Naringenin Derivative Diethyl (5,4'-dihydroxy flavanone-7-yl)Phosphate Inhibits Cell Growth and Induces Apoptosis inA549 Human Lung Cancer Cells

Jung-Hee Kim · Heejong Kim · Yesol Bak · Jeong-Woo Kang · Dong Hun Lee · Man Sub Kim · Yun Sun Park · Eun-Jin Kim · Kang-Yeoun Jung ·Yoongho Lim · Jintae Hong · Do-Young Yoon

Received: 28 October 2011 / Accepted: 10 December 2011 / Published Online: 29 February 2012

© The Korean Society for Applied Biological Chemistry and Springer 2012

Abstract Anti-cancer effects of naringenin derivative diethyl

(5,4'-dihydroxy flavanone-7-yl) phosphate were evaluated in

human lung cancer cells. The effect of diethyl (5,4'-dihydroxy

flavanone-7-yl) phosphate (dEdHF-7-p) on A549 cell viability

was measured using MTS assay and cell counting. Morphological

changes were detected using phase-contrast microscopy.

Apoptosis was analyzed using Hoechst staining. The influence of

dEdHF-7-p on cell cycle distribution was determined using

propidium iodide (PI) staining, and protein expression was

determined by Western blot analysis. A newly synthesized

naringenin derivative dEdHF-7-p suppressed cell growth of A549

though mechanisms including inhibition of cell cycle and

increased apoptosis. Apoptotic and cell cycle modulators were

changed by dEdHF-7-p in A549 cells; cyclins, ppRB, and anti-

apoptotic factor Bcl-2 were down-regulated, whereas apoptotic

factor Bax and cyclin-dependent kinase inhibitors p21 and p53

were enhanced, thereby releasing cytochrome c into the cytosol of

dEdHF-7-p -treated-A549 cells. dEdHF-7-p treatment processed

caspases-3/-8/-9 and cleavage of poly ADP-ribose polymerase.

The dEdHF-7-p treatment enhanced Fas expression and decreased

expression of cell survival factors such as PI3K and p-Akt in a

dose-dependent manner. Taken together, dEdHF-7-p induces

apoptosis by inhibiting the PI3K/Akt survival signaling pathway

and modulating mitochondria-emanated intrinsic and Fas extrinsic

pathways in A549 cells.

Keywords apoptosis · flavonoid · lung cancer · naringenin ·

naringenin derivative

Introduction

Lung cancer, one of the most common cancers worldwide and a

major cause of cancer-related death is classified into two

categories: small cell lung cancer (SCLC) and non-small cell lung

cancer (NSCLC). NSCLC, which is composed of several histological

types such as squamous cell carcinoma (SC), adenocarcinoma

(AC), and bronchoalveolar carcinoma (BAC), constitutes the

majority of cases, accounting for approximately 80% of all lung

cancers (Wolf et al., 1997). Despite aggressive therapeutic

approaches such as surgery, chemotherapy, and radiotherapy,

NSCLCs easily develop resistance to such therapeutics (Krzakowski,

2001). Thus, the development of more effective anti-cancer drugs

is rightly regarded as an important and urgent issue.

Flavonoids are a class of plant secondary metabolites that are

ubiquitously present in fruit- and vegetable-derived foods (Chen et

al., 2000; Kandaswami et al., 2005). Among them, naringenin is

considered to have bioactive effects on human health (Szejtli and

Szente, 2005; Chen et al., 2007). Naringenin has been reported to

have pharmacological effects, including anti-cancer, anti-mutagenic,

and anti-inflammatory activities (Francis et al., 1989; So et al.,

J.-H. Kim and H. Kim contributed equally.

