Induction of apoptosis by quercetin: different response of human ...

1 Molecular and Cellular Biochemistry 296: 137–149, 2007. DOI: 10.1007/s11010-006-9307-3


Springer 2006

Induction of apoptosis by quercetin: different response of human chronic myeloid (K562) and acute lymphoblastic (HSB-2) leukemia cells Fabrizia Brisdelli,1 Cristina Coccia,1 Benedetta Cinque,2 Maria Grazia Cifone2 and Argante Bozzi1 1 2

Department of Biochemical Sciences and Technologies, University of L’Aquila, Via Vetoio, Coppito 2, 67100 L’Aquila, Italy; Department of Experimental Medicine, University of L’Aquila, Via Vetoio, Coppito 2, 67100 L’Aquila, Italy

Received 5 June 2006; accepted 10 August 2006

Abstract This work shows that 25 lM quercetin caused a marked inhibition of K562 cells growth together with a mild cytotoxicity, while HSB-2 cells were practically unaffected. Moreover, quercetin induced caspase-3 and cytochrome c-dependent apoptosis almost exclusively in the former cell line. Exposure of K562 cells to quercetin caused also a significant increase of cells in G2/ M phase that reached the maximum peak at 24 h (4-fold with respect to the basal value). The major sensitivity exhibited by K562 cells was only in part imputable to their higher glutathione content, as compared to HSB-2 cells, thus confirming previous reports describing the formation of intracellular quercetin–thiol toxic adducts in cells exposed to the flavonoid. In fact, after induction of intracellular glutathione increase we detected in both cell lines a significant rise of apoptotic cells, again more marked in K562 cells. By contrast, glutathione-depleted cells, failed to show a decrease of apoptosis in both cell lines, thus contradicting our previous findings and literature data. Since the yet unresolved question about the anti-oxidant or the prooxidant capacity of quercetin, we investigated which of these two properties worked in our experimental model. Interestingly, not only quercetin did not produce reactive oxygen species but also prevented their formation, as observed in cells exposed to the oxidizing agent ter-butylhydroperoxide, acting as an efficient oxygen radicals scavenger. This result indicates that quercetin exhibited, in these cell lines, anti-oxidant more than pro-oxidant ability. (Mol Cell Biochem 296: 137–149, 2007) Key words: quercetin, apoptosis, glutathione, cytochrome c, caspases, ROS Abbreviations: Ac-DEVD-AMC, Acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin; BSO, buthionine-L-sulphoximine; DCFHDA, 2¢,7¢-dichlorofluorescein diacetate; DEM, diethyl maleate; ECL, enhanced chemiluminescence; NAC, N-acetyl-L-cysteine; ROS, reactive oxygen species; t-BHP, tert-butylhydroperoxide.

Introduction Quercetin (3,3¢,4¢,5,7-pentahydroxyflavone), one of the main flavonoids widely distributed in the plant kingdom, is particularly abundant in many edible fruits and vegetables.

In the European countries, a daily intake of about 16 mg has been estimated [1]. Some beverages, like red wine and green tea may contain quercetin concentrations up to 150 lM [2]. Since quercetin derived from the diet is mainly present as glycosylated derivatives, its anti-oxidant efficacy is notably

Address for offprints: Argante Bozzi, Department of Biochemical Sciences and Technologies, University of L’Aquila, Via Vetoio, Coppito 2, 67100 L’Aquila, Italy (E-mail: [email protected])

138 dependent from the position of the sugar linked to the diphenylpropane moiety. Humans consume approximately 1 g of flavonoids daily in the diet, and quercetin is relatively well absorbed through the gastrointestinal tract [3]. Nevertheless, quercetin and other flavonoids are metabolically converted by the epithelial intestinal cells before reaching blood flow and liver [4]. These reactions are either devoted to improve its absorption (hydrolysis to quercetin aglycone by enterobacterial flora), and addressed to structurally modify its solubility (O-methylation or glucuronidation/sulfatation by small intestine and liver) [5]. Few studies have been published on the potential biological effects of quercetin–thiol conjugates. In particular, the formation of adducts with glutathione has been reported either in normal human fibroblasts [6] and in B16F-10 melanoma cells [7]. Recently, in the in vitro experiments, it has been shown that quercetin reacts with cysteine faster than with GSH and NAC [8]. However, since the physiological concentrations of GSH are far higher than those of cysteine, in vivo, the formation of quercetin–thiol conjugates seems to favour the adducts with GSH. The covalent addition of quercetin to –SH groups of proteins or enzymes, thus impairing their function, cannot be discarded. The cytotoxicity of quercetin–thiol adducts could be due either to the reduction of cellular –SH groups, and to the binding of quercetin semiquinone intermediates to cysteine residues present in the active site of some pivotal enzymes. Concerning the intracellular metabolism of quercetin mediated by cytochrome-P450, there are only few informations to date, even if it has been reported that this enzymatic system can oxidize the flavonoids [9], but glucuronidation and sulfation are more efficient than P450mediated oxidation [10]. The biological activities of quercetin include, besides cytotoxic effects like induction of apoptosis, cell cycle arrest and anti-proliferative activity, inhibition of apoptosis, anti-inflammatory effects and protection from oxidative stress. Moreover, it has been reported that quercetin is an anti-tumoral agent since its cytostatic effects towards several cancer cell lines [11]. The induction of apoptosis by quercetin has been widely investigated and its onset has been ascribed to caspase-3 activation in HPBALL tumor cells [12], to activation of caspase-3, -9 and cytochrome c release in HL-60 cells [13], while the final effects are evidentiated as chromatin fragmentation and condensation in human colon cancer cells [14]. In spite of the wide literature on the cytostatic and pro-apoptotic effects of quercetin, its mechanism of action is not yet fully clarified. In the light of these considerations, it seemed interesting to further investigate the molecular processes that characterize the anti-cancer properties of this flavonoid. To this purpose, apoptosis induced by 25 lM quercetin exposure for up to 72 h of human chronic myeloid (K562) and acute lymphoblastic (HSB-2) leukaemia cells, was studied

