The role of activated MEK-ERK pathway in quercetin-induced growth inhibition and apoptosis in A549...

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The role of activated MEK-ERK pathway in quercetin-induced growth inhibition and apoptosis in A549 lung cancer cells.

Nguyen, T.T.T., Tran, E., Nguyen, T.H., Do, P.T., Huynh, T.H., and Huynh*, H.

Laboratory of Molecular Endocrinology, Division of Cellular and Molecular Research, National Cancer Centre of Singapore, Singapore 169610

*Correspondence to:Hung HuynhLaboratory of Molecular EndocrinologyDivision of Cellular and Molecular ResearchNational Cancer Centre of SingaporeSingapore 169610Phone: +65 6436 8347Fax: +65 6226 5694E-mail: [email protected]

Running Title: Quercetin induces apoptosis in A549 cells.Key words: Activated ERK, growth inhibition, apoptosis, quercetin

; a .# Oxford University Press ll rights reserved2003arcinogenesisC

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Abstract

Dietary phytochemicals have been shown to be protective against various types of cancers.

However, the precise underlying protective mechanisms are poorly understood. In the present study,

we report that treatment of A549 cells with quercetin resulted in a dose-dependent reduction in cell

viability and DNA synthesis with the rate of apoptosis equivalent to 1.2 ± 0.8, 6.3 ± 0.9, 16.5 ± 1.5,

36.4 ± 2.6, and 42.5 ± 5.8 % on treatment with 0.1% DMSO, 14.5, 29.0, 43.5 and 58.0 µM

quercetin, respectively. Concomitantly, quercetin treatments led to a 1.1-, 1.1-, 2.5- and 3.5-fold

increase in Bax. Similar elevations were also observed in Bad, which increased 1.1-, 2.1-, 2.2- and

2.3-fold respectively as compared to control. While Bcl-2 was decreased by 30%, Bcl-xL was

elevated in a dose-dependent fashion. Quercetin also induced the cleavage of caspase-3, caspase-7

and PARP (poly ADP-ribose polymerase). While Akt-1 and phosphorylated Akt-1 were inhibited,

the extracellular signal-regulated kinase (ERK) was phosphorylated following quercetin treatment

in a dose-dependent fashion. Phosphorylation of ERK and c-Jun occurred at 3 h and was sustained

over 14 h. Phosphorylation of MEK1/2 was increased in concordance with ERK activation.

Quercetin-induced phosphorylation of JNK and cleavage of caspase-3 occurred 6 h after quercetin

exposure and before cleavage of caspase-7 and PARP was detected. Inhibition of MEK1/2 but not

PI-3 kinase, p38 kinase or JNK abolished quercetin-induced phosphorylation of c-Jun, cleavage of

caspase-3 and -7, cleavage of PARP, and apoptosis. Inhibition of caspase activation completely

blocked quercetin-induced apoptosis. Expression of constitutively activated MEK1 in A549 cells

led to activation of caspase-3 and apoptosis. The results suggest that in addition to inactivation of

Akt-1 and alteration in the expression of the Bcl-2 family of proteins, activation of MEK-ERK is

required for quercetin-induced apoptosis in A549 lung carcinoma cells.

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Introduction

Lung cancer is one of the most common cancers in many countries and accounts for 28% of all

cancer death (Reviewed in (1,2)). Approximately 75-85% of lung cancer is non-small cell lung

cancer and the rest is small cell lung cancer (3). The average five-year survival rate for localized

lung cancer was 48% compared to 14.5 % overall and 2.5 % for a metastatic lung cancer. Five-year

survival rate after surgical resection was approximately 60% for stage I lung cancer patients (4).

Unfortunately, only 15% of people are diagnosed at an early, localized stage because most lung

cancer begins to grow silently without any symptoms until the cancer is in an advanced stage (5).

Thus, there is an urgent need for novel diagnosis, prevention and/or treatment of lung cancer.

Epidemiological studies have shown that the consumption of vegetables, fruits and tea is associated

with a low risk of cancer (6). Many natural dietary phytochemicals found in fruits, vegetables,

spices and tea have been shown to be protective against cancer in various animal models (7,8). The

most common flavonoid glycones found in the diet are quercetin, rutin and robinin (9). Although

the mechanisms by which quercetin exerts its anti-proliferative and apoptotic activities remain to be

elucidated, there is evidence suggesting that the action of flavonoids is probably mediated by either

interaction with the type II estrogen binding sites (10) or aryl hydrocarbon receptor (11). Quercetin

has been shown to inhibit the enzymes involving in proliferation and signal transduction pathway

including protein kinase C (12), tyrosine kinase (13), cdc25 phosphatase (14), PI-3 kinase (15),

DNA topoisomerase II (16), proline-directed protein kinase fatty acid in human prostate carcinoma

cells (17), Na+K+ATPase (18) and JNK (19). Quercetin has a wide range of biological activities

including inhibition of mutant p53 expression (20), and androgen receptor expression and function

in LnCap cells (21). It has anti-proliferative activity in vitro against several cancer cells of human

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origin (22-26). It blocks the cell cycle at the G1/S transition in colon and gastric cancer as well as in

leukaemic cells (27,28) but causes a G2/M block in breast and laryngeal cancer cell lines or in non-

oncogenic fibroblasts (20,29,30). Quercetin potentiates the cytotoxic action of 1-ß-D-

arabinofuranosylcytosine (31). Quercetin also inhibits cell invasion and induces apoptosis through a

pathway involving heat shock proteins (32). These activities of quercetin make it a promising

candidate for treatment and prevention of various cancers.