J.-H. Kim · H. Kim · Y. Bak · J.-W. Kang · D. H. Lee · M. S. Kim · Y. S. Park· E.-J. Kim · Y. Lim · D.-Y. Yoon ( )Department of Bioscience and Biotechnology, Bio/Molecular InformaticsCenter, Konkuk University, Seoul 143-701, Republic of KoreaE-mail: ydy4218@konkuk.ac.kr

K.-Y. JungDepartment of Biochemical Engineering, College of Engineering,Gangneung-Wonju National University, Gangneung 210-702, Republic ofKorea

J. HongCollege of Pharmacy and Medical Research Center, Chungbuk NationalUniversity, Cheongju 361-763, Republic of Korea

ORIGINAL ARTICLE

J Korean Soc Appl Biol Chem (2012) 55, 75−82

DOI 10.1007/s13765-012-0013-4

76 J Korean Soc Appl Biol Chem (2012) 55, 75−82

1997). Based on these beneficial activities of naringenin, many

scientists have focused on constructing synthetic naringenin

derivatives, and various effects have been elucidated. In our recent

studies, naringenin was derivatized to produce more effective

small-molecule inhibiting cancer cell proliferation, and the

anticancer effects of its derivative, 5-hydroxy-7,40-diacetyloxy

flavanone-N-phenyl hydrazone (N101-43) were investigated in

NSCLC cell lines (Bak et al., 2011). In the present study, to

compare the effects of the derivatives, naringenin derivative,

diethyl (5,4'-dihydroxy flavanone-7-yl) phosphate (named as

dEdHF-7-p), was synthesized and the effects on the growth of

A549 human lung cancer cells and the underlying intracellular

signal transduction pathways involved in regulating apoptosis

were investigated. Compared to naringenin itself, dEdHF-7-p

suppressed cell proliferation, provoked G0/G1 cell cycle arrest,

and induced apoptotic cell death via mitochondria-emanated

intrinsic pathways and inhibition of the PI3K/Akt pathway in

A549 human lung cancer cells.

Materials and Methods

Reagents. CellTiter 96 AQueous One Solution Cell Proliferation

Assay reagent 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-

phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased

from Promega (Madison, WI). Phenazine methosulfate (PMS),

Hoechst stain solution, and propidium iodide were from Sigma (St

Louis, MO). Antibodies specific to cyclin A, cyclin D1, and p-

pRb were purchased from BD Biosciences (San Diego, CA).

Antibodies specific to poly (ADP-ribose) polymerase (PARP),

caspases-3/-8/-9, Bax, Bcl-2, cytochrome c, phospho-p53 (Ser-

15), and anti-mouse IgG-horseradish peroxidases were purchased

from Cell Signaling Technology (Beverly, MA). Antibodies

specific to p21, p27, GAPDH, phospho-Akt1/2/3 (Ser-473), Akt1,

PI3K p85α, and anti-rabbit IgG-HRP were from Santa Cruz

Biotechnology (Santa Cruz, CA). Anti-p53 was from Oncogene

(Cambridge, MA). Naringenin derivative, diethyl (5,4'-dihydroxy

flavanone-7-yl) phosphate, was synthesized as described below

and named as DEDHF-7-p (Fig. 1A).

Synthesis of DEDHF-7-p. DEDHF-7-p is a newly synthesized

naringenin derivative (Fig. 1A). To the solution of naringenin

(0.2 g, 0.74 mmol) in dichloromethane (5 mL) was added Et3N

(0.31 mL, 2.22 mmol) under argon atmosphere. After stirring for

10 min at room temperature, diethyl chlorophosphate (0.14 mL,

0.74 mmol) was added slowly. After 2 h, when thin layer

chromatography (TLC) analysis showed complete disappearance

of the starting material, distilled water was used to quench the

reaction. Crude product was purified by flash column chromato-

graphy using a gradient solvent system with ethyl acetate and

dichloromethane to obtain the desired product DEDHF-7-p as a

white solid (0.16 g, 0.38 mmol, 52.2% yield): 1H-NMR δ 11.92 (s,

1H), 7.25 (d, J=8.61 Hz, 2H), 6.86 (d, J=8.58 Hz, 2H), 6.41 (dd,

J=2.19, 0.90 Hz, 1H), 6.36 (dd, J=2.22, 0.91 Hz, 1H), 5.29 (dd,

J=13.17, 2.85 Hz, 1H), 4.25 (m, 4H), 3.09 (dd, J=17.16, 13.17

Hz, 1H), 2.79 (dd, J=17.17, 2.87 Hz, 1H), 1.39 (dt, J=12.09, 0.72

Hz, 6H); 13C-NMR δ 197.2, 163.6, 162.9, 157.9 (J c-p=6.75 Hz),

157.2, 129.1, 127.8, 115.8, 105.7, 101.3 (J c-p=6.23 Hz), 99.9 (J

c-p=4.95 Hz), 79.6, 65.3 (J c-p=6.15 Hz), 16.0 (J c-p=6.38 Hz);