in detail. In particular, K562 cells, usually reported to be quite resistant to conventional anti-tumor drugs, showed a surprising sensitivity to quercetin, with respect to HSB-2 cells. Apoptosis has been evaluated by monitoring the chromatin condensation and fragmentation, DNA ladder, caspase-3 activation and cytochrome c release from mitochondria. Cell cycle distribution, monitored by flow cytometric analysis, showed a different pattern in the two quercetin-treated cell lines. Since quercetin has been described as a radical scavenger, the inhibition of ROS was also checked. The possible role exerted by glutathione was studied after either intracellular GSH depletion and GSH enrichment. The results obtained are reported and discussed in the present paper.

Materials and methods Materials RPMI 1640 medium, foetal calf serum and proteinase K were from Labtek-Eurobio. Acridine orange, buthionine-L-sulphoximine, 1-chloro-2,4-dinitrobenzene, diethyl maleate, 5,5¢-dithiobis(2-nitrobenzoic acid), ethidium bromide, reduced (GSH) and oxidized (GSSG) glutathione, glutathione reductase, N-acetyl-L-cysteine, NADPH, Nonidet P-40, propidium iodide, RNase A, sulfosalicilic acid, tert-butylhydroperoxide (t-BHP) and 2-vinylpyridine were purchased from Sigma Chemical Co. Fluorogenic caspase substrates Ac-DEVD-AMC (acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin), Ac-IETD-AMC (acetyl-Ile-GluThr-Asp-aminomethylcoumarin) and Ac-LEHD-AMC (acetyl-Leu-Glu-His-Asp-aminomethylcoumarin) were from Alexis Biochemicals. Monoclonal anti-actin, anti-Bax and anti-cytochrome c antibodies were purchased from Roche Molecular Biochemicals, Santa Cruz Biotechnology and PharMingen, respectively. Reagents for enhanced chemiluminescence (ECL) detection were obtained from Amersham Life Science. 2¢,7¢-Dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes, Inc. All other chemicals were reagent grade. Stock solutions of quercetin were prepared in DMSO and stored in the dark at )20 C. The DMSO concentration in all drug-treated cells was always less than 0.01 (v/v).

Cell cultures Human chronic myeloid (K562) and human acute lymphoblastic (HSB-2) leukemia cells were obtained from the American Type Culture Collectin (ATCC), maintained in exponential growth in RPMI 1640 medium supplemented with 10% heat-inactivated foetal bovine serum, 2 mM

139 glutamine, streptomycin and penicillin and kept at 37 C in a humidified atmosphere of 5% CO2 in air.

for 1 h. DNA was analysed by 2% (w/v) agarose gel electrophoresis.

Analysis of cell proliferation and viability

Caspase activity

Cells were seeded at a density of 1 · 105 per ml and incubated in the absence or in the presence of 25 lM quercetin. After 24, 48 and 72 h, cells were counted and viability determined by trypan blue exclusion assay.

The activities of caspase-3, caspase-8 and caspase-9 were determined as previously described [16]. Briefly, cells were collected by centrifugation and suspended in extraction buffer (50 mM Tris–HCl, pH 7.4, 10 mM EGTA, 1 mM EDTA, 10 mM DTT, 1% (v/v) Triton X-100). The supernatants were incubated with 20 lM fluorogenic peptide substrates, Ac-DEVD-AMC (caspase-3), Ac-IETD-AMC (caspase-8) and Ac-LEHD-AMC (caspase-9), in reaction buffer for 30 min at 37 C. Fluorescence was monitored on a Perkin–Elmer LS-50B spectrofluorimeter, setting excitation at 380 nm and emission at 460 nm. The increase of protease activities was determined by comparing the levels of the quercetin-treated cells with untreated controls.

Assays for detection of apoptosis Cells were monitored for apoptosis during an extended time interval (24–72 h) and the best points for detection of apoptosis for each cell line were determined in both cell lines.