One of the most frequent targets downstream of receptor and non-receptor tyrosine kinases and the

ras family of GTP-binding proteins is the extracellular related kinase (ERK) signal transduction

pathway (33). Constitutive MEK1 activation contributes to cell survival (33), migration (34),

transformation of fibroblasts and epithelial cells (35). Studies with small molecule inhibitors of

MEK activity (36) demonstrate a role for MEK in mediating expression of proteinases implicated in

invasion and metastasis (37), and disruption of normal epithelial morphology (38). Treatment of

cells with various growth factors or chemotherapeutic agents produces activation of MEK1/2 and

its downstream target, ERK, resulting in proliferation and differentiation (Reviewed in (33)). ERK

activation may exert either anti-apoptotic (Reviewed in (33)) or pro-apoptotic (39) influence

depending upon the cellular context.

Regulation of apoptosis is a complex process and involves a number of cellular genes, including

Bcl-2 (40), and Bcl-2 related family members such as Bcl-xL, Bcl-xs, Bad, and Bax (41). The Bcl-2

gene product protects cells against apoptosis in a variety of experimental systems. Suppression of

Bcl-2 has been shown to promote apoptosis in response to a number of stimuli, including anti-

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cancer drugs (40). Bcl-2 and Bcl-xL exert their anti-apoptotic effect, at least in part by binding to

Bax and related pro-apoptotic proteins. They also prevent Bax and pro-apoptotic proteins from

inducing the release of cytochrome c and activation of the caspase-9. Recent work into apoptosis

has demonstrated the importance of PI-3 kinase and its downstream substrate protein kinase B

(Akt) (42). Akt exerts an anti-apoptosis effect against various stimuli (42) and confers resistance to

taxol (43). A direct link between the PI-3 kinase and apoptosis-regulating proteins was established

through Akt phosphorylation of Bad (44,45).

We demonstrate herein that quercetin inhibited the A549 cell growth and induced apoptosis.

Quercetin-induced apoptosis was associated with inactivation of Akt-1 protein, altered expression

of Bcl-2 family of proteins and phosphorylation of ERK. Our studies, employing transfection study

and pharmacological inhibitors for MEK-ERK and caspase revealed that MEK-ERK activation was

required to trigger caspase-3 activation and that MEK-ERK blockage modified the cytotoxicity of

quercetin, indicating that MEK-ERK activation in A549 cells linked to cell death.

Materials and Methods

Reagents: U0126, PD98059, LY294002, rabbit anti-phospho MEK1/2 (Ser217/221), rabbit anti-

cleaved caspase-7 (20 kDa), rabbit anti-caspase-3, rabbit anti-phospho Akt (Ser473), mouse anti-

phospho p44/42 ERK (Thr202/Tyr204), rabbit anti-Akt, mouse anti-ERK and rabbit anti-cleaved

PARP antibodies were purchased from New England Biolabs, Beverly, MA. Caspase inhibitor Z-

VAD-FMK was from Promega, Madison, MI. A c-Jun N-terminal kinase (JNK) specific inhibitor

SP600125, a p38 kinase inhibitor SB203580 and G418 were supplied by Calbiochem, La Jolla, CA.

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Mouse anti-Bax, mouse anti-phospho-c-Jun (Ser63), mouse anti-phospho-JNK (Thr183/Tyr185),

mouse anti-α-tubulin, rabbit anti-Bcl-2, rabbit anti-Bcl-xL, rabbit anti-Bad antibodies were obtained

from Santa Cruz Inc., Santa Cruz, CA. Horseradish peroxidase-conjugated donkey anti-mouse or

anti-rabbit secondary antibodies were purchased from Pierce, Rockford, Illinois. Chemiluminescent

detection system was supplied from Amersham, Pharmacia Biotech, Arlington Heights, IL. Tissue

culture petri-dishes, 6-well plates, 96-well plates, 100 mm tissue culture petri-dish, and 8-chamber

slides were purchased from Nunc Inc. Naperville, IL. Quercetin and 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma, Saint Louis, MO. Cell

Proliferation ELISA Kit (BrdU, colorimetric assay) and In Situ Cell Death Detection Kit

(Fluorescein) were supplied from Roche Diagnostics Corporation, Indianapolis, IN. RPMI 1640

medium, fetal bovine serum (FBS), Lipofectamine reagent and penicillin-streptomycin were from

Gibco-BRL, Grand Island, NY.

U0126, PD98059, SP600125, SB203580, LY294002 and caspase inhibitor Z-VAD-FMK

compounds were dissolved in dimethylsulfoxide (DMSO) with final concentration never exceeding

0.1%. Quercetin was dissolved in DMSO at a concentration of 100 mg/ml. They were stored frozen

under light-protected conditions at -20 0C.

Cell culture and treatment: Human A549 lung epithelial cells were obtained from American Type

Culture Collection and cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin

and streptomycin (growth medium) at 37 0C in a 5% CO2 incubator. It has been documented that

A549 cells are pRB-positive (46) and express wild-type p53 (47). To study the effects of quercetin

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on cell morphology, A549 cells were seeded onto 6-well plates at a density of 5 x 104 per well in

the growth medium. Confluent cells were washed with serum-free RPMI 1640 (SRF) medium and

then incubated further with SRF medium for 6 h. Cells were then treated with 14.5, 29.0 and 58.0

µM of quercetin in SRF medium for 14 h. Photographs were taken using the inverse microscope

(Nikon TMS, Tokyo, Japan).