31P NMR δ −7.71 ppm; IR (cm−1) 3264, 2958, 1632, 1582, 1519,

1442, 1371, 1342, 1265, 1184, 1145, 1092, 1016; HRFABMS

Calcd for C20H14N2O7 (M+Na)+ 431.0888, found 431.0882.

Cell culture. Human NSCLC cell lines, A549 (p53 +/+) and NCI-

H1299 (p53 −/−), were purchased from the American Type

Culture Collection (Rockville, MD) and cultured in RPMI-1640

medium (Hyclone Laboratories, Logan, UT) supplemented with

2 mM L-glutamine, 100 U/mL of penicillin, 100 mg/mL of

streptomycin, and 10% heat-inactivated fetal bovine serum (FBS)

(Hyclone Laboratories). The cells were incubated at 37oC in a 5%

CO2 humidified atmosphere.

Cell viability assay and cell morphological assessment. To

examine the effects of DEDHF-7-p on cell growth, approximately

0.5×104 cells per well were plated onto 96-well microtiter plates,

grown overnight, and treated with DEDHF-7-p dissolved in

DMSO (final concentration 0.05%) for 24 and 48 h. Cell viabilities

were analyzed by MTS assay according to the manufacturer’s

instructions. Optical absorbance was measured at 492 nm using a

microplate reader (Apollo LB 9110, Berthold Technologies

GmbH, Bad Wildbad, Germany). Morphology of the cells was

observed under an inverted phase-contrast microscope. Apoptotic

morphological changes were detected by Hoechst staining. Cells

plated on coverslips were treated with DEDHF-7-p and incubated

for 48 h. The coverslips were then washed with phosphate-

buffered saline (PBS) and fixed with 4% paraformaldehyde for

1 h at room temperature. After washing with PBS, the cells fixed

on the coverslips were stained for 20 min with Hoechst staining

solution at 37oC. The coverslips were then washed with PBS,

completely dried, and mounted on microscope slides. The slides

were then observed under a fluorescent microscope.

Western blot analysis. The cells were lysed in lysis buffer

containing 20 mM Tris pH 7.4, 0.5% NP-40, 200 mM sodium

chloride, 1 mM EDTA, 10 mM β-glycerophosphate, 10 mM

sodium pyrophosphate, 0.05% sodium deoxycholic acid, and

protease inhibitor cocktail. Cytosolic fractions were obtained

using cytosolic lysis buffer containing 10 mM HEPES-KOH (pH

7.9), 10 mM KCl, 2 mM MaCl2, 0.1 mM EDTA, 0.2 mM NaF,

0.4 mM PMSF, 0.1 mM Na3VO4, and 1 mM DTT. Protein

concentrations were determined using a Bio-Rad protein assay kit

(Bio-Rad Laboratories, Hercules, CA), followed by 12% sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

and transfer to polyvinylidene difluoride membranes (Millipore,

Billerica, MA). The membranes were then blocked with 5% non-

fat dry milk dissolved in PBST containing 140 mM NaCl, 27 mM

KCl, 10 mM Na2HPO4 · 12H2O, 1.8 mM KH2PO4, and 0.05%

Tween-20 and then incubated with primary antibodies against

PARP, caspases-3/-8/-9, Bax, Bcl-2, cytochrome c, phospho-p53

(Ser-15), p53, p21, p27, phospho-Akt1/2/3 (Ser-473), Akt1, PI3K

J Korean Soc Appl Biol Chem (2012) 55, 75−82 77

p85α or GAPDH (1:1000) for overnight at 4oC. After three

washes, the proteins were visualized by staining the membrane

with horseradish peroxidase-conjugated goat anti-mouse or goat

anti-rabbit secondary antibodies (1:5000) for 1 h at room

temperature. The blots were used to visualize the bound primary

antibodies with the Westzol® plus Western Blot detection system

(iNtRON Biotechnology, Seoul, Korea).