Chromatin condensation Cytochrome c release Nuclear morphology was analysed by double acridine orange- and ethidium bromide staining. After mixing with an equal volume of a solution containing 100 lg/ml ethidium bromide and 100 lg/ml acridine orange, the cell suspension was examined with a fluorescence microscope [15].

Apoptosis and cell cycle analysis by flow cytometry Control and quercetin-treated cells were collected, washed twice with ice-cold PBS and fixed in 70% ethanol at 4 C for at least 30 min. The fixed cells were then washed twice with ice-cold PBS and stained with 50 lg/ml of propidium iodide in the presence of 25 lg/ml of RNase A. Cell cycle phase-distribution was analysed in three different experiments using flow cytometry (FACScan flow cytometry, Becton Dickinson Immunocytometry System, San Jose, CA, USA). Data from 10,000 events per sample were collected and analysed using the cell cycle analysis software (Modfit LT for Mac V 3.0). Apoptotic cells were determined by their hypochromic subdiploid staining profiles and analysed using Cell Quest software.

Internucleosomal fragmentation of chromatin Cells (1.0 · 106) were washed with PBS and resuspended in 100 ll of lysis buffer (50 mM Tris–HCl, pH 8.0, 10 mM EDTA, 0.25% (v/v) Nonidet P-40, 1 mg/ml RNase A). After 1 h incubation at 37 C the suspension was supplemented with 1 mg/ml proteinase K and further incubated at 37 C

Cells were washed twice with cold PBS and pellets were resuspended in permeabilization buffer (0.25 M Sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM Na–EGTA, 1.5 mM Na–EDTA, 1 mM MgCl2, 1 mM DTT, 50 lg/ml digitonin, pH 7.4, containing a cocktail of protease inhibitors) for 10 min at 4 C. Permeabilized cells were centrifuged (800 · g, 15 min) at 4 C. The clear supernatants were centrifuged again (18,000 · g, 30 min) at the same temperature. Supernatant proteins were separated on a 15% SDS-PAGE, transferred to PVDF membranes, probed with anti-cytochrome c antibody (7H8.2C12) and visualized with an ECL detection system.

Glutathione determination Cells were washed with PBS and resuspended in 5 mM EDTA and 5% (w/v) sulfosalicilic acid. Total glutathione and GSSG concentrations were measured as previously reported [17]. An aliquot of cell extract was added to 0.7 ml of 0.143 M sodium phosphate buffer (pH 7.5), containing 6.3 mM EDTA and 0.2 mM NADPH and to 0.1 ml of 6 mM 5,5¢-dithiobis(2-nitrobenzoic acid). The assay was initiated by addition of 5 ll of glutathione reductase (470 U/ml). The rate of formation of 5-thio-2-nitrobenzoic acid, proportional to total glutathione concentration, was followed at 412 nm for 3 min at 25 C in a Perkin–Elmer LS5 spectrophotometer. GSH concentration values were calculated from a standard curve and expressed as nmol/106 cells. GSSG concentration was determinated under the same

140 condition after adjusting pH to 6–7 with triethanolamine and GSH derivatization by 2-vinylpyridine.

Results Cell growth and viability

K562 cells were washed with cold PBS, resuspended in 50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1% (v/v) Triton X100, and then disrupted by three consecutive cycles of freeze–thawing. After centrifugation at 17,000 · g for 10 min at 4 C, the supernatant was collected for protein determination [18] and subsequently analysed. The glutathione reductase activity of cell extracts was assayed spectrophotometrically by following NADPH oxidation at 340 nm at 25 C [19]. The assay mixture consisted of 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM GSSG, 0.2 mM NADPH and the appropriate amount of the extracted proteins. Total (Se-dependent and Se-independent) glutathione peroxidase activity was evaluated specrophotometrically by measuring the NADPH oxidation at 340 nm. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 0.2 mM NADPH, 1 mM GSH, 0.01 U/ ml GSH reductase, 70 lM tert-butylhydroperoxide (t-BHP) and the appropriate amount of the extracted proteins [20]. The glutathione transferase activity was assayed by measuring the rate of GSH conjugation to 1-chloro-2,4dinitrobenzene at 340 nm. The reaction mixture contained 0.1 M potassium phosphate buffer, pH 6.5, 2 mM GSH, 1 mM 1-chloro-2,4-dinitrobenzene and aliquots of supernatant [21]. The enzymatic activities were expressed as nmol/min/mg protein.