Cell transfection: To examine whether constitutively activated MEK1 had any impact on the

survival of lung cancer cells, A549 cells were transfected with 10 µg of the pß-MEKDD or

pcDNA-3 control plasmid DNA and 28 µl of Lipofectamine reagent following the manufacturer’s

recommendation. The pß-MEKDD was the constitutively activated MEK1 cDNA, kindly provided

by Professor Phillip Leder (48). Forty-eight hours post-transfection, cells were subcultured at a ratio

of 1:10 and replaced with selective growth medium containing 800 µg/ml G418. Three weeks post-

transfection, pooled clones were expanded. To study the effects activated MEK1 on quercetin-

induced apoptosis, constitutively activated MEK1-transfected or pcDNA-transfected cells were

seeded at a density of 1 x 106 cells per 100 mm petri-dish and allowed to grow in growth medium

for 24 h. Cells were treated with indicated concentrations of quercetin in serum free RPMI for 14 h

and then harvested for analysis.

Detection of apoptosis: A549 cells were plated onto 8-chamber slides at a density of 5 x 103 cells

per well and allowed to grow in the growth medium for 24 h. Cells were then washed once with

SRF medium and treated with indicated concentrations of quercetin in fresh SRF medium for 14 h.

Cells were fixed with phosphate buffer saline (PBS) containing 4% formaldehyde for 1 h at room

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temperature, washed with PBS and stored at -80 0C until analysis. Apoptosis was detected by the

terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay using the

In Situ Cell Death Detection Kit (Roche) as described by the manufacturer. Slides were visualized

under fluorescent microscope (Olympus BX60) equipped with an FITC filter. Labelling indices

were obtained by counting cell number of labelled cells among at least 500 cells per region and

expressed as a percentage values.

Cell viability and proliferation: To study the effects of quercetin on cell proliferation and

viability, A549 cells were plated at 1x104 cells per well in 96-well plates and allowed to grow in the

growth medium for 24 h. Cells were then washed once with SRF medium and allowed to grow in

SRF medium for another 24 h. Cells then treated with indicated concentrations of quercetin in fresh

SRF medium for 24 or 48 h. Cell proliferation and cell viability were determined at 24 and 48 h

after treatment using the Cell Proliferation ELISA Kit and the MTT assay respectively as described

(49). Experiments were repeated at least 3 times, and the data were expressed as the mean ± SD.

Western blot analysis: To examine the effects of quercetin on Bax, Bad, Bcl-2, Bcl-xL, cleaved

caspase-3 and -7, cleaved PARP, PI-3 kinase, Akt, and phosphorylation of ERK, c-Jun, JNK, and

Akt. A549 cells were plated at a density of 5 x 106 cells per 100 mm petri-dish in the growth media.

After 24 h, the cell monolayer was washed and then treated with indicated concentrations of

quercetin in SRF medium as described above. Following the treatment, cells were harvested at

indicated times, lysed in a lysis buffer and Western blot analysis was performed using total cell

lysate as described (50). For each lane 100 µg of protein were loaded. Blots were incubated with the

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indicated antibodies and 1:7500 horseradish peroxidase-conjugated donkey anti-mouse or anti-

rabbit secondary antibody. All the primary antibodies were used at the final concentration of 1

µg/ml. Blots were then visualized with a chemiluminescent detection system as described by the

manufacturer.

Statistical analysis: For quantitation analysis, the sum of the density of bands corresponding to

protein blotting with the antibody under study was calculated, and normalized to the amount of α-

tubulin. After normalisation with α-tubulin, changes in the expression of the protein under study in

treated samples were expressed relative to the basal levels of this protein in untreated sample.

Differences in cell proliferation, apoptosis and the levels of proteins under study were analyzed by

the Kruskal-Wallis test.

Results

For the time-course and dose-response experiments, human A549 lung cancer cells were treated

with 14.5, 29.0, 43.5 and 58.0 µM of quercetin for 24 and 48 h. Cell viability and cell growth were

the assessed by the MTT assay and BrdU incorporation, respectively. Control cells were treated

with 0.1% DMSO alone. Figure 1 shows that quercetin caused a dose-dependent reduction in DNA

synthesis (Fig. 1A) and cell viability (Fig. 1B). Significant inhibition in BrdU incorporation was

observed as early as 24 hours post-treatment (p<0.01). Fifty percent reduction in cell viability was

seen at a dose of 29.0 µM after 48 h incubation (Fig. 1B). To determine if quercetin reduced cell

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number by inducing apoptosis, TUNEL assay was performed. Figure 2A shows that DNA

fragmentation was detected in quercetin-treated cells. In cells treated with 0.1% DMSO, 14.5, 29.0,

43.5 and 58.0 µM of quercetin for 24 h, approximately 1.2 ± 0.8, 6.3 ± 0.9, 16.5 ± 1.5, 36.4 ± 2.6,

and 42.5 ± 5.8% of apoptotic cells were observed, respectively (Fig. 2B). The percentage of

apoptotic cells in the treatment groups compared to the control group were statistically significant at

p<0.01 as determined by the Kruskal-Wallis test.

Because apoptosis in mammalian cells has been shown to be regulated by Bax, Bcl-xL, Bad, Bcl-2

(41), we determined whether quercetin-induced apoptosis in A549 cells was also associated with

the modulation of these proteins. To test this possibility, cell lysate from A549 cells treated with

different concentrations of quercetin was examined by Western blot analysis. As shown in figure 3,

quercetin induced a significant elevation in the expression of pro-apoptotic Bax and Bad. Treatment

of A549 cells with 14.5, 29.0, 43.5 and 58.0 µM of quercetin for 14 h led to 1.1-, 1.1-, 2.5- and 3.5-

fold increase in Bax. Similar elevations were observed in Bad levels, which increased 1.1-, 2.1-,

2.2- and 2.3-fold respectively in the quercetin treatment as compared to controls. While Bcl-2

expression was decreased by approximate 30%, the anti-apoptotic Bcl-xL expression was increased

by 2.0-, 2.3-, 2.4-, and 2.5-fold on treatment with 14.5, 29.0, 43.5 and 58.0 µM of quercetin,

respectively (Fig. 3D). Because phosphorylation of Bad at Serine 112 and 136 created consensus

sites for interaction with 14-3-3 protein; phosphorylated Bad then bound to 14-3-3 instead of Bcl-2

or Bcl-xL, resulting in the liberation of the anti-apoptotic proteins and the consequent promotion of

cell survival (51), we determined the phosphorylation status of Bad following quercetin treatment.