Cell cycle analysis by flow cytometry. Approximately 2×105

cells per well were plated onto 6-well plates and incubated

overnight to allow adhesion. After treatment with DEDHF-7-p for

48 h, the cells were harvested and fixed with 70% ethanol at –20oC. Subsequently, the fixed cells were washed with PBS and were

then stained for 30 min with PBS containing 50 µg/mL of

propidium iodide and 100 µg/mL of RNase A. The proportion of

apoptotic cells was analyzed by a FACScalibur and CellQuest

software (BD Bioscience, San Jose, CA).

Flow cytometry analysis after Anexin V and PI staining.

Apoptosis was detected by FACS Calibur flow cytometry (Becton

Dickinson & Co., Franklin Lakes, NJ) and Cell Quest Pro

software using Annexin V-FITC Apoptosis Detection Kit (BD

Pharmigen, San Diego, CA). Briefly, 1.5×105 cells were seeded in

6-well plates and allowed to adhere overnight to the wells of

culture plates. Cells were treated with various concentrations of

DEDHF-7-p for 48 h, harvested, and washed with PBS. The cell

pellets were re-suspended in Annexin binding buffer and were

double stained with annexin V-FITC and propidium iodide (PI)

following manufacturer's instruction.

Reverse transcription-polymerase chain reaction (RT-PCR).

Total RNA was isolated using an easy-BLUE™ total RNA

extraction kit (iNtRon Biotechnology) as described by the

manufacturer’s protocol. Reverse transcription was performed

using M-MuLV reverse transcriptase (New England Biolabs,

Beverly, MA). RT-PCR analysis was conducted using a PCR

thermal cycler Device (TaKaRa, Shiga, Japan) with the following

primer sets: Fas (1005 bp): 5'-TGA AGG ACA TGG CTT AGA

AGT G -3' (forward), 5'-GGT GCA AGG GTC ACA GTG TT-3'

(reverse), GAPDH (240 bp): 5'-TCC ACC ACC CTG TTG CT

TA-3' (forward), 5'-ACC ACA GTC CAT GCC ATC AC-3'

(reverse), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

as an internal control. The PCR was performed as follows: (1) 15

min, 95oC; (2) n cycles 30 s, 95oC; (3) 30 s, 57oC; and (4) 30 s,

72oC. After cycling, the sample was heated (10 min, 72oC) and

cooled (4oC; total number of cycles: n=35 for Fas; n=25 for

GAPDH). The PCR products were electrophoresed on 1%

agarose gels and detected by ethidium bromide staining.

Statistical analysis. All presented data and results were confirmed

by at least three independent experiments. The data are expressed

as the means ± SD. Statistical analysis was performed using the

Student’s t-test, with the following significance levels: ap <0.05,bp <0.005, cp <0.001.

Results

Anti-proliferative and apoptotic effects of naringenin and its

derivative DEDHF-7-p on human lung cancer cells. The anti-

proliferative effects of a newly synthesized naringenin derivative,

DEDHF-7-p, was first examined on human NSCLC cells, A549

(p53 +/+) and NCN-H1299 (p53 −/−) (Fig. 1B). The cells were

exposed to different concentrations of naringenin and DEDHF-7-

p for 24 and 48 h, and cell viability was measured by MTS assay.

The naringenin derivative DEDHF-7-p significantly inhibited

growth of A549 human non-small cell lung carcinoma cells in

time- and dose- dependent manners (Fig. 1B), whereas there was

no anti-proliferative effect of DEDHF-7-p observed on human

non-small cell lung carcinoma NCI-H1299 (p53 −/−) cells.

Moreover, naringenin did not show any cytotoxic effects on A549

cells (Fig. 1B). Morphological changes were apparent under

inverted phase-contrast microscopy (Fig. 1C upper panel). These

results show that p53-dependent pathway and/or other pathways

may be involved in the inhibition of A549 NSCLC cell growth.