In preliminary experiments aimed at assessing the best concentration of quercetin to be used in our two cell lines, different concentrations, ranging from 5 to 50 lM, have been tested on both K562 and HSB-2 cells. Cell growth and viability, evaluated by trypan blue exclusion test, were determined after 24, 48, 72 h exposure to quercetin and are shown in Figs. 1 and 2. As can be seen, quercetin 25 lM induced an almost full block of growth only in K562 cells (Fig. 1A) while the amount of dead cells was around 18% after 72 h esposure (Fig. 2A). On the contrary, HSB-2 cell mortality was far less, about 9%. Among leukemic cell

A 1400

control quercetin 5 µM

1200 nr of viable cells/ml (x 103)

Enzymatic assays

quercetin 10 µM

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quercetin 25 µM 800

quercetin 50 µM

600 400 200 0 0

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The generation of reactive oxygen species was detected by DCF fluorescence [22]. Cells, suspended in culture medium, were loaded with DCFH-DA to a final concentration of 10 lM and incubated at 37 C for 30 min. After the addition of quercetin (or t-BHP as positive control), or quercetin plus t-BHP, cells were incubated at 37 C for short (10– 120 min) or more prolonged (12–72 h) incubation times, washed with PBS and the fluorescence intensity was monitored with a Perkin–Elmer LS-50B spectrofluorometer, setting the excitation at 485 nm and emission at 530 nm.

control

B 1400 nr of viable cells/ml (x 103)

Determination of reactive oxygen species (ROS)

quercetin 5 µM

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quercetin 25 µM

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Statistical analysis Data are reported as means ± SD. Statistical differences between control and quercetin-treated cells were calculated using the Student’s t-test. Differences were considered significant at P < 0.05 versus each respective untreated control (*).

0

24 h

Fig. 1. Effect of quercetin concentration (ranging from 5 to 50 lM) on K562 (A) and HSB-2 (B) cells growth. Untreated or quercetin-treated cells were counted at the indicated time. Results represent the mean ± SD of three independent experiments.

141 Effect of quercetin on cell cycle distribution and apoptosis in K562 and HSB-2 cells

80

In order to investigate the mechanisms involved in growth inhibition induced by quercetin, we next evaluated whether the drug could induce cell cycle arrest in K562 and HSB-2 cells using flow cytometric analyses. Untreated cells or cells treated with 25 lM quercetin were submitted to cell cycle analysis at 24, 48 and 72 h by FACS. As shown in Fig. 3A, quercetin had no significant influence on the HSB-2 cell cycle at any time. On the contrary, K562 cells treated with quercetin showed a cell cycle profile significantly different from that of the control cells between 24 and 72 h (Fig. 3A). A representative example depicting the effect of quercetin treatment for 24, 48 and 72 h on cell cycle distribution in the K562 cells is shown in Fig. 3B. Exposure of K562 cells to quercetin induced a significant increase in the percentage of cells in G2/M phase at all analysed times. Interestingly, the accumulation in the G2/M phase reached the maximum peak at 24 h (4-fold increase, with respect to the basal value). Compared with the untreated control cells, 25 lM quercetin induced a decrease in the G0/G1 phase at 24, 48 and 72 h; this decrease was associated to a concomitant reduction of the S phase cell percentage at all incubation times. However, although the flow cytometry analysis showed the decrease of S peak, this reduction was statistically significant only at 48 h upon quercetin treatment. To confirm the different sensitivity of these two cell lines to quercetin, apoptosis was evaluated by measuring cell cycle kinetics, nuclear morphology and DNA fragmentation. Figure 4 shows that K562 cells exhibited a progressive chromatin condensation (10%) upon 24 h of quercetin treatment, which reached a 30% value after 72 h. The insert represents a typical picture of chromatin condensation after staining with AO and EB either in control or in quercetin-treated K562 cells. On the contrary, in HSB-2-treated cells, chromatin condensation appeared much less pronounced, reaching a 12% value only after prolonged (72 h) exposure times. According to these results, we examined sub-G1 apoptotic fraction in both cell lines untreated or quercetin-treated, by flow cytometry. Figure 5A shows the effect of quercetin on K562 apoptotic levels. The results indicate that a typical sub-G1 fraction, representing the apoptotic cell population, start to appear at 24 h, while the apoptotic cell percentage increased in a time-related manner, reaching a 15% value after 72 h. By contrast, HSB-2 cells exposed to quercetin, did not exhibit any substantial increase of hypodiploidy level at all times, as shown in Fig. 5B. A characteristic feature of apoptosis is the degradation of chromatin DNA at the internucleosomal linkages. To determine whether the flavonoid induces DNA fragmentation, K562 and HSB-2 cells were treated with 25 lM

% of viable cells

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control quercetin 5 µM

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quercetin 10 µM quercetin 25 µM

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% of viable cells

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60 control quercetin 5 µM

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quercetin 10 µM quercetin 25 µM

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quercetin 50 µM 0 0

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h Fig. 2. Trypan blue exclusion test. The percentage of trypan blue-negative K562 (A) and HSB-2 (B) cells after exposure to concentrations ranging from 5 to 50 lM of quercetin was determined at the indicated times. Results represent the mean ± SD of three independent experiments.

types, K562 cells display the higher resistance towards several anti-tumor drugs since these cells express the p210BCR-ABL fusion protein that reportedly confers resistance to drug-induced apoptosis [23–26]. Interestingly, in our experimental conditions, K562 cells showed lower resistance to quercetin as inducer of programmed cell death. Thus, it seemed interesting to better investigate the mechanism/s underlying the different sensitivity of these two cell lines to 25 lM quercetin. However, it has to be recalled that quercetin is not usually found in plasma as the free form or as the parent glucoside [3]. By contrast, after a meal rich of polyphenols, quercetin aglycone could reach a blood concentration ranging between 1 and 5 lM, and it is reported that an intake of 50 mg quercetin would lead to plasma concentrations of up to 0.75–1.5 lM [27, 28].

142 K562 50

** *

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Fig. 3. Effect of quercetin on cell cycle distribution. (A) Cell cycle analysis of K562 and HSB-2 cells, by propidium iodide staining and flow cytometry after treatment with 25 lM quercetin for 24, 48 and 72 h. Values are means ± SEM, n = 3. *Data are significantly different from untreated control (p < 0.05). (B) A pattern representative of three similar profiles of cell cycle distribution in K562 cells population.

quercetin for 24, 48, 72 h and DNA was fractionated by agarose gel electrophoresis. A typical ladder pattern of internucleosomal fragmentation was evidenced at 48 h with a progressive increase at 72 h only in K562 cells (Fig. 6). On the contrary, HSB-2-treated cells failed to show any appearance of DNA ladder irrespective of the exposure time, thus paralleling the results obtained for cell cycle distribution and for chromatin condensation.

Caspase activation and cytochrome c release induced by quercetin The effects of quercetin on caspase-3, caspase-8 and caspase-9 activities were studied in K562 and HSB-2 cells. The

latter two enzymatic activities were scarcely affected in both cell types exposed to quercetin for up to 72 h (data not shown). It has to be underlined that caspase-8 (IETDdependent) is reported as an early marker of the apoptotic pathway and its activation is dependent on the ligation of death receptors. This ‘‘initiator’’ caspase-8 activates, in turn, down-stream or ‘‘effector’’ caspases such as caspase-3 [29, 30]. Moreover, caspase-9 (LEHD-dependent) is also described as an early apoptotic marker that is activated when apoptosis follows the mitochondrial pathway. In this route, many cellular stress cause mitochondrial alterations, like mitochondrial membrane depolarization and release of cytochrome c, which in turn activates caspase-9 and effector caspases [31, 32]. However, caspase-3 (DEVD-dependent)

K562 control

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1,3 1,1 Control

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Time (h)

Fig. 4. Time course of nuclear morphology alterations in untreated K562 and HSB-2 cells, or treated with 25 lM quercetin. The percentage of condensed and fragmented nuclei was estimated by fluorescence microscope on acridine orange and ethidium bromide double-stained cells examined at the indicated times. At least 400 cells were counted for each determination. Results represent the mean ± SD of three independent experiments. *Data are significantly different from untreated control (p < 0.05). The insert shows a fluorescence microscope picture of control and quercetin-treated (72 h) K562 cells.

activity showed a progressive increase, as compared to control untreated cells, that reached a 4- to 5-fold value after 72 h of quercetin exposure in K562 cells (Fig. 7). In HSB-2 cells, caspase-3 activity increased only by a factor 1.5 at 24 and 48 h then decreasing to a value similar to control after 72 h of quercetin treatment (Fig. 7). Moreover, the drug caused cytochrome c release, after 48 h, only in K562treated cells (Fig. 8). This pattern is in line with the above reported data, which show the different sensitivity of the two cell lines to quercetin. On the whole, our results indicate a major sensitivity of K562 cells to apoptosis induced by quercetin, with respect to HSB-2 cells.

Fig. 5. Effect of quercetin on apoptosis in K562 (A) and HSB-2 (B). Cells were treated with 25 lM quercetin for the indicated time and analysed by flow cytometry. The number of sub-G1 cells was expressed as a percentage of the total cells and shown as a graph. Values are means ± SEM, n = 4. *Data are significantly different from untreated control (p < 0.02).

generation of ROS, K562 and HSB-2 cells, suspended in the culture medium, were exposed to the drug for short (10–180 min) and prolonged (12–72 h) incubation times

A 48 h 72 h M C Q C Q

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2500 bp 1800 bp

Quercetin do not produce ROS but is an efficient ROS scavenger 100 bp

Several biological activities of flavonoids have been ascribed to their anti-oxidant properties. Nevertheless, it is still under debate if this is the unique function since, as reported by some authors [33], the flavonoids behave also like pro-oxidant agents. The precise mechanism(s) by which these molecules exert their protective (or toxic) effects are not yet fully clarified [34]. To verify if quercetin induced the

B

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Fig. 6. Agarose gel electrophoresis of genomic DNA isolated from untreated or 25 lM quercetin treated K562 (A) and HSB-2 (B) cells. Cell preparations were analysed at the indicated times. C, Control cells; Q, quercetin treated cells; M, DNA molecular weight markers.

144 K562 (control) K562 (quercetin) HSB-2 (control) HSB-2 (quercetin)

caspase -3-like activity (arbitrary fluorescence units)

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Fig. 7. Time course of caspase-3-like activity. The caspase activity was measured in the cytosol of untreated and 25 lM quercetin treated cells by using DEVD-aminomethylcoumarin as a substrate. Results represent the mean ± SD of three independent experiments. *Data are significantly different from untreated control (p < 0.05). 0

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Fig. 8. Cytochrome c release in the cytosol of 25 lM quercetin treated K562 cells by immunoblot analysis with anti-cytochrome c antibody. No cytochrome c band could be detected in control cells at any time of incubation.

and then analysed for DCF fluorescence. Interestingly, not only quercetin did not produce ROS up to 72 h (data not shown), but also efficiently prevented their formation, as observed in cell samples treated with the oxidizing agent t-BHP (Fig. 9). This result, in agreement to a previous paper

1200

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HSB-2

DCF fluorescence

(arbitrary units)

1000 800 600 400

describing quercetin as an oxygen radical scavenger [35], indicates that the flavonol exhibit, in these cell lines, antioxidant more than pro-oxidant properties. Quercetin causes alterations of intracellular glutathione concentration Glutathione is involved in signal transduction, cell proliferation and apoptosis [36–38]. We found a marked difference in the basal content of glutathione in the two cell types, K562 cells exhibiting a glutathione concentration almost 10-fold higher than HSB-2 cells, irrespective of quercetin treatment (Table 1). It has to be recalled that total glutathione practically identifies reduced glutathione, being this intracellular metabolite almost completely in the reduced state (GSH) [39]. Quercetin-treated K562 cells exhibited a substantial (about 3-fold) increase of total glutathione at 24 h, followed by a decline to basal values at 48 h and by a drop to half of the control concentration at 72 h (Table 1). In HSB-2 cells, quercetin treatment induced a less marked glutathione increase (about 2-fold) in the time Table 1. Levels of total glutathione in untreated (control) and quercetintreated (25 lM) K562 and HSB-2 cells determined at the indicated times

* 200

Total glutathione (nmol/106 cells) 0 l

ro

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Fig. 9. ROS production in K562 and HSB-2 cells untreated or treated for 1 h at 37 C with 25 lM quercetin or 100 lM tert-butylhydroperoxide (t-BHP) or quercetin plus t-BHP, after incubation with 10 lM DCFH2-DA for 30 min. DCF fluorescence was determined by spectrofluorimetric analysis. Results represent the mean ± SD of three independent experiments. *Data are significantly different from untreated control (p < 0.02).

K562

HSB-2

Incubation time (h)

Control

Quercetin

Control

Quercetin

24

12.9 ± 1.20

37.7 ± 2.76*

2.29 ± 0.30

4.62 ± 0.65*

48

13.9 ± 6.79

16.5 ± 7.78

1.56 ± 0.14

3.47 ± 0.26*

72

13.3 ± 3.61

7.2 ± 2.75

2.03 ± 0.5

2.99 ± 0.30

Results represent the mean ± SD of triplicate experiments.*Data are significantly different from untreated control (p < 0.05).

145 K562

50

% of apoptotic cells

40

*

30 * 20

*

10

50

Several biological effects of quercetin, including antiinflammatory, anti-viral and anti-tumoral activities have been previously described [40, 41]. Moreover, flavonoids behave like natural anti-oxidants thus contributing, at least in part, to the defence from many pathologies depending on a dietary intake rich in fruits and vegetables [42, 43]. Table 2. Variations of GSH levels in K562 and HSB-2 cells not treated (control) or incubated 48 h in culture medium containing the indicated drugs Total glutathione (nmol/106 cells) Treatment

K562

HSB-2

Control BSO 50 lM

8.29 ± 0.06 2.80 ± 0.81

1.44 ± 0.38 0.10 ± 0.02

Quercetin 25 lM

13.4 ± 1.54

3.28 ± 0.46

BSO + quercetin

3.16 ± 0.23

0.16 ± 0.004

Results represent the mean ± SD of triplicate experiments.

t in ce er

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HSB-2

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0

% of apoptotic cells

interval of 24–48 h upon drug exposure, with a decrease at 72 h, even if the concentration was higher than that of the control cells. Moreover, in both cell lines, quercetin did not induce significant alterations of glutathione transferase, peroxidase and reductase activities, irrespective of the exposure times (data not shown). These results prompted us to better investigate the role, if any, exerted by glutathione in the apoptotic cascade triggered by quercetin. When K562- and HSB-2 cells were incubated with 50 lM BSO (a powerful and selective inhibitor of c-glutamyl cysteine synthase activity, the key enzyme of glutathione synthesis), the GSH content decreased to concentrations lower than those of control cells (Table 2). However, the marked depletion of glutathione did not cause any significant increase of apoptotic cells (Fig. 10). The simultaneous addition of BSO and quercetin caused a dramatic drop of GSH increment induced by the flavonoid (Table 2), while the amount of apoptosis increased in both cell types, but more markedly in K562 cells (Fig. 10). It is noteworthy that also diethylmaleate (a well-known agent used for intracellular GSH depletion), together with quercetin, did not modify the number of apoptotic cells (data not shown). Surprisingly, when K562 and HSB-2 cells were enriched in glutathione, upon incubation with NAC (the esterified permeable form of cysteine, an essential amino acid residue for glutathione synthesis), apoptosis induced by quercetin significantly increased in both cell lines (Fig. 10), thus confirming previous data on the formation of quercetin–thiol toxic adducts [6–8].

