depolarization in normal human peripheral blood mononuclear cells (Supplementary Fig. 1E), nor did it induce PARP cleavage (Supplementary Fig. 1F).

Over-expression of Mcl-1 substantially attenuated quercetin-mediated mitochondrial injury, caspase activation, and apoptosis

If down-regulation of Mcl-1 is responsible for the subsequent induction of apoptosis, then maintenance of Mcl-1 levels is expected to prevent apoptosis. To test this hypothesis, studies were done by employing U937 cells stably over-expressing Mcl-1 (30). Enforced Mcl-1 expression decreased lethal effects of quercetin (Fig. 4A), whereas empty vector controls (pCEP) U937 cells were approximately as sensitive as parental cells. As shown in Fig. 4B, treatment with quercetin diminished Mcl-1 expression in U937/pCEP cells but failed to do so in their U937/Mcl-1 counterparts. Bcl-2 and Bcl-xL expression in U937/Mcl-1 were roughly equivalent to those in U937/pCEP cells. Moreover, enforced expression of Mcl-1 blocked quercetin-mediated mitochondrial membrane depolarization (Supplementary Fig. 2A) and cytochrome c release (Supplementary Fig. 2B). In addition, exposure to quercetin failed to promote caspase-9, -3 and -7 cleavages and PARP degradation (Supplementary Fig. 2C) in Mcl-1 over-expressed U937 cells. To determine whether over-expression of Bcl-2 and Bcl-xL could compensate the reduction of Mcl-1 at the mitochondrial outer membrane and prevent apoptosis (41), we compared the sensitivity to quercetin of the HL-60/Bcl-2 and HL-60/Bcl-xL cell lines to the parental HL-60 cell line. Over-expressing Bcl-2 or Bcl-xL did not either protect cells from quercetin-mediated lethality (Supplementary Fig. 2D) or protect Mcl-1 and PARP from degradation (data not shown). These data indicate that it is the down-regulation of Mcl-1 expression that may play a major role in lowering the threshold of apoptosis and eventually inducing it.

Figure 4.

Figure 4

Open in a new tab

Ectopic expression of Mcl-1 markedly protects cells from quercetin-induced apoptosis, while Mcl-1 siRNA transfection renders cells more susceptible to quercetin-induced apoptosis. U937 cells stably transfected with an empty vector (pCEP) and Mcl-1 construct were performed. (A) Cells were treated with 0, 20, 40, and 60 µM of quercetin for 9 h, after which apoptosis was analysed using Annexin V/PI assay. *Values for Mcl-1 cells were significantly decreased compared to those for pCEP cells after quercetin treatment at concentrations of 40 and 60 µM by ANOVA; p < 0.05. (B) Cells were treated with 0, 10, 20, 30, and 40 µM of quercetin for 9 h, after which total cellular extracts were prepared and subjected to Western blot analysis using antibodies against Mcl-1, Bcl-2, and Bcl-xL. Blots were subsequently stripped and reprobed with an antibody against β-actin to ensure equivalent loading. (C) and (D) U937 cells were transfected with Mcl-1 siRNA or Control siRNA and incubated for 24 h at 37°C, after which cells were treated with 20 µM of quercetin for additional 9 h, after which (C) the percentage of apoptotic cells was determined using the Annexin V/PI assay by flow cytometry. *Values for quercetin-treated cells were significantly increased compared to those for untreated cells after transfected with Mcl-1 siRNA by ANOVA; p < 0.05. (D) cells were lysed and subjected to immunoblot analysis using antibodies against Mcl-1, Bcl-2, and Bcl-xL. For Western blot analysis blots were subsequently stripped and reprobed with an antibody against β-actin to ensure equivalent loading.