To ascertain whether DEDHF-7-p could induce cell apoptosis,

apoptotic nuclei were visualized by Hoechst staining. Apoptotic

nuclei, characterized by distinct condensed chromatin and DNA

fragmentation, were prominent in a dose-dependent manner in

A549 cells (Fig. 1C down panel). These results indicate that

DEDHF-7-p inhibits cell proliferation and induces apoptotic death

of A549 human lung cancer cells.

Cell-cycle arrest effects of DEDHF-7-p on A549 human lung

cancer cells. To examine whether the anti-proliferative effect of

DEDHF-7-p is related to cell-cycle arrest, A549 cells were treated

with different concentrations of DEDHF-7-p for 48 h, after which

cell cycle distribution was measured by flow cytometry. The

number of A549 cells accumulated in G0/G1 phase increased

from 59.5 to 74.8% upon DEDHF-7-P treatment for 48 h, whereas

there were slight decreases in the populations of cells in S phase

and G2/M phase (Figs. 2A and B). These results led to the

hypothesis that DEDHF-7-p induces apoptotic cell death through

cell-cycle arrest in G0/G1 phase. To confirm apoptosis, flow

cytometry was performed using Annexin V-FITC Apoptosis

Detection Kit. DEDHF-7-p induced late apoptosis defined by

Annexin V+/PI+ staining in A549 cells, in a dose response manner

(Fig. 2C).

Based on the results of cell cycle dynamics, the regulation of

G1 phase-associated molecules was analyzed by Western blotting.

As shown in Fig. 2D, the expression levels of cyclin D1 and

cyclin A were reduced in DEDHF-7-p-treated cells. The expression

level of p-pRb protein, which is regulated by G1 cyclins/cyclin-

dependent kinases (CDKs), was also reduced. Next, two upstream

cell cycle regulators, tumor suppressors p53 and p21, a regulator

of G1/S cyclins as a CDK inhibitor, were examined. DEDHF-7-

p treatment clearly elevated p21 and p53 expressions in A549

78 J Korean Soc Appl Biol Chem (2012) 55, 75−82

cells. To investigate whether DEDHF-7-p could induce p21

expression through a p53-independent pathway, NCI-H1299 (p53

−/−) lung cancer cells were used. DEDHF-7-p did not inhibit the

growth of NCI-H1299 cells (Fig. 1B). Moreover, neither p53 nor

p21 were expressed in NCI-H1299 cells (Fig. 2D). DEDHF-7-p

treatment markedly induced the elevation of p21 and activation of

p53 (p-p53) in A549 cells (Fig. 2D). These results suggest that

DEDHF-7-p induces down-regulation of G1/S cyclins and p-pRb

and up-regulates p21 expression via a p53-dependent pathway,

thereby provoking G0/G1 arrest in A549 cells.

Effects of DEDHF-7-p on apoptotic signaling pathways. The

caspase cascade is a critical signaling pathway that mediates cell

apoptosis. Therefore, we investigated whether activation of

caspases, accompanied by PARP processing, is induced by

DEDHF-7-p. PARP (89 kDa) cleavage was increased in DEDHF-

7-p-treated A549 cells (Fig. 3A). Additionally, treatment of A549

cells with DEDHF-7-p led to a decrease in the expressions of pro-

caspase-9 (47 kDa) and pro-caspase-3 (35 kDa). Cleaved forms of

caspase-3 (17 and 19 kDa) were increased in a dose-dependent

manner.