Using anti-phospho-specific Bad (Ser112) and Bad (Ser136) antibodies, we observed that Bad was

not phosphorylated at these positions (data not shown). The results indicate that quercetin-induced

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apoptosis in A549 cells is associated with the accumulation on Bax, Bad, Bcl-xL and the decrease in

Bcl-2 proteins.

It has been demonstrated that the proteolytic cleavage of PARP, which synthesizes poly(ADP-ribo)

from ß-nicotinamide adenine dinucleotide (NDA) in response to DNA strand breaks, is an

biochemical event during apoptosis (52). Since PARP cleavage is a hallmark of caspase activation,

we determined whether the apoptosis machinery was activated by quercetin treatment, using an

anti-specific-cleaved PARP antibody that detects only cleaved products of PARP. As shown in

Figure 4E, the 89 kDa cleaved PARP fragment was detected in quercetin-treated samples. Since

caspase-3 and -7 have a central role in PARP cleavage (52), we determined the activation of these

two caspases by Western blot analysis using antibodies capable of detecting activated (cleaved)

caspase-3 and -7. Figure 4C shows that the cleaved forms of caspase-3 (19 and 17 kDa fragments)

were readily detectable at the dose as low as 14.5 µM of quercetin and reached high levels at the

dose of 29.0 µM. Cleaved caspase-7 fragments (19 and 20 kDa) were also observed in concordance

with caspase-3 activation (Fig. 4D). The data indicate that quercetin induced the activation of

caspase -3 and -7.

PI-3 kinase pathway is regulated by a variety of growth factors (43,44). Recent work on apoptosis

signalling has demonstrated the importance of PI-3 kinase and its downstream substrate, Akt

(42,53). In addition, a direct link between PI-3 kinase and apoptosis-regulating protein Bcl-2 family

of proteins has been established through Akt phosphorylation of Bad (44,45). We determined the

effects of quercetin on the levels of p85 subunit of PI-3 kinase, Akt-1 and phosphorylated Akt-1 in

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A549 cells. Figure 5B shows that the expression of p85 subunit of PI-3 kinase was not altered

following quercetin treatment. However, treatment of A549 cells with 14.5, 29.0, 43.5 and 58.0 µM

of quercetin for 14 h resulted in a 30, 44, 48 and 58% decrease in total Akt protein (Fig. 5D). The

basal phosphorylation of Akt-1 was sharply reduced and barely detectable in cells treated with 29.0

µM of quercetin (Fig. 5C). The results indicate that quercetin was more effective in inhibiting the

phosphorylation of Akt-1 than Akt-1 expression. Subsequent blotting with anti-α-tubulin antibody

showed relatively equal amounts of total protein loaded per lane.

It has been demonstrated that the ERK signalling pathway is activated in response to certain cellular

stresses (Reviewed in (54)). To investigate whether quercetin induced growth arrest and apoptosis

in A549 cell was associated with the activation of ERK, cell lysate from quercetin-treated cells at

different times were subjected to Western blot analysis using an anti-phospho-ERK antibody to

detect phosphorylated (and therefore activated) ERK. The same blots were subsequently stripped

and reblotted with an antibody that recognized total ERK to verify equal amounts of the protein in

various samples. As shown in figure 6, treatment of A549 cell with 14.5, 29.0, 43.5 and 58.0 µM of

quercetin, all of which induced apoptosis, led to a dose-dependent activation of ERK (Fig. 6D). The

phosphorylation of MEK1/2 was increased in a dose-dependent manner following quercetin

treatment over the same time frame as seen for ERK (Fig. 6B). Because c-Jun is a target for ERK

activity, the levels of c-Jun phosphorylation were determined. Figure 6F shows that c-Jun was

phosphorylated following quercetin treatment and the pattern of c-Jun phosphorylation was similar

to ERK activation suggesting that phosphorylation of ERK by MEK1/2 increased ERK activity.

Since JNK activation is thought to be a determining factor in cell cycle arrest and apoptosis (55),

we set out to determine whether quercetin-induced apoptosis in A549 lung cancer cells was also

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associated with the activation of JNK/SAPK pathway. The activation of JNK was determined by

examining its degree of phosphorylation by Western blot analysis with anti-phospho-JNK1/2

antibody. Figure 6G shows that quercetin significantly enhanced the phosphorylation of a 54 kDa

JNK2 without significant increase in the basal phosphorylation of 46 kDa JNK1. Blocking of JNK

activity with JNK inhibitor SP600125 did not prevent quercetin-induced apoptosis (Fig. 9)

suggesting that JNK is not a mediator of quercetin-induced apoptosis.

To study the time-course of MEK1/2, ERK, c-Jun, caspase-3, caspase-7 and JNK activation, cells

were treated with 0.1% DMSO or 58.0 µM of quercetin and then harvested at different times for

Western blot analysis. As shown in Figure 7D, activation of ERK was detected as early as 3 hours,

reached maximum levels 6 h after quercetin treatment and sustained over the 14 h period (Fig. 7D).

The result was corroborated by demonstrating that ERK kinase activity was increased by 4-fold 6 h

after quercetin treatment as determined by the levels of c-Jun phosphorylation (Fig. 7F). MEK1/2

was also phosphorylated by quercetin treatment over the same time frame as seen for ERK (Fig.

7B). Phosphorylation of the 54 kDa JNK2 was detectable 6 h after quercetin exposure (Fig. 7G).

Cleaved forms of caspase-3 and -7 were clearly detected 9 h after quercetin treatment and became

more pronounced at 14 h (Fig. 7H and 7I). Significant PARP (89 kDa) cleavage was detected 14 h

after quercetin treatment (Fig. 7K).

It has been demonstrated that ERK activation may exert pro-apoptotic influence depending upon

the cellular context (39,55). To determine whether quercetin-induced apoptosis is mediated by the

activation of ERK, two approaches were used. First strategy was to treat the cells with

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pharmacologic blockers of MEK1/2 to inhibit quercetin-induced ERK activation and their

downstream effects. Second approach was to express the constitutively activated MEK1 in these

cells. Quercetin, in combination with MEK1/2 inhibitors U0126 and PD98059, or a PI-3 kinase

inhibitor LY294002, or p38 kinase inhibitor SB203580, or JNK inhibitor SP600125 or caspase

inhibitor Z-VAD-FMK were also used to treat human A549 lung carcinoma cells. The apoptosis

was measured by TUNEL assay. The levels of cleaved caspase-3, cleaved caspase-7, and cleaved

PARP were also investigated. TUNEL assay showed that quercetin and combined quercetin-

LY294002 caused apoptosis in A549 cells (Fig. 8D & 8F). Co-treatment of A549 cells with

quercetin and U0126 or PD98059 completely blocked quercetin-induced apoptosis as determined

by TUNEL assay (Fig. 8E). Western blot analysis revealed that quercetin alone significantly

increased phosphorylation of ERK, c-Jun, cleaved PARP, cleaved caspase-3 and cleaved caspase-7

(Fig 9). Like quercetin, both LY294002 and U0126 were capable of inducing 54 kDa JNK2

phosphorylation. Unlike U0126 and PD98059, LY294002 alone caused a mild increase in cleaved

PARP, cleaved caspase-7, cleaved caspase-3 (Fig. 9) and apoptosis (Fig. 8C). Co-treatment of cells

with quercetin and U0126 or PD98059 prevented quercetin-induced phosphorylation of ERK,

phosphorylation of c-Jun, cleavage of caspase-3 and-7 and cleavage of PARP (Fig. 9). Quercetin-

induced 54 kDa JNK2 phosphorylation was also attenuated by U0126-quercetin or PD98059-

quercetin treatment (Fig. 9E). Blocking PI-3 kinase, p38 kinase and JNK activity neither potentiated

nor prevented quercetin-induced apoptosis, cleavage of caspase-3 and -7 and cleavage of PARP

(Fig. 9). Inhibition of caspase activation by Z-VAD-FMK abolished quercetin-induced cleavage of

caspase-3 and PARP and apoptosis (Fig. 9). The results suggest that activation of MEK-ERK plays

an important role in quercetin-induced apoptosis and ERK acts upstream of caspase-3 and -7 to

exert its apoptotic influence in the quercetin-treated A549 cells.

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Since blocking of MEK activity by chemical inhibitors prevented quercetin-induced apoptosis in

A549 cells, we wished to determine if constitutive activation of MEK1 in A549 cells would make

the cells more sensitive to quercetin-induced apoptosis. A549 cells were transfected with pcDNA-3

or constitutively activated MEK1 (48). After 3 weeks of selection in G418, pooled mock-

transfected and constitutively activated MEK1-transfected cells were treated with 0.1% DMSO or

58 µM of quercetin for 14 h and harvested for analysis. Because cleaved PARP by activated

caspase-3 and -7 has been proposed as one of the events in the execution phase of apoptosis, the

levels of cleaved caspase-3, -7 and PARP were used as markers for apoptosis. As shown in figure

10, cleaved caspase-3, -7 and PARP were detected in activated MEK1-transfected cells grown in

serum free medium. In contrast, these apoptotic markers were not detected in mock-transfected

cells in the absence of quercetin. Furthermore, activated MEK1-transfected cells were more

susceptible to quercetin-induced apoptosis than mock-transfected cells as determined by the

increased levels of apoptotic markers and TUNEL assay (Fig. 10G). The results further support the

important role of activated MEK-ERK in quercetin-induced apoptosis in A549 cells.

Discussion

The relationship between diet and cancers has been implicated in several epidemiological studies

(6). The cancer incidence is significantly lower in people whom diet consists largely of fruits and

vegetables than people whom diet consists mainly of animal products (6). The results from several

studies indicate that vegetables and fruits contain components that have anti-proliferative and anti-

neoplastic properties (56,57). In the present study, we have shown that quercetin inhibits human

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A549 lung cancer cell proliferation and induces apoptosis. TUNEL assay clearly shows DNA

fragmentation. In addition to the inhibition of Akt-1 phosphorylation, alteration in the expression of

Bcl-2 family of proteins, sustained activation of ERK is required for quercetin-induced apoptosis in

A549 cells. Quercetin treatment results in dose- and time-dependent activation of ERK. The

elevated ERK activity contributed to apoptosis by quercetin is supported by the observations that

activation of ERK by expression of activated MEK1 induces apoptosis while inhibition of ERK by

MEK inhibitors blocks quercetin-induced cell death. Quercetin-induced apoptosis is associated with

phosphorylation of c-Jun, PARP cleavage, caspase -3 and -7 cleavage, all of which can be reduced

by treatment of A549 cells with the MEK1/2 inhibitor. Our findings suggest that in addition to the

inhibition of Akt activation and alteration in the ratio between the anti-apoptotic and pro-apoptotic

proteins, ERK activation plays an important role in mediating quercetin-induced apoptosis in A549

cells and ERK functions upstream of the caspase activation to initiate the apoptotic signal.

After ingestion, the major circulating metabolites in rat blood are quercetin glycosides, quercetin

glucoronides and quercetin sulfates. It remains to be determined whether quercetin is metabolised

to more or less active metabolites. Our preliminary data indicate that the concentration of quercetin

and its metabolites was 25.1, 43.3 and 54.3 µM after 10 days of treatment with 50, 100 and 150 mg

of quercetin per kg body weight respectively (our unpublished data). The results suggest that the

concentrations of quercetin employed in the present study can be achieved in vivo.

Two major distinct apoptotic pathways have been described for mammalian cells. One involves

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caspase-8, which is recruited by the adapter molecule Fas/APO-1 associated death domain protein

to death receptors upon extracellular ligand binding (58). We do not observe any change in either

Fas or FasL expression in quercetin-treated A549 cells (data not shown). Administration of

quercetin results in a significant increase in the expression of Bax and Bad. Furthermore, the levels

of Bcl-xL are elevated while the expression of Bcl-2 protein is reduced by quercetin. Overall, there

is a shift in the ratio between the anti-apoptotic and pro-apoptotic proteins following quercetin

treatment. Although the contribution of Bcl-2 family of proteins to quercetin-induced apoptosis is

not examined in the present study, it is possible that the reduction in Bcl-2 by quercetin allows less

Bcl-2-Bax complex. Furthermore, inactivation of Akt prevents Akt-1 from phosphorylating Bad on

the serine 112 and serine 136. As a result, Bad becomes bound to Bcl-2, and its pro-apoptotic

activity is effectively increased from the death-regulation equation. Elevation of non-

phosphorylated Bad by quercetin further helps to sequester more Bcl-2 and Bcl-xL. The net effect is

more free Bax which then translocates to the mitochondrial membrane and induces the opening of

the mitochondrial permeability transition pore, a critical event in the loss of cell viability (59).

Anti-apoptotic effects of PI-3K are due to its activation of serine/threonine protein kinase Akt. This

kinase has been shown to block apoptosis via several mechanisms (60). In the present report, we

observe that quercetin inhibits Akt and Akt phosphorylation. Because Akt is a downstream target of

PI-3 kinase, the observed inhibition of Akt phosphorylation suggests that quercetin also inhibits PI-

3 kinase. This argument is supported by previous studies showing that quercetin is an inhibitor of

PI-3 kinase and serine/threonine protein kinases (12,13,15,19). By suppressing the activation of

Akt-1, quercetin can promote apoptosis via several pathways. Inactivation of Akt would prevent

Akt-1 from phosphorylating Bad on the serine 136. As a result, Bad becomes bound to Bcl-2, and

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its pro-apoptotic activity is effectively increased in the death-regulation equation. Although the

effects of quercetin on the gene encoding A1/Bf11 protein is not examined in the present study, it is

possible that inhibition of Akt-1 phosphorylation by quercetin might impair the ability of Akt to

transactivate the gene encoding A1/Bf11 protein which, under certain experimental conditions, is

essential for the release of cytochrome c and/or AIF from the mitochondria (61).

Previous studies indicate that JNK/SAPK activation is associated with apoptosis (55,62). In our

own study using A549 lung cancer epithelial cells, we also find that 54 kDa JNK2 is activated in

response to quercetin, LY294002 and U0126 treatments. Our observation is different from the

previous study (19) showing that JNK activation by angiotensin II in cultured rat aortic smooth

muscle cells is inhibited by quercetin. Therefore, the effects quercetin on JNK may differ

significantly depending on the cell context. In U0126- and LY294002-treated cells, phosphorylation

of 54 kDa JNK2 takes place in the absence and in the presence of mild apoptosis respectively.

Furthermore, inhibition of JNK activity by SP600125 did not prevent quercetin-induced apoptosis.

Therefore, it is unlikely that activation of JNK plays an important role in quercetin-induced

apoptosis of A549 cells.

In the present study, we have provided the evidence that activation of MEK-ERK plays a dominant

role in quercetin-induced activation of caspase-3 and -7 which is necessary for cleavage of PARP

and apoptosis in A549 lung cancer cells. Quercetin treatment results in high and sustained

activation of ERK in A549 cells. One important difference between the quercetin and IGF-I

induced ERK activation is the time and duration of activity (data not shown). In the case of IGF-I,

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ERK activation is rapid, occurring within minutes of treatment, and transient. With quercetin,

significant activation occurs at 3 h, but the activity remains highly elevated throughout the

experiment (up to 14 h). Utilizing U0126 or PD98059 to modulate ERK activity, we find that

inhibition of MEK-ERK activation abolishes quercetin-induced apoptosis. Our results are similar to

other studies which demonstrate that abrogation of ERK pathway by taxol delayed or failed to

prevent taxol-induced apoptosis (63,64). However, it is not a universal feature of mammalian cells

as activation of MEK-ERK pathway has been shown to contribute to cell proliferation and survival

(33), migration (34) and transformation (35). Furthermore, inhibition of stress-induced signalling

via the MEK-ERK pathway can potentiate the toxic effects of chemotherapic drugs and irradiation

(65). Therefore, the ability of MEK-ERK pathway to regulate proliferation versus survival appears

to depend on cell types and the amplitude and duration of ERK activation. A short activation of

MEK-ERK cascade by growth factors such as IGF-I is associated with proliferation while

prolonged activation of ERK activity inhibits DNA synthesis.