Fig. 10. Effects of glutathione modulation by BSO and NAC on apoptosis in K562 and HSB-2 cells treated with 25 lM quercetin for 48 h. Results represent the mean ± SD of three independent experiments. *Data are significantly different from untreated control (p < 0.02).

In a previous paper, we have shown the different sensitivity of chronic myeloid leukemia (K562) and acute lymphoblastic leukemia (HSB-2) cells to apoptosis induced by the polyphenolic agent resveratrol [39]. The major sensitivity of resveratrol-treated HSB-2 cells was attributed to an enhanced Bax expression and caspase-3 activity, as well as to cytochrome c release and possibly to a minor intracellular content of glutathione, with respect to K562 cells. In this study, we show that quercetin, at 25 lM concentration, causes a marked inhibition of K562 cells growth together with a mild cytotoxicity, while HSB-2 cells are practically unaffected. The anti-proliferative action is possibly related to the ability of the flavonoid to induce programmed cell death. The results obtained by flow cytometry indicated a significant accumulation of K562 cells in G2/M phase after 24 h of quercetin treatment. Similar results were observed when K562 were exposed to the flavonoid for 48

146 and 72 h. The G2/M phase arrest was associated with a significant decrease of cell percentage in G1 and S phases. This peculiar cell cycle kinetic, evident even at 24 h, occurred again at 48 and 72 h of quercetin treatment. Concerning HSB-2 cell cycle kinetics, the results obtained upon quercetin treatment, suggest that this cell line was mainly drug-resistant since the cell cycle profile, irrespective on the exposure time, did not change significantly, as compared with control untreated cells. Our results were similar to other previous studies, in which quercetin treatment induced a cell cycle arrest in the G2/M phase in various cell types [44, 45]. By contrast, other studies reported that the drug blocked the cell cycle at the G1/S transition in human colon cancer cells or in late G1 in human leukemic T-cells [46, 47], suggesting that quercetin induces cell cycle arrest depending on cell type. Therefore, the mechanism of quercetin is not the same in all cell types. Incubation for up to 72 h of both cell lines with 25 lM quercetin, a concentration much higher than that found in the blood of normal subjects following a Mediterranean diet [48, 49], caused apoptosis with different potency and different time-course. In particular, the higher sensitivity of K562 cells to apoptosis induced by the drug, seems consistent with the simultaneous increase of caspase-3 activity, as well as with the cytochrome c release, but appears independent from ROS production. It has to be noticed that caspase-3 is not included in the early events associated to the apoptotic process but it is responsible for DNA fragmentation at the internucleosomal linkages. Since both caspases-8 and -9 were not significantly increased in the first 24 h of quercetin exposure, the onset of apoptosis could be not dependent from the intrinsic (mitochondrial) pathway. On the other hand, mitochondria play a key role in the propagation of the apoptotic signals since cytochrome c is released in the cytosol in response to many apoptotic inducers [50]. The ‘‘free’’ cytochrome c induces the formation of a complex with Apaf-1 which binds to pro-caspase-9; this complex, in turn, can activate the other pro-caspases, including caspase3 [51]. These results were also confirmed by flow cytometry either in K562 and HSB-2 cells treated with quercetin. Indeed, K562 cells showed a time-dependent increase of subG1 DNA content whereas no apoptosis was detected in HSB-2 cell line. Moreover, DNA ladder could be evidenced only in K562 cells and upon at least 48 h quercetin exposure. Interestingly, K562-quercetin treated cells exhibited a marked increase of intracellular GSH concentration at 24 h (approximately 3-fold the basal value), then followed by a decline to half of the basal value at 72 h, while HSB-2 treated cells displayed GSH levels higher, compared to control, irrespective of the exposure time (Table 1). On the whole, these results indicate a major resistance of HSB-2 cells to apoptosis induced by

quercetin. It is noteworthy to underline that K562 cells are usually reported as a cell type resistant to apoptosis induced by conventional anti-cancer drugs, since the increased expression of Bcl-2 family proteins which display anti-apoptotic potential [38, 52, 53]. Another interesting observation is the intracellular content of GSH of the two cell lines, K562 cells exhibiting a concentration 10-fold higher than HSB-2 counterpart (Table 1). As a consequence, K562 cells should better counteract the eventual oxidative damages, like those induced by some chemical agents or radiations used in anti-tumoral therapy. Surprisingly, in our experimental conditions, the apoptotic propensity of the two cell lines exposed to quercetin exhibited a result opposite to that expected. However, previous reports show that quercetin is able to form toxic adducts with thiol compounds, in particular GSH [6–8]. Therefore, the enrichment of cells in glutathione should enhance their sensitivity to quercetin-induced apoptosis, while the opposite effect should be observed after intracellular glutathione depletion. Our results (shown in Fig. 10) confirm only in part the above-mentioned hypothesis. In fact, GSH depletion not only did not induce a decrease of apoptosis in quercetin-treated cells, but caused instead a mild increase, thus contradicting our belief. On the contrary, glutathione enrichment sensitized both cell lines to apoptosis induced by quercetin, in agreement with literature data [6–8] and with our hypothesis. In the light of these results, it is reasonable that another mechanism of action of the flavonol is working in the two cell types. The conclusion is that glutathione is not the main responsible (or not the alone) for the different sensitivity of K562- and HSB-2 cells to quercetin treatment. On the other hand, it has been reported that the addition of NAC enhances the apoptosis induced by hypoxic conditions on four different tumor cell lines [54]. The authors demonstrate that NAC interferes with the NFjB-DNA binding, thus blocking the transcriptional activation of NFjB induced by hypoxia; as a consequence, caspase-3 activity and increase of apoptosis are triggered. A similar mechanism of enhancement of apoptosis induced by quercetin could be in action also in our experimental conditions, since K562-treated cells which show higher sensitivity as compared to HSB-2 cells, exhibit higher levels of either caspase-3 activity and of GSH. However, it has to be underlined that in that work [54], the researchers used 20 mM NAC, a concentration 20-fold higher than that used in our experiments. In fact, we used 1 mM NAC because it induced the milder apoptotic effect (as reported in Fig. 10), but it caused a significant GSH enrichment in both cell lines. By contrast, a marked apoptosis (together with a large number of necrotic cells) was observed at higher NAC concentrations; therefore they were not used. Quercetin has also been reported as an oxygen-, nitric oxide- and peroxy-radicals scavenger, in addition to

147 display iron chelating ability [43, 55–58]. Examples of the chemico-biological potential properties of quercetin as anti-oxidant are: protection (IC50 = 0.1 lM) of lymphoid cells from cytotoxic effects of oxidized LDL [59], protection (quercetin ‡10 lM) of human normal lymphocytes from H2O2-induced DNA damage [60], protection (EC50 = 20–40 lM) of GSH-depleted cells from oxidative damage [61]. To demonstrate that even in our experimental model quercetin behave as anti-oxidant, K562- and HSB-2 cells were loaded with H2DCF-DA, able to reveal ROS, but unable to discriminate among the various reactive oxygen species. As shown in Fig. 9, both cell lines exhibited a significant decrease of ROS, upon 1 h quercetin exposure that persisted, even if less markedly, up to 24 h of treatment. The attenuation of the inhibition of ROS production by quercetin, at prolonged incubation times, is probably related to chemical modifications of the flavonoid (like glycosylation, methylation and other conjugation reactions) that reduce its anti-oxidant capacity. The simultaneous addition of quercetin and t-BHP (a well-known oxidizing agent) to both cell lines, confirmed the drastic inhibition of ROS exerted by the flavonoid (Fig. 9). Therefore, according to previous reports [5, 56–59], it can be concluded that quercetin exhibit anti-oxidant properties acting as an oxygen radicals scavenger. A recent paper [62] demonstrates that quercetin-treated colon cancer (HT-29) cells show a decrease of Bcl-2 expression together with a down-regulation of ErbB2, ErbB3 and Akt proteins, thus explaining the pro-apoptotic effects of the flavonoid. In addition, it has been shown that proteasome inhibition seems to be responsible for the induction of apoptosis in quercetintreated Jurkat T cells [63]. Finally, a novel hypothesis suggest that the anti-cancer/toxicological potential of quercetin is correlated with changes in the mitochondrial energetics [64]. Therefore, several factors may contribute to the cellular response to quercetin treatment, including the cells/ drug ratio, the cell strain, the degree of metabolic conversion of the flavonoid, the nature and the severity of the injury inferred to cells, the type and the conditions of culture medium [65–67]. The contrasting (opposite) effects of quercetin on DNA integrity as well as on cell membrane function, suggest that the intracellular distribution of the flavonoid, together its concentration, can be pivotal for the cell response to this agent. The multiplicity of effects exerted by quercetin may rise doubts on efforts to exactly define all the patho-preventive functions or the therapeutic advantages, not only of quercetin but also of other flavonoids. Therefore, it needs great caution about the role of these compounds as dietary agents universally effective towards different cellular strains and/or tissue types, such as those characterizing the human organism. However, it is well established that quercetin, as well as other flavonoids display a selective cytotoxicity

against several tumor cell lines, while they are only mildly cytostatic towards normal cells. In conclusion, the results of the present work demonstrate that quercetin triggers the onset and progression of caspase-3 and cytochrome c-dependent apoptosis together with a significant accumulation in G2/M phase in K562 cells, while is almost uneffective in HSB-2 cells. The different response of the two cell types to quercetin exposure appears only partially related to their different glutathione content and other factors possibly involved require further studies to be elucidated. Since quercetin exhibits anti-oxidant capacity in either K562 or HSB-2 cells, this property could be, for humans, of physiological relevance depending on a dietary intake rich in fruits and vegetables.

Acknowledgment This work was partially supported by the ‘‘Ministero dell’Universita` e della Ricerca Scientifica’’ (PRIN 2004) to AB.

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