Knockdown of Mcl-1 via RNA interference enhanced quercetin-mediated cell death

To further confirm the functional role of Mcl-1 in quercetin-mediated apoptosis, RNA interference of Mcl-1 was employed to U937 cells. A great reduction in Mcl-1 protein levels was detected by immunoblot analysis after U937 cells were transfected with Mcl-1 siRNA (Fig. 4D). Although residual Mcl-1 expression was very low after Mcl-1 siRNA transfection, a further decline could be discerned after quercetin exposure (Fig. 4D), which sensitized U937 cells to quercetin lethality (p < 0.01, compared with those untreated cells tranfected with Mcl-1 siRNA, see Fig. 4C). The siRNA transfection had no effect on Bcl-xL protein levels, but it decreased Bcl-2 expression in the presence of quercetin. Taken together, these findings suggest that the reduction in Mcl-1 levels following quercetin exposure may be a critical factor contributing to the induction of apoptosis.

Knockdown of Bax substantially diminished the lethality of quercetin, and knockout of Bax completely abrogated quercetin-induced cell death

Our study showed that Bax activation occurred in quercetin-treated cells. We therefore tested the role of Bax in the lethality by quercetin. RNA interference was used to knockdown Bax in U937 cells prior to treatment with quercetin. Transfection with Bax siRNA yielded a sharp reduction of Bax protein level (Fig. 5B). Apoptosis induced by quercetin was significantly reduced from 59% to 21% by knockdown of Bax (Fig. 5A). The Mcl-1 protein level was unperturbed by Bax siRNA transfection compared with control siRNA treated cells, and exposure to quercetin resulted in equivalent decrease in Mcl-1 expression (Fig. 5B), arguing against the possibility that quercetin down-regulates Mcl-1 through Bax. We further used HCT116 Bax+/− and HCT 116 Bax−/− cells to investigate the role of Bax in quercetin-induced cell death. Firstly, we compared the cell viability upon quercetin exposure and found that administration of quercetin was still able to induce cell death dramatically in Bax+/− cells, but failed to increase the lethality in Bax−/− cells (Supplementary Fig. 3A). Also, we observed that PARP cleavage occurred in Bax +/− cells, but not in Bax −/− cells during treatment with quercetin (Supplementary Fig. 3B). The level of Mcl-1 and changes of Mcl-1 expression were similar between Bax+/− and Bax−/ − cell types. Bcl-2 and Bcl-xL levels were roughly equivalent in these two cell types in the absence or presence of quercetin (Supplementary Fig. 3B). As Supplementary Fig. 3C showed, in the absence of quercetin, Mcl-1 siRNA decreased cell survival in Bax+/− cells but had no effect on cells lacking Bax, suggesting that the presence of Bax is required for Mcl-1 to exert its anti-apoptotic activity. Collectively, these results indicate that apoptosis induced by quercetin is dependent upon the presence and activation of Bax.

Figure 5.

Figure 5

Open in a new tab

(A) and (B) Down-regualation of Bax successfully protects cells from cell death induced by quercetin. U937 cells were transfected with either Bax siRNA or Control siRNA for 24 h, after which cells were treated with 40 µM of quercetin for additional 9 h., after which (A) apoptosis was determined using the Annexin V/PI assay by flow cytometry. *Values for Bax siRNA-treated cells were significantly decreased compared to those for Control siRNA-treated cells after treatment with quercetin by ANOVA; p < 0.05, (B) total cellular extracts were prepared and subjected to immunoblot analysis using antibodies against Bax, Mcl-1, and PARP. Blots were subsequently stripped and reprobed with an antibody against β-actin to ensure equivalent loading. (C), (D) and (E) Mcl-1 inhibits Bax transformational change and translocation. (C) WT U937, U937 stably-over-expressing Mcl-1, and U937 transfected with Mcl-1 siRNA cells were treated with 40 µM quercetin for 9 h, after which the percentage of 6A7 Bax positive cells (U937 WT untreated cells were set up as 100%) was determined by flow cytometry. *Values for U937/Mcl-1 cells were significantly decreased compared to those for U937 WT cells after treatment with quercetin by ANOVA; p < 0.05. *Values for Mcl-1 siRNA-treated cells were significantly increased compared to those for U937 WT cells after treatment with quercetin by ANOVA; p < 0.05. U937/Mcl-1 and its empty-vector control (U937/pCEP) cells (D) and U937 transfected with Mcl-1 siRNA or Control siRNA cells (E) were treated with 0 and 40 µM of quercetin, after which cytosolic (designated as C) and mitochondrial (designated as M) fractions were prepared and subjected to immunoblot analysis using an anti-Bax antibody. For western blot analysis, blots were subsequently stripped and reprobed with an antibody against β-actin (cytosolic fraction) or Cox IV (mitochondrial fraction) to ensure equivalent loading.

Mcl-1 inhibited conformational change and translocation of Bax without direct interaction

Recent studies suggest that Mcl-1 anti-apoptotic activity may be related to its inactivation of Bax (42), which prompted our investigation of the regulation of Bax by Mcl-1. The effects of Bax by Mcl-1 were addressed using cells that are either over-expressing or knocking-down Mcl-1. Wild type U937 cells treated with quercetin displayed a rapid increase in conformationally-changed Bax (Fig. 5C). In contrast, over-expression of Mcl-1 partially reduced Bax conformational change after exposure to quercetin (Fig. 5C). Knockdown of Mcl-1 expression by siRNA exhibited an increase in Bax conformational change upon querectin treatment compared with wild type cells. Taken together, these results provide clear evidences that Mcl-1 down-regulation is a critical determinant of quercetin-mediated Bax conformational change.

To test whether Mcl-1 affects the translocation of Bax to mitochondria, U937 cells stably over-expressing Mcl-1 or vector alone were treated with quercetin, and the mitochondria and cytosol were isolated. As shown in Fig. 5D, Bax underwent a shift from cytosol to mitochondria in U937/pCEP cells, whereas enforced expression of Mcl-1 prevented Bax translocation from cytosol to mitochondria following quercetin treatment. On the other hand, siRNA knock-down of Mcl-1 enhanced quercetin-induced Bax translocation to mitochondria (Fig. 5E). These data indicate that the Bax redistribution following quercetin treatment is regulated by Mcl-1.

One possible explanation for the inhibition of Bax by Mcl-1 is that Bax is sequestered in a Bax/Mcl-1 complex. To test this possibility, immunoprecipitation was performed to determine the interaction between these two proteins. As shown in Supplementary Fig. 3D, no direct interaction between Bax and Mcl-1 was detected in either control or quercetin treatment conditions. Taken together, it may be concluded that Mcl-1 inhibits Bax activation without a direction interaction between these two proteins.

Quercetin exhibited antitumor activity in xenografts of human leukemia U937 cells

The in vitro data described above prompted us to further test the anti-cancer efficacy of quercetin in an in vivo model, i.e., in mice xenografted subcutaneously with U937 cells. Treatment of mice with 20 and 40 mg/kg quercetin resulted in 53% and 90% inhibition of tumor growth compared with controls in day 16 (Figs. 6A and 6B). As Figure 6C showed, the body weights of the xenograft mice were not significantly variable between different treatment groups after 16 days. We further determined apoptotic cells in tumor tissue by TUNEL assay. Our data showed that TUNEL positive apoptotic cells of tumor sections significantly increased in quercetin (20 and 40 mg/kg) treated-U937 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-1 expression in tumor samples decreased upon quercetin treatment, as Fig. 6E immunofluorescence results shown. Bax activation was observed in quercetin exposed xenografts (Fig. 6F). These data implicate a therapeutic value of quercetin in preventing or eradicating tumor growth in xenograft models of human hematologic malignancies.

Figure 6.

Figure 6

Open in a new tab

Quercetin inhibits tumor growth and induces apoptosis in the xenograft animal model. 6- to 8-week-old nude mice received subcutaneous transplants of 6 × 106 U937 cells. From the second day, mice were randomized into a control group (6 mice/group) and two treated groups (6 mice/group, quercetin 20 mg/kg and 40 mg/kg). Quercetin i.p. administration and tumor volume assessment were conducted as described in “Methods”. Representive animals with solid tumor volume were shown in (A), tumor volume measured in day 11 and day 16 was shown in (B), body weight was shown in (C), and representative IHC images for TUNEL staining and percentage of apoptotic cells (TNUNEL positive cells) in tumor tissue were shown in (D), representative IF images for Mcl-1 expression were shown in (E) and Bax activation using IP (anti-Bax 6A7) following immunoblotting (anti-Bax N-20) was shown in (F) in tumor samples. *Values of tumor volumn for quercetin treatment groups were significantly decreased compared to those for non-treatment group by Student’s t-test; p < 0.05. *Values of TUNEL positive cells for quercetin treatment groups were significantly increased compared to those for non-treatment group by Student’s t-test; p < 0.05.