Apoptosis is known to be regulated by mitochondrial-related

proteins composed of anti-apoptotic and pro-apoptotic members

(Boise et al., 1993). Therefore, the modulation of Bcl-2 family

proteins and cytochrome c release during DEDHF-7-p-induced

apoptosis by Western blotting were evaluated. The level of pro-

apoptotic Bax was elevated, whereas the level of pro-survival Bcl-

2 was markedly reduced in response to DEDHF-7-p treatment,

and release of cytochrome c into the cytosol was increased as

expected (Fig. 3B). The expression level of the extrinsic death

receptor Fas (CD95) was also increased (Fig. 3C), and moderate

cleavage of the downstream effector molecule pro-caspase-8 (57

kDa) was observed by DEDHF-7-p treatment (Fig. 3A), suggesting

that Fas extrinsic death receptor was involved. These results

suggest that DEDHF-7-p induces apoptosis through pathways

accompanying the processing of caspases-9/-8/-3 and PARP, and

activation of the mitochondrial pathway may be involved in the

Fig. 1 Anti-proliferative and apoptotic effects of naringenin and its derivative DEDHF-7-p in A549 (p53 +/+) and NCI-H1299 (p53 −/−) human lungcancer cells. (A) Synthesis and chemical structure of DEDHF-7-p. (B) Human NSCLC cell lines, NCSLC A549 (p53 +/+) and NCI-H1299 (p53 −/−),were treated with the indicated concentrations of naringenin and its derivative DEDHF-7-p for 24 and 48 h, and cell viabilities were measured by MTSassay. Data are presented as means ± SD (n=3). (C) A549 cells treated with different concentrations of DEDHF-7-p for 48 h were observed under aphase-contrast microscope (×40) and photographed (upper panel), and nuclear morphologic changes were observed under a fluorescent microscope byHoechst staining (×100) (down panel).

J Korean Soc Appl Biol Chem (2012) 55, 75−82 79

apoptotic process mediated by DEDHF-7-p in A549 cells.

Inhibition of the PI3K/Akt-mediated pathway by DEDHF-7-

p. The phosphatidyl inositol-3 kinase (PI3K) pathway is regulated

by various growth factors (Datta et al., 1997; Page et al., 2000).

Recent studies have demonstrated that PI3K and its downstream

substrate Akt are related to apoptosis signaling (Franke et al.,

1995; Kulik et al., 1997). Therefore, the effects of DEDHF-7-p on

the PI3K/Akt pathway were examined, and the result showed

DEDHF-7-p treatment gradually reduced the level of PI3K in

A549 cells (Fig. 4). Subsequently, DEDHF-7-p suppressed the

phosphorylation of Akt in a dose-dependent manner. However, no

significant changes in total Akt protein levels were observed,

indicating that DEDHF-7-p had no effect on total Akt protein

stability. These results show that blockade of the PI3K/Akt

pathway is involved in DEDHF-7-P-induced apoptosis.

Discussion

Previous studies have shown that a naringenin derivative could

induce apoptosis in several cancer cells (Totta et al., 2004;

Morikawa et al., 2008; Park et al., 2008; Sabarinathan et al.,

2010), and thus could be a promising anti-cancer drug. Based on

the chemical structure of naringenin, many scientists have

attempted to synthesize derivatives for collecting effective anti-

cancer candidate drugs. Lee et al. (2007) and Jin et al. (2011)

reported that synthetic naringenin derivatives, specifically

substituted at carbon-7 of naringenin, 7-O-benzyl naringenin

(KUF-1) or 7-O-(MeO-L-Leu-D-Pro-carbonylmethyl) naringenin

(KUF-7), induce apoptotic cell death in lung cancer cells. KUF-1

and 7-O-(m-metoxybenzyl) naringenin (KUF-2) have apoptotic

effects on human colorectal carcinoma RKO cells while

provoking intracellular reactive oxygen species (ROS) production

Fig. 2 Effect of DEDHF-7-p on cell cycle progression of A549 cells. The cells were treated with different concentrations of DEDHF-7-p for 48 h. (A)The cells were fixed with 70% ethanol and stained with PI staining solution, and 10,000 events per experiments were analyzed by flow cytometry inA549 cells. Data are presented as means ± SD (n=3). (B) The values in (A) were graphed. (C) Flow cytometric analysis was preformed, and the datashown are representative of three separate experiments. The lower right quadrants represent early apoptotic cells that were stained by annexin V but notby propidium iodide. The upper right quadrants represent late apoptotic cells that were stained by both annexin V and propidium iodide. (D) Theexpression levels of cell cycle regulation factors (cyclin A/D1, p21, p27, p-p53, p53, and pRb) were detected by Western blot analyses in A549 (p53 +/+) cells. Expression levels of p53 and p21 were detected by Western blot analyses in NCI-H1299 (p53 −/−) cells. GAPDH was used as an internalcontrol. The intensity of the control was normalized to 1, and the intensity of each band from DEDHF-7-p treated cells was compared to that of thecontrol. Densitometric quantification was performed using ImageJ densitometry (NIH, Bethesda, MA; http://rsbweb.nih.gov/ij/).

80 J Korean Soc Appl Biol Chem (2012) 55, 75−82

coupled with the concomitant activation of the caspase cascade

signaling pathway (Lee et al., 2008).

These results were similar to those of N101-43 (Bak et al.,

2011), suggesting that 3-N-phenyl hydrazone, 7, 4' acetyl moieties

has no significant effects on A549 cells.

In the present study, a synthetic naringenin derivative, diethyl

(5,4'-dihydroxy flavanone-7-yl) phosphate (DEDHF-7-p), was

applied to A549 NSCLC cells. DEDHF-7-p was synthesized by

adding diethyl chlorophosphate to naringenin, inducing a cytotoxic

effect accompanied by apoptotic nuclei formation in A549 cells in

a dose-dependent manner. However, DEDHF-7-p had no obvious

inhibitory effect on the growth of NCI-H1299 (p53 −/−) lung

cancer cells and HaCaT human normal epithelial cells (data not

shown). In contrast, naringenin did not show any comparable

cytotoxic effects on A549 cells. Consistent with these anti-

proliferative and apoptotic phenomena, a connection between

DEDHF-7-p and cell-cycle progression was demonstrated. Cell

cycle progression is elaborately regulated through a complex

network of cell-cycle-associated molecules. Tumor suppressor

protein p53 controls the cell-cycle G1-checkpoint while promoting

induction of p21, a CDK inhibitor, thereby regulating cell-cycle

factors such as cyclins and pRb (Sherr, 1996; Clarke and Allan,

2009). In the present study, DEDHF-7-p provoked cell-cycle G0/

G1 arrest via up-regulation of p53 and p21 as well as down-

regulation of cyclin A /D1 and p-pRb.

Subsequently, DEDHF-7-p was predicted to induce death of

A549 cells via apoptosis-related signaling. Caspases, a family of

aspartate-specific systeine proteases, along with PARP, a diverse

cellular substrate, are critical signal factors in apoptosis triggered

by various pro-apoptotic signals (Nunez et al., 1998). Treatment

of DEDHF-7-p to A549 cells induced processing of caspases-8/-

9/-3 as well as proteolytic cleavage of PARP. Additionally, the

expression level of anti-apoptotic factor Bcl-2 was decreased,

whereas that of apoptotic factor Bax was increased, thereby

releasing cytochrome c into cytosol. The PI3K/Akt pathway has

been widely implicated in cell growth, cell cycle arrest, and cell

death in many cancer cells (Luo et al., 2003; Bellacosa et al.,

2005; Bussink et al., 2008). It serves as a direct link between

Fig. 2 Continued.

J Korean Soc Appl Biol Chem (2012) 55, 75−82 81

PI3K/Akt and the apoptosis-regulating Bcl-2 protein family (Datta

et al., 1997). Furthermore, a recent study reported that down-

regulation of Akt is involved in human leukemia THP-1 cells

undergoing apoptosis by naringenin (Park et al., 2008). Therefore,

the effects of DEDHF-7-p on the levels of the p85 subunit of

phosphatidyl inositol 3-kinase (PI3K) and phosphorylation of Akt

in A549 cells were determined, and DEDHF-7-p was found to

inhibit PI3K/Akt signaling, thereby inducing apoptosis. Taken

together, results of the present study implied that a newly

synthesized naringenin derivative, diethyl (5,4’-dihydroxy

flavanone-7-yl) phosphate (DEDHF-7-p), could be a potential

anti-cancer agent for treatment of lung cancer.

Acknowledgments This research work was supported by a Korea Research

Foundation (KRF) grant funded by the Korean government (2010-0019306,

2009-0072028). D.Y is partially supported from Priority Research Centers

Program (2009-0093824).

Fig. 3 Effects of DEDHF-7-p on the processing of caspