Effect of flavonoids on cell cycle progression inprostate cancer cells

Takashi Kobayashia, Teruhiro Nakatab, Takejiro Kuzumakia,*

aDepartment of Biochemistry, Yamagata University School of Medicine, Yamagata 990-9585, JapanbDepartment of Urology, Yamagata University School of Medicine, Yamagata 990-9585, Japan

Received 22 August 2001; received in revised form 24 August 2001; accepted 24 August 2001

Abstract

The effect of some flavonoids, which are components of fruits, vegetables, and peas, on the cell cycle progression of human

LNCaP prostate cancer cells has been investigated in this study. Genistein arrested the cell cycle at the G2/M phases, which is

attributed to the suppression of cyclin B expression. In addition, genistein induced the cyclin-dependent kinase inhibitor p21,

which does not depend on p53 activation. Apigenin and luteolin also increased p21 levels, but quercetin did not. Apigenin

induced p21 production through a p53-dependent pathway, but luteolin did so in a p53-independent manner. These results

suggest that flavonoids are potent regulators of cyclin B and p21 for cell cycle progression, which may play some roles in

prevention of carcinogenesis. q 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Genistein; Apigenin; Luteolin; Cyclin; Cell cycle; Prostate

1. Introduction

Flavonoids are structurally related to flavone and

are contained in many fruits, vegetables, and peas.

Epidemiological studies suggest that flavonoids play

an important role in the prevention of carcinogenesis.

The isoflavone genistein, which is the principal isofla-

vonoid contained in soybeans, is thought to be parti-

cularly effective in the prevention of prostate, breast,

and colon cancers [1]. A considerable body of

evidence has been accumulated to indicate that genis-

tein inhibits tumor cell growth in experimental models

[1,2]. Flavonoids are biosynthesized by plants and

have strong antioxidant activity for scavenging free

radicals which are involved in cell damage and tumor

promotion [3]. In addition to its antioxidant activity,

genistein has been reported to have some biochemical

activities; for example, the inhibition of activity of

several protein–tyrosine kinases, including epidermal

growth factor receptor and src tyrosine kinase [4], the

inhibition of topoisomerases and S6 kinase [5,6],

estrogenic/antiestrogenic activities [1,5], and anti-

angiogenetic activity [7]. Apigenin is a member of

the flavone family and is found in high levels in

many vegetables. Apigenin has been reported to inhi-

bit ornithine decarboxylase activity and to inhibit

chemically induced skin tumorigenesis when applied

topically in mice [8].

Flavonoids are known to suppress tumor cell

growth which is mediated by different types of cell

cycle arrest and the induction of apoptosis in several

tumor cell lines. Genistein arrests the cell cycle at the

Cancer Letters 176 (2002) 17–23

0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0304-3835(01)00738-8

www.elsevier.com/locate/canlet

* Corresponding author. Tel.: 181-23-628-5229; fax: 181-23-

628-5230.

E-mail address: kuzumaki@med.id.yamagata-u.ac.jp

(T. Kuzumaki).

G2/M phases and induces apoptosis in gastric cancer

cells, breast cancer cells, prostate cancer cells, and

lung cancer cells [9–13]. In addition, genistein arrests

the cell cycle at G0/G1 in fibroblasts and melanoma

cells [14]. Apigenin, luteolin, and quercetin also arrest

the cell cycle at either the G1 or G2 phase in different

types of cells [15–19]. In terms of regulation of the

cell cycle, cyclin-dependent kinases play a most criti-

cal role. In the G1 and S phases, cdk4 and cdk2

kinases are activated by binding to cyclin D and cyclin

E/A, respectively, and in the G2 and M phases, cdc2

kinase is activated by binding to cyclin B [20]. Cdk4

and cdk2 activities are inhibited by the binding of

inhibitors p21 and p27 [21]. It has been reported

that p21 is potently transactivated by the tumor

suppressor gene product p53, and suppresses tumor-

igenicity in vivo in experimental systems [22–24]. We

report here that cyclin B and p21 are regulated by

flavonoids, and p21 is induced via both p53-depen-

dent and p53-independent pathways.

2. Materials and methods

2.1. Cell culture

Human LNCaP prostate carcinoma cells were

cultured with an RPMI 1640 medium containing 1.5

g/l sodium bicarbonate. Mouse BALB/c 3T3 and SV-

T2 (SV40-transformed BALB/c 3T3) fibroblasts were

cultured in Dulbecco’s modified Eagle’s medium

(DMEM). All media used were supplemented with

200 U/ml penicillin G, 0.1 mg/ml streptomycin

sulfate, and 10% fetal calf serum. The viable cell

number was determined by counting cells that

excluded trypan blue with a microscope. Genistein,

apigenin, luteolin, and quercetin were purchased

from Funakoshi Chemical Co. (Tokyo, Japan) and

dissolved in dimethylsulfoxide. Cultured cells in the

control dishes were treated with the same amount of

dimethylsulfoxide as that in the dishes treated with the

reagents.

2.2. Flow cytometry

The percentage of the cells in each phase of the cell

cycle was determined by means of a flow cytometer

using procedures that have been previously described

[14].

2.3. Immunoblot analysis

Immunoblot analysis using total protein extracts

from cultured cells was performed as previously

described [25]. Antibodies specific for p27 (K25020,

monoclonal), cdc2 (C12720, monoclonal), and cyclin

B (C23420, monoclonal) were purchased from Trans-

duction Laboratories, Inc. (Lexington, KY). Antibody

specific for p21 (sc-397, polyclonal) was from Santa

Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody

specific for p53 (PAb421, monoclonal) was from

Oncogene Research Products, Inc. (Cambridge, MA).

2.4. Immunoprecipitation and cyclin B/cdc2 kinase

activity

Cyclin B-associated and cdc2-associated histone

H1 kinase activities in the proteins which were immu-

noprecipitated with the anti-cyclin B and anti-cdc2

antibodies were measured using previously described

procedures [25].

2.5. Gel retardation assay

Isolation of nuclei from cultured cells and the

extraction of nuclear proteins were performed by the

procedures described by Rana et al. [26]. The gel

retardation assay was performed using nuclear

extracts prepared by the procedures described by

Takano et al. [27]. The sequence of the double-

stranded oligonucleotide used as a probe, which

contains a p53-binding site located in the 5 0-upstream

region of mouse p21 gene [22], is as follows:

TTCAGGAACATGTCTTGACATGTTCAGCCC.

3. Results

3.1. G2/M arrest of the cell cycle and inhibition of

cyclin B/cdc2 kinase activity by genistein

The structures of flavonoids used in our experi-

ments are shown in Fig. 1. Genistein is a derivative

of isoflavone. Apigenin and luteolin are derivatives of

flavone, and quercetin is the member of the flavonol

family. Human prostate cancer LNCaP cells were

used for experiments in this study because prostate

cancer is suggested to be prevented by flavonoids

[1]. Genistein was found to inhibit the cell prolifera-

T. Kobayashi et al. / Cancer Letters 176 (2002) 17–2318

tion of LNCaP cells in a dose-dependent manner (Fig.

2A). High doses of genistein (180 mM) were observed

to increase dead cells, which are, presumably, apop-

totic cells [11]. A flow cytometric analysis showed

that doses of genistein in excess of 60 mM increased

the number of cells at the G2/M phases, which indi-

cates that genistein arrests the cell cycle at G2/M (Fig.

2B). Since it has been established that cyclin B/cdc2

kinase activation is critical for the progression of the

G2/M phases [28], the effect of genistein on cyclin B/

cdc2 kinase activity was examined. Although the

amount of cdc2 protein was only slightly suppressed

by genistein, the amount of cyclin B protein was

strongly suppressed by genistein (Fig. 3A). Both

cyclin B-associated and cdc2-associated histone H1

kinase activities were significantly inhibited by genis-

tein (Fig. 3B). These results suggest that genistein

suppressed cyclin B expression, followed by the inhi-

bition of cyclin B/cdc2 kinase activity, and conse-

quently arrested the cell cycle at G2/M. On the

other hand, apigenin, luteolin, and quercetin did not

significantly affect the cyclin B expression (data not

shown).

3.2. Induction of cyclin-dependent kinase inhibitor

p21 by genistein, which does not depend on p53

activation

We next examined the effect of genistein on the

cyclin-dependent kinase inhibitors p21 and p27. p21

and p27 are key regulators of cell cycle progression

and function by inhibiting the cyclin/cdk activities

T. Kobayashi et al. / Cancer Letters 176 (2002) 17–23 19

Fig. 2. Effect of genistein on proliferation of the cells and the

percentage of cells in G0/G1, S, G2/M phases in human prostate

cancer LNCaP cells. (A) Cell proliferation. Cells (1 £ 105) were

seeded in 35-mm dishes and the exponentially growing cells were

exposed to each concentration of genistein for 72 h, and then the

number of viable cells was determined. The values represent rela-

tive cell numbers. The cell number at the time 0 h was taken as

100%. The values represent the means ^ SD (n ¼ 8). (B) Flow

cytometric analysis. Exponentially growing cells were exposed to

each concentration of genistein for 48 h. A DNA histogram of the

cells stained with propidium iodide was obtained by flow cytometric

analysis. The percentage of cells in each phase was calculated based

on the DNA histogram. The values represent the means ^ SD

(n ¼ 3–4). (W) G0/G1 phase; (X), S phase; (A), G2/M phase.

Fig. 1. Chemical structures of genistein, apigenin, luteolin, and

quercetin.

[21,29–31]. Genistein was found to induce p21

expression in LNCaP cells in a dose-dependent

manner (Fig. 4A). The amounts of p27 protein were

not significantly affected by genistein. It has been

established that p53 is a potent transcriptional activa-

tor of p21 [22]. However, the amount of p53 protein

remained unchanged by genistein treatment (Fig. 4A).

In addition, the gel retardation assay shows that the

DNA-binding activity of p53 to the p53-binding

sequence, which is located in the promoter region of

p21, was not changed by genistein (Fig. 4B). The

shifted bands were competed out by the addition of

an excess of cold oligonucleotide probes. The same

result was obtained in the experiment in which the

synthetic oligonucleotide containing the p53-binding

consensus sequence was used as a probe (data not

shown). These results suggest that, in LNCaP cells,

genistein induces p21 in a p53-independent manner.

3.3. Induction of p21 by apigenin through a p53-

dependent pathway and by luteolin in a p53-

independent manner

Apigenin, luteolin, and quercetin are structurally

T. Kobayashi et al. / Cancer Letters 176 (2002) 17–2320

Fig. 4. Effect of genistein on the amounts of p21, p27, and p53

proteins and DNA-binding activity of p53 in LNCaP cells. (A)

Immunoblot analysis. Exponentially growing cells were exposed

to each concentration of genistein for 16 h. Total proteins were

extracted and analyzed by immunoblot analysis with anti-p21,

anti-p27, and anti-p53 antibodies. (B) Gel retardation assay. Cells

were exposed to 60 mM of genistein for the indicated time. Nuclear

proteins were extracted and incubated with the 32P-labeled double-

stranded oligonucleotide which contains the p53-binding sequence

located in the 5 0-upstream region of the mouse p21 gene. The

protein–oligonucleotide complexes were shifted to the upper

portion of oligonucleotides which did not bind proteins in sodium

dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).

Shifted bands are shown by the arrow. G, genistein.

Fig. 3. Effect of genistein on the amounts of cyclin B and cdc2

proteins, and cyclin B/cdc2 kinase activity in LNCaP cells. (A)

Immunoblot analysis. Exponentially growing cells were exposed

to 60 mM of genistein for the indicated time. Total proteins were

extracted and analyzed by immunoblot analysis with anti-cyclin B

and anti-cdc2 antibodies. (B) Cyclin B/cdc2 kinase activity. Cells

were exposed to 60 mM of genistein for 24 h, and total cellular

proteins were extracted. Immunoprecipitates (IP) formed with

anti-cyclin B antibodies (IP: a-cyclin B) or with anti-cdc2 antibo-

dies (IP: a-cdc2) were analyzed for cyclin B-associated and cdc2-

associated histone H1 kinase activity. C, control; G, genistein.

similar to the isoflavone genistein (Fig. 1), which are

generally contained in fruits and vegetables. In addi-

tion to genistein, apigenin and luteolin induced p21

(Fig. 5A), but quercetin did not. The amounts of p27

protein were not changed by apigenin, luteolin, and

quercetin. Apigenin and luteolin also induced p21 in

mouse BALB/c 3T3 fibroblasts (Fig. 5B). Both

LNCaP cells and BALB/c 3T3 cells produce wild-

type p53 [32,33]. SV-T2 is the established cell line

that was generated by the infection of simian virus

SV40 to BALB/c 3T3 cells. The fact that SV40-

large and small T antigens are produced in SV-T2

cells was confirmed, but was not for BALB/c 3T3

cells (data not shown). It has been reported that

SV40-large T antigen binds to and inactivates p53

[34]. In SV-T2 cells, luteolin was found to induce

p21, but apigenin did not (Fig. 5B). This suggests

that apigenin induced p21 through a p53-dependent

pathway and that luteolin did so in a p53-independent

manner.

4. Discussion

Flavonoids, multi-functional bioactive compounds,

are widely distributed in many plants and are useful

for protection from active oxygen radicals generated

by ultraviolet radiation. Flavonoids have potent anti-

oxidant activity, and it has been suggested that they

prevent chronic diseases, such as cancer, atherosclero-

sis, and allergies. It is noteworthy that evidence has

accumulated to suggest that flavonoids potently

T. Kobayashi et al. / Cancer Letters 176 (2002) 17–23 21

Fig. 5. Effect of apigenin, luteolin, and quercetin on: (A), the amounts of p21 and p27 proteins in LNCaP cells; and (B), the amounts of p21

protein in mouse BALB/c 3T3 and SV-T2 fibroblasts. Exponentially growing cells were exposed to each concentration of apigenin, luteolin, and

quercetin for 24 h. Total proteins were extracted and analyzed by immunoblot analysis with anti-p21 and anti-p27 antibodies. A, apigenin; L,

luteolin; Q, quercetin.

suppress tumor cell proliferation. We showed in this

paper that genistein arrests the cell cycle at the G2/M

phases and suppresses cyclin B expression in LNCaP

cells. It has also been reported that genistein

suppresses cyclin B expression in breast and lung

cancer cells, in addition to prostate cancer cells [10–

13]. The amounts of cyclin B protein in the cells are

regulated both by the transcriptional level of the

cyclin B gene and by the degradation rate of protein.

As the mRNA level of cyclin B is decreased by genis-

tein in breast cancer cells [10], it is possible that genis-

tein decreases cyclin B protein levels by suppressing

the transcription of its gene. The suppression of cyclin

B expression by genistein is thought to be a main

reason for G2/M arrest. It has been established that

p21 regulates G1 and S phase progression by inhibit-

ing cdk4 and cdk2 activities [21]. However, in addi-

tion to this, evidences have recently been accumulated

to suggest that p21 is involved in the suppression of

G2/M phase progression [29–31]. p21 may be

involved in the G2/M arrest triggered by genistein.

We previously reported that genistein arrests the cell

cycle at the G0/G1 phase, accompanied by an increase

in p21 in melanoma cells and fibroblasts [14]. In

LNCaP cells, it is possible that genistein arrests the

cell cycle at G0/G1 because the cell number of S

phase cells decreased in the flow cytometric analysis.

However, we could not confirm by additional experi-

ments that genistein arrests the cell cycle at G0/G1 in

this cell line.

p21 is transactivated via both p53-dependent and

p53-independent pathways [22,35]. Apigenin and

luteolin were also found to induce p21, as well as

genistein. The findings herein show that genistein

and luteolin induce p21 in a p53-independent manner,

but that apigenin did so through a p53-dependent

pathway. p53 is frequently mutated in human cancers

and the mutation rate of p53 in human cancers is over

50% [34]. Our findings suggest that flavonoids

suppress tumor cell growth, not only in tumor cells

that produce a wild-type p53, but also in tumor cells

that produce a mutated p53. Even if the activity of p53

is lost in cancer cells, flavonoids are suggested to be

able to rescue a part of the function of p53 to arrest the

cell cycle. Flavonoids may effectively work as chemo-

preventive agents against carcinogenesis in human

beings. The molecular mechanism for induction of

p21 by flavonoids is not clearly known. Consensus

sequences of the transcription factor Sp1/Sp3-binding

site are located in the promoter region of the p21 gene

[22]. It has been reported that transforming growth

factor b induces p21 by promoting the binding of

the Sp1 and Sp3 to their responsive elements [35]. It

is possible that genistein and luteolin induce p21 via a

Sp1/Sp3-dependent pathway. The activity of p53 is

increased by the protein modification, such as protein

phosphorylation of p53 [34]. It is possible that

apigenin binds to some kinases to suppress their activ-

ities. The different functions of flavonoids may be due

to affinities to some kinases and transcription factors.

Further studies will be required to understand the

functions of flavonoids in the regulation of cell growth

and carcinogenesis.

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