Discussion

The present study is focused on the tumor-selective apoptosis induced by quercetin, a prospective anti-cancer drug that has been done with pre-clinical and small phase I clinical evaluation (43).

In view of the extensive evidence that Mcl-1 plays an important role in the survival of malignant hematopoietic cells (25, 44–45), the development of anti-cancer compounds that can diminish Mcl-1 protein levels has been the focus of intense interest. Since Mcl-1 protein has a short half-life (30 min) (46–47), it is particularly susceptible to down-regulation by agents. Here we discovered that quercetin is efficient in killing tumor cells exhibiting relatively high levels of Mcl-1. The findings that over-expression of Mcl-1 diminished quercetin lethality highlight the central role of Mcl-1 down-regulation. This interpretation is further supported by the results showing that knockdown of Mcl-1 was able to enhance querctin-mediated apoptosis. It is noteworthy that over-expression of Bcl-2 or Bcl-xL failed to protect leukemia cells from quercetin-induced cell death, reflecting the important contribution of Mcl-1 down-regulation to the lethality of this drug. Our unpublished data indicate that quercetin-induced MnSOD down-regulation, Akt inactivation as well as a translational initiation factor eIF4E-mediated translational mechanism are responsible for this Mcl-1 reduction. Therefore, 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 cell death, suggesting that elimination of Mcl-1 may be necessary but not sufficient to trigger apoptosis, and other apoptosis inducing actions might be required.

In our study although the overall Bax protein levels are not altered during quercetin-induced apoptosis, our data clearly demonstrate that Bax conformational change is one of the early steps in drug-induced apoptosis. Our data also show that an increase in Bax translocation which leads to mitochondria-mediated caspase activation: mitochondria dysfunction promoted by Bax translocation leads to leakage of cytochrome c from mitochondria, which subsequently activates caspase-9 and -3. The importance of Bax in quercetin lethality is confirmed by the observation that knocking down of Bax effectively protects cells from quercetin-mediated cell death, and that cells lacking Bax displayed complete resistance to quercetin. These results suggest that the presence of Bax is essential for quercetin lethality. These findings provide strong support for the notion that activation of Bax represents a highly potent apoptotic stimulus in the presence of quercetin.

Mcl-1 has previously been shown to inhibit Bax activation when over-expressed at high levels (36). In accord with this notion, our findings that enforced expression of Mcl-1, essentially diminished quercetin-induced Bax activation, strongly support that Mcl-1 plays an important role in regulating the function of Bax. Conversely, low levels of Mcl-1 enhanced the formation of active Bax by quercetin. These findings are consistent with the results described by Nijhawan et al (27) that Mcl-1 operates upstream of Bax and Bcl-xL translocation to the mitochondria in UV-treated Hela cells. The inhibition of Bax by Mcl-1 in the absence of direct interactions between the two proteins could occur through several possible mechanisms. One would be that Mcl-1 acts through other multi-domain pro-apoptotic Bcl-2 family proteins, such as active Bak in an as yet to be defined way. A second possibility is that Mcl-1 inhibits Bax through a process involving the Bax “activator”. BH3-only proteins, such as Bid or Bim, could be possible targets for Mcl-1 in this regard (48) . Very recent studies from our group have uncovered a novel mechanism of quercetin-mediated Bax activation which involves Bid cleavage (unpublished data). This unique mechanism could have important implications for the development of combination strategies involving agents with different apoptosis targets to achieve the additive or synergistic effects against hematopoietic malignancies.

It has been reported that administration of quercetin eliminates colorectal cancer xenografts (49) as well as human breast cancer MDA-mB-435 cells xenografts (50). In our in vivo studies using a nude mice U937 xenograft model, tumor volume was reduced compared to control after treatment of quercetin, indicating an anti-tumor activity of this compound. To further support the apoptotic mechanism found in vitro, we next examined the TUNEL staining, Mcl-1 expression and Bax activation in tumor specimens obtained from control and quercetin-treated animals. The increase of TUNEL positive cells and Bax activation, as well as the decrease of Mcl-1 expression were detected in the quercetin-treated xenografts compared with the control group. This is the first report that describes an effective extrapolation of the in vitro apoptosis-inducing effects of quercetin on the leukemia cells to the in vivo situation.

In summary, the present findings indicate that quercetin effectively induces cell death in human leukemia cells, including primary leukemia blasts, as well as in leukemia xenografts. This effect occurs in association with the rapid down-regulation of Mcl-1. Moreover, this process is accompanied by the conformational change and translocation of Bax, which are dependent on Mcl-1 degradation. The novel mode of action and the potent antitumor activity of quercetin both in vitro and in vivo found by this study make it attractive as an antitumor agent for hematologic malignancies. In addition, this work also identifies Mcl-1 and Bax as potential biomarkers of quercetin activity that directly relate to its mechanism of action. Such biomarkers may be useful to show the mode of action of quercetin in vivo using patient biopsy samples. Further efforts are warranted elucidate the mechanism(s) by which quercetin inhibits Mcl-1, as well as the other possible factors that contribute to Bax activation during quercetin treatment. This study could provide a better understanding of how this compound exerts its anti-tumor activity in vivo and aid in developing this compound either alone or in combination with established chemotherapeutic agents to treat leukemia and potentially other hematologic malignancies.

Statement of Translational Relevance

The hematological malignancies are the types of cancer that arise from malignant transformation of cells derived from peripheral blood, lymphatic system, and bone marrow. Because of the increase in the morbidity and mortality of human leukemia in recent years, chemoprevention or intervention towards the control of human leukemia is highly desirable. The present study has provided evidences that the natural product quercetin induces human leukemia cell death through down-regulation of Mcl-1 and activation of Bax. The data presented here suggest that the two Bcl-2 family proteins, Mcl-1 and Bax, may represent attractive targets for quercetin-induced apoptosis in human leukemia cells. In vivo data confirmed the anti-tumor efficacy of quercetin via induction of apoptosis by targeting Mcl-1 and Bax in xenografts of leukemia cells. The results of this study could have implications for the incorporation of agents such as quercetin into the chemoprevention or therapeutic intervention against hematologic malignancies.

Supplementary Material

1
2
3
4

Acknowledgments

Grant support: NIH Grant RO1 ES015375 (X.Shi)

References

  • 1.Granado-Serrano AB, Martin MA, Bravo L, Goya L, Ramos S. Quercetin induces apoptosis via caspase activation, regulation of Bcl-2, and inhibition of PI-3-kinase/Akt and ERK pathways in a human hepatoma cell line (HepG2) J Nutr. 2006;136:2715–2721. doi: 10.1093/jn/136.11.2715. [DOI] [PubMed] [Google Scholar]
  • 2.van Erk MJ, Roepman P, van der Lende TR, et al. Integrated assessment by multiple gene expression analysis of quercetin bioactivity on anticancer-related mechanisms in colon cancer cells in vitro. Eur J Nutr. 2005;44:143–156. doi: 10.1007/s00394-004-0503-1. [DOI] [PubMed] [Google Scholar]
  • 3.Nguyen TT, Tran E, Nguyen TH, Do PT, Huynh TH, Huynh H. The role of activated MEK-ERK pathway in quercetin-induced growth inhibition and apoptosis in A549 lung cancer cells. Carcinogenesis. 2004;25:647–659. doi: 10.1093/carcin/bgh052. [DOI] [PubMed] [Google Scholar]
  • 4.Shen SC, Chen YC, Hsu FL, Lee WR. Differential apoptosis-inducing effect of quercetin and its glycosides in human promyeloleukemic HL-60 cells by alternative activation of the caspase 3 cascade. J Cell Biochem. 2003;89:1044–1055. doi: 10.1002/jcb.10559. [DOI] [PubMed] [Google Scholar]
  • 5.Reed JC. Apoptosis-regulating proteins as targets for drug discovery. Trends Mol Med. 2001;7:314–319. doi: 10.1016/s1471-4914(01)02026-3. [DOI] [PubMed] [Google Scholar]
  • 6.Saikumar P, Mikhailova M, Pandeswara SL. Regulation of caspase-9 activity by differential binding to the apoptosome complex. Front Biosci. 2007;12:3343–3354. doi: 10.2741/2317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Herr I, Debatin KM. Cellular stress response and apoptosis in cancer therapy. Blood. 2001;98:2603–2614. doi: 10.1182/blood.v98.9.2603. [DOI] [PubMed] [Google Scholar]
  • 8.Slee EA, Harte MT, Kluck RM, et al. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol. 1999;144:281–292. doi: 10.1083/jcb.144.2.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Green DR. At the gates of death. Cancer Cell. 2006;9:328–330. doi: 10.1016/j.ccr.2006.05.004. [DOI] [PubMed] [Google Scholar]
  • 10.Letai A. BCL-2: found bound and drugged! Trends Mol Med. 2005;11:442–444. doi: 10.1016/j.molmed.2005.08.007. [DOI] [PubMed] [Google Scholar]
  • 11.Sharpe JC, Arnoult D, Youle RJ. Control of mitochondrial permeability by Bcl-2 family members. Biochim Biophys Acta. 2004;1644:107–113. doi: 10.1016/j.bbamcr.2003.10.016. [DOI] [PubMed] [Google Scholar]
  • 12.Sattler M, Liang H, Nettesheim D, et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science. 1997;275:983–986. doi: 10.1126/science.275.5302.983. [DOI] [PubMed] [Google Scholar]
  • 13.Jin Z, El-Deiry WS. Overview of cell death signaling pathways. Cancer Biol Ther. 2005;4:139–163. doi: 10.4161/cbt.4.2.1508. [DOI] [PubMed] [Google Scholar]
  • 14.Labi V, Erlacher M, Kiessling S, Villunger A. BH3-only proteins in cell death initiation, malignant disease and anticancer therapy. Cell Death Differ. 2006;13:1325–1338. doi: 10.1038/sj.cdd.4401940. [DOI] [PubMed] [Google Scholar]
  • 15.Leber B, Lin J, Andrews DW. Embedded together: the life and death consequences of interaction of the Bcl-2 family with membranes. Apoptosis. 2007;12:897–911. doi: 10.1007/s10495-007-0746-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Wei MC, Zong WX, Cheng EH, et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science. 2001;292:727–730. doi: 10.1126/science.1059108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Gomez-Bougie P, Bataille R, Amiot M. Endogenous association of Bim BH3-only protein with Mcl-1, Bcl-xL and Bcl-2 on mitochondria in human B cells. Eur J Immunol. 2005;35:971–976. doi: 10.1002/eji.200425878. [DOI] [PubMed] [Google Scholar]
  • 18.Zhu Y, Swanson BJ, Wang M, et al. Constitutive association of the proapoptotic protein Bim with Bcl-2-related proteins on mitochondria in T cells. Proc Natl Acad Sci U S A. 2004;101:7681–7686. doi: 10.1073/pnas.0402293101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Choi EJ, Bae SM, Ahn WS. Antiproliferative effects of quercetin through cell cycle arrest and apoptosis in human breast cancer MDA-MB-453 cells. Arch Pharm Res. 2008;31:1281–1285. doi: 10.1007/s12272-001-2107-0. [DOI] [PubMed] [Google Scholar]
  • 20.Hsu CL, Yen GC. Induction of cell apoptosis in 3T3-L1 pre-adipocytes by flavonoids is associated with their antioxidant activity. Mol Nutr Food Res. 2006;50:1072–1079. doi: 10.1002/mnfr.200600040. [DOI] [PubMed] [Google Scholar]
  • 21.Lee TJ, Kim OH, Kim OH, et al. Quercetin arrests G2/M phase and induces caspase-dependent cell death in U937 cells. Cancer Lett. 2006;240:234–242. doi: 10.1016/j.canlet.2005.09.013. [DOI] [PubMed] [Google Scholar]
  • 22.Vijayababu MR, Kanagaraj P, Arunkumar A, Ilangovan R, Aruldhas MM, Aruldhas MM. Quercetin-induced growth inhibition and cell death in prostatic carcinoma cells (PC-3) are associated with increase in p21 and hypophosphorylated retinoblastoma proteins expression. J Cancer Res Clin Oncol. 2005;131:765–771. doi: 10.1007/s00432-005-0005-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Kaufmann SH, Aruldhas MM, Svingen PA, et al. Elevated expression of the apoptotic regulator Mcl-1 at the time of leukemic relapse. Blood. 1998;91:991–1000. [PubMed] [Google Scholar]
  • 24.Derenne S, Monia B, Dean NM, et al. Antisense strategy shows that Mcl-1 rather than Bcl-2 or Bcl-x(L) is an essential survival protein of human myeloma cells. Blood. 2002;100:194–199. doi: 10.1182/blood.v100.1.194. [DOI] [PubMed] [Google Scholar]
  • 25.Moulding DA, Giles RV, Spiller DG, White MR, Tidd DM, Edwards SW. Apoptosis is rapidly triggered by antisense depletion of MCL-1 in differentiating U937 cells. Blood. 2000;96:1756–1763. [PubMed] [Google Scholar]
  • 26.Zhou P, Qian L, Bieszczad CK, et al. Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineage. Blood. 1998;92:3226–3239. [PubMed] [Google Scholar]
  • 27.Nijhawan D, Fang M, Traer E, et al. Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation. Genes Dev. 2003;17:1475–1486. doi: 10.1101/gad.1093903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Weng C, Li Y, Xu D, Shi Y, Tang H. Specific cleavage of Mcl-1 by caspase-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in Jurkat leukemia T cells. J Biol Chem. 2005;280:10491–10500. doi: 10.1074/jbc.M412819200. [DOI] [PubMed] [Google Scholar]
  • 29.Zhang B, Gojo I, Fenton RG. Myeloid cell factor-1 is a critical survival factor for multiple myeloma. Blood. 2002;99:1885–1893. doi: 10.1182/blood.v99.6.1885. [DOI] [PubMed] [Google Scholar]
  • 30.Zhou P, Qian L, Kozopas KM, Craig RW. Mcl-1, a Bcl-2 family member, delays the death of hematopoietic cells under a variety of apoptosis-inducing conditions. Blood. 1997;89:630–643. [PubMed] [Google Scholar]
  • 31.Morikawa K, Ikeda C, Nonaka M, Suzuki I. Growth arrest and apoptosis induced by quercetin is not linked to adipogenic conversion of human preadipocytes. Metabolism. 2007;56:1656–1665. doi: 10.1016/j.metabol.2007.07.008. [DOI] [PubMed] [Google Scholar]
  • 32.Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 1999;13:1899–1911. doi: 10.1101/gad.13.15.1899. [DOI] [PubMed] [Google Scholar]
  • 33.Dewson G, Snowden RT, Almond JB, Dyer MJ, Cohen GM. Conformational change and mitochondrial translocation of Bax accompany proteasome inhibitor-induced apoptosis of chronic lymphocytic leukemic cells. Oncogene. 2003;22:2643–2654. doi: 10.1038/sj.onc.1206326. [DOI] [PubMed] [Google Scholar]
  • 34.Gross A, Jockel J, Wei MC, Korsmeyer SJ. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 1998;17:3878–3885. doi: 10.1093/emboj/17.14.3878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Nechushtan A, Smith CL, Hsu YT, Youle RJ. Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO J. 1999;18:2330–2341. doi: 10.1093/emboj/18.9.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Antonsson B, Montessuit S, Sanchez B, Martinou JC. Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J Biol Chem. 2001;276:11615–11623. doi: 10.1074/jbc.M010810200. [DOI] [PubMed] [Google Scholar]
  • 37.George NM, Targy N, Evans JJ, Zhang L, Luo X. Bax contains two functional mitochondrial targeting sequences and translocates to mitochondria in a conformational change- and homo-oligomerization-driven process. J Biol Chem. 2010;285:1384–1392. doi: 10.1074/jbc.M109.049924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Yang JH, Hsia TC, Kuo HM, et al. Inhibition of lung cancer cell growth by quercetin glucuronides via G2/M arrest and induction of apoptosis. Drug Metab Dispos. 2006;34:296–304. doi: 10.1124/dmd.105.005280. [DOI] [PubMed] [Google Scholar]
  • 39.Lee DH, Szczepanski M, Lee YJ. Role of Bax in quercetin-induced apoptosis in human prostate cancer cells. Biochem Pharmacol. 2008;75:2345–2355. doi: 10.1016/j.bcp.2008.03.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Gao N, Budhraja A, Cheng S, Yao H, Zhang Z, Shi X. Induction of apoptosis in human leukemia cells by grape seed extract occurs via activation of c-Jun NH2-terminal kinase. Clin Cancer Res. 2009;15:140–149. doi: 10.1158/1078-0432.CCR-08-1447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Chen L, Willis SN, Wei A, et al. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell. 2005;17:393–403. doi: 10.1016/j.molcel.2004.12.030. [DOI] [PubMed] [Google Scholar]
  • 42.Menoret E, Gomez-Bougie P, Surget S, et al. Mcl-1(128–350) fragment induces apoptosis through direct interaction with Bax. FEBS Lett. 2010;584:487–492. doi: 10.1016/j.febslet.2009.11.094. [DOI] [PubMed] [Google Scholar]
  • 43.Mulholland PJ, Ferry DR, Anderson D, et al. Pre-clinical and clinical study of QC12, a water-soluble, pro-drug of quercetin. Ann Oncol. 2001;12:245–248. doi: 10.1023/a:1008372017097. [DOI] [PubMed] [Google Scholar]
  • 44.Danial NN, Korsmeyer SJ. Cell death: critical control points. Cell. 2004;116:205–219. doi: 10.1016/s0092-8674(04)00046-7. [DOI] [PubMed] [Google Scholar]
  • 45.Moulding DA, Akgul C, Derouet M, White MR, Edwards SW. BCL-2 family expression in human neutrophils during delayed and accelerated apoptosis. J Leukoc Biol. 2001;70:783–792. [PubMed] [Google Scholar]
  • 46.Opferman JT, Iwasaki H, Ong CC, et al. Obligate role of anti-apoptotic MCL-1 in the survival of hematopoietic stem cells. Science. 2005;307:1101–1104. doi: 10.1126/science.1106114. [DOI] [PubMed] [Google Scholar]
  • 47.Yang-Yen HF. Mcl-1: a highly regulated cell death and survival controller. J Biomed Sci. 2006;13:201–204. doi: 10.1007/s11373-005-9064-4. [DOI] [PubMed] [Google Scholar]
  • 48.Letai A, Bassik MC, Walensky LD, Sorcinelli MD, Weiler S, Korsmeyer SJ. Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell. 2002;2:183–192. doi: 10.1016/s1535-6108(02)00127-7. [DOI] [PubMed] [Google Scholar]
  • 49.Priego S, Feddi F, Ferrer P, et al. Natural polyphenols facilitate elimination of HT-29 colorectal cancer xenografts by chemoradiotherapy: a Bcl-2- and superoxide dismutase 2-dependent mechanism. Mol Cancer Ther. 2008;7:3330–3342. doi: 10.1158/1535-7163.MCT-08-0363. [DOI] [PubMed] [Google Scholar]
  • 50.Dechsupa S, Kothan S, Vergote J, et al. Quercetin, Siamois 1 and Siamois 2 induce apoptosis in human breast cancer MDA-mB-435 cells xenograft in vivo. Cancer Biol Ther. 2007;6:56–61. doi: 10.4161/cbt.6.1.3548. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS246540-supplement-1.doc (37.5KB, doc)
2
NIHMS246540-supplement-2.ppt (923KB, ppt)
3
NIHMS246540-supplement-3.ppt (383.5KB, ppt)
4
NIHMS246540-supplement-4.ppt (1.2MB, ppt)

RESOURCES