The questions remaining are: how does quercetin induce apoptosis of A549 cells and what are the

mechanisms responsible for transmitting the signal to the cell nucleus. It has been proposed that the

anti-proliferative and apoptotic effects of quercetin are also mediated via non-estrogen receptor

regulated mechanisms (20,29,30). Although the precise mechanisms of the anti-proliferation and

apoptosis of quercetin are unknown, there is evidence suggesting that the action of flavonoids is

probably mediated by interaction with the aryl hydrocarbon receptor (11) or type II estrogen binding

sites (10) which have been shown to occupy by a flavonoid-like molecule with growth inhibitory

properties (66). In addition to activation of MEK-ERK as we report in this study, quercetin is

shown to induce apoptosis through a pathway involving heat shock proteins (32). Other effects of

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quercetin may also contribute to its anti-proliferative activity seen in certain experimental systems

including inhibition of mutant p53 expression in breast cancer cells (20) and blockage of the cell

cycle at the G1/S or G2/M transition (27,28). Quercetin also inhibits various enzymes involved in

proliferation and apoptosis including protein kinase C (12), tyrosine kinase (13), cdc25 phosphatase

(14), PI-3 kinase (12,15), and DNA topoisomerase II (16). This suggests that quercetin exerts

multiple effects on cellular growth and apoptosis. The target proteins observed under one

experimental condition or cell type may differ from one another depending on cell context.

Our data support a role for ERK activation in quercetin-induced growth inhibition and apoptosis in

lung cancer cells. Since quercetin can act in synergy with cisplatin to inhibit lung cancer xenograft

growth (67) and potentiate the cytotoxic action of 1-ß-D-arabinofuranosylcytosine (31), it raises the

possibility that quercetin can be used either as a single agent or in combination with other

chemotherapeutic agents such as cisplatin or 1-ß-D-arabinofuranosylcytosine in treatment and/or

prevention of lung cancer.

Acknowledgements

We thank Professor Phillip Leder (Department of Genetics, Harvard Medical School) for the

plasmid pß-MEKDD plasmid. This work was supported by grants from National Medical Research

Council of Singapore (NMRC/0541/2001), A*STAR-BMRC (LS/00/019) and A*STAR-BMRC

(LS/00/017) to Huynh Hung.

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Figure Legends

Figure 1. Effects of quercetin on the viability and proliferation of A549 cells. A549 lung cancer

cells were grown and treated with serum free RPMI 1640 (SRF) medium containing either 0.1%

DMSO or indicated doses of quercetin for 24 h and 48 h as described under Materials and Methods.

Cell proliferation (A) and cell viability (B) were determined by Bromo-uridine incorporation and

MTT assay respectively as described under Materials and Methods. Experiments were performed in

quadruplicate, with the results reflecting the mean and standard deviation of the quadruplicate of

each group. Bars with different letters are significantly different from one another at p<0.01 as

determined by the Kruskal-Wallis test. The experiments were repeated 3 times with similar results.

Figure 2. Induction of apoptosis by quercetin in A549 cells. A549 cells were grown and treated

with escalating doses of quercetin in SRF medium for 14 h. Apoptotic cells were determined by

TUNEL assay as described in Materials and Methods. (A) Apoptotic cells were visualized under a

fluorescent microscope. The rate of apoptosis expressed as percentage of total cells counted is

shown in (B). Bars with different letters are significantly different from one another at p<0.01 as

determined by the Kruskal-Wallis test. Experiments were repeated 3 times with similar results.

Figure 3. Effects of quercetin on the levels of Bcl-2, Bax, Bad and Bcl-xL in A549 cells. A549

cells were cultured as described under Materials and Methods. Cells were treated with 0.1% DMSO

or indicated concentrations of quercetin in SRF medium for 14 h. Cells were harvested and lysed

for Western blot analysis as described under Materials and Methods. Blots were incubated with

mouse anti-α-tubulin (A), mouse anti-Bax (B), rabbit anti-Bad (C), rabbit anti-Bcl-xL (D) and

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rabbit anti-Bcl-2 (E) antibodies. Changes in the levels of Bax, Bad, Bcl-2 and Bcl-xL proteins after

being normalized to the levels of α-tubulin are shown below each blot. Experiments were repeated

3 times with similar results.

Figure 4. Effects of quercetin on cleavage of caspase-3 and -7 and PARP in A549 cells. A549

cells were cultured as described under Materials and Methods. Cells were treated with SRF medium

containing 0.1% DMSO or indicated concentrations of quercetin for 14 h. Cells were harvested and

lysed for Western blot analysis as described under Materials and Methods. Blots were incubated

with mouse anti-α-tubulin (A), rabbit anti-cleaved caspase-3 (B), rabbit anti-cleaved caspase-7 (20

kDa) (C) and rabbit anti-cleaved PARP (D) antibodies. Changes in the levels of cleaved caspase-3,

cleaved caspase-7 and cleaved PARP after being normalized to the levels of α-tubulin are shown

below each blot. Experiments were repeated 3 times with similar results.

Figure 5. Effects of quercetin on the basal levels of p85 subunit of PI-3 kinase, Akt-1 and

phosphorylated Akt (Ser473) in A549 cells. A549 cells were cultured as described in Materials

and Methods. Cells were treated with SRF medium containing 0.1% DMSO or indicated

concentrations of quercetin for 14 h. Cells were harvested and lysed for Western blot analysis as

described under Materials and Methods. Blots were incubated with mouse anti-α-tubulin (A), rabbit

anti- p85 subunit of PI-3 kinase (B), rabbit anti-phospho Akt-1 (Ser473) (C), and rabbit anti-Akt-1

(D) antibodies. Changes in the levels of the Akt-1 and phospho-Akt-1 after being normalized to the

levels of α-tubulin are shown below each blot. Experiments were repeated 3 times with similar

results.

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Figure 6. Effects of quercetin on the levels of MEK1, ERK, and phosphorylated MEK1/2

(Ser217/221), phosphorylated ERK (Thr202/Tyr204), phosphorylated JNK (Thr183/Tyr185),

and phosphorylated c-Jun (Ser63) in A549 cells. A549 cells were cultured as described under

Materials and Methods. Cells were treated with SRF medium containing 0.1% DMSO or indicated

concentrations of quercetin for 14 h. Cells were harvested and lysed for Western blot analysis as

described under Materials and Methods. Blots were incubated with mouse anti-α-tubulin (A), rabbit

anti-phospho MEK1/2 (Ser217/221) (B), rabbit anti-MEK1 (C), mouse anti-phospho p44/42 ERK

(Thr202/Tyr204) (D), rabbit anti-ERK (E), and mouse anti-phospho c-Jun (Ser63) (F) and mouse

anti-phospho JNK (Thr183/Tyr185) (G). Changes in the levels of phosphorylated ERK,

phosphorylated MEK1/2, phosphorylated JNK and phosphorylated c-Jun after being normalized to

the levels of a-tubulin are shown below each blot. Experiments were repeated 3 times with similar

results.

Figure 7. Time-dependent phosphorylation of MEK1/2, ERK, JNK and c-Jun, and cleavage

of caspase-3 and -7 and PARP in A549 cells. A549 cells were cultured as described under

Materials and Methods. Cells were treated with SRF medium containing 0.1% DMSO or 58.0 µM

of quercetin for 3, 6, 9 and 14 h. Cells were harvested at indicated time and lysed for Western blot

analysis as described under Materials and Methods. Blots were incubated with mouse anti-α-

tubulin (A), rabbit anti-phospho MEK 1/2 (Ser217/221) (B), rabbit anti-MEK1 (C), mouse anti-

phospho p44/42 ERK (Thr202/Tyr204) (D), rabbit anti-ERK (E), mouse anti-phospho c-Jun

(Ser63) (F), mouse anti-phospho JNK (Thr183/Tyr185) (G), rabbit anti-cleaved caspase-3 (H),

rabbit anti-cleaved caspase-7 (I) and rabbit anti-cleaved PARP (K) antibodies. Experiments were

repeated 3 times with similar results.

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Figure 8. Effects of MEK1/2 inhibitor U0126 and PI-3 kinase inhibitor LY294002 on

quercetin-induced apoptosis in A549 cells. A549 lung cancer cells were grown and treated with

SRF medium containing 0.1% DMSO (A), 10.0 µM of U0126 (B), 10.0 µM of LY294002 (C),

58.0 µM of quercetin (D), 58.0 µM of quercetin plus 10.0 µM of U0126 (E), and 58.0 µM of

quercetin plus 10.0 µM of LY294002 (F) for 14 h. Cells were subjected to TUNEL assay as

described under Materials and Methods. Apoptotic cells were visualized under a fluorescent

microscope. Original magnification, X 200.

Figure 9. Effects of MEK1/2 inhibitor U0126 or PD98059, p38 kinase inhibitor SB203580,

JNK inhibitor SP600125, caspase inhibitor Z-VAD-FMK and PI-3 kinase inhibitor LY294002

on quercetin-induced phosphorylation of ERK, c-Jun, JNK, and cleavage of caspase-3, -7,

and PARP in A549 cells. A549 lung cancer cells were grown and treated with SRF medium

containing 0.1% DMSO and indicated concentration of drugs for 14 h. Cells were harvested and

lysed for Western blot analysis as described under Materials and Methods. Blots were incubated

with mouse anti-α-tubulin (A), mouse anti-phospho p44/42 ERK (Thr202/Tyr204) (B), rabbit anti-

ERK (C), mouse phospho c-Jun (Ser63) (D), mouse anti-phospho JNK (Thr183/Tyr185) (E), rabbit

anti-cleaved caspase-3 (F), rabbit anti-cleaved caspase-7 (G) and rabbit anti-cleaved PARP (H)

antibodies. Experiments were repeated 3 times with similar results.

Figure 10. Effects of constitutively activated MEK1 on quercetin-induced ERK, phospho-

ERK (Thr202/Tyr204), cleavage caspase-3, caspase-7 and PARP and apoptosis in mock-

transfected and activated MEK1-transfected A549 cells. Mock-transfected and constitutively

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activated MEK1-transfected A549 cells were cultured as described under Materials and Methods.

Cells were incubated with PSF medium containing 0.1% DMSO or indicated concentrations of

quercetin for 14 h. Total cell lysate was prepared for Western blot analysis as described under

Materials and Methods. Blots were incubated with mouse anti-∝-tubulin (A), mouse anti-phospho-

ERK (Thr202/Tyr204) (B), mouse anti-ERK (C), rabbit anti-cleaved caspase-3 (D), rabbit anti-

cleaved caspase-7 (E) and rabbit anti-cleaved PARP (F) antibodies. All the antibodies were used at

a final concentration of 1 µg per ml. Apoptotic cells were determined by TUNEL assay as described

in Materials and Methods. The rate of apoptosis expressed as percentage of total cells counted is

shown in (G). Bars with different letters are significantly different from one another at p<0.01 as

determined by the Kruskal-Wallis test. Experiments were repeated 3 times with similar results.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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Figure 10

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The role of activated MEK-ERK pathway in quercetin-induced growth inhibition and apoptosis in A549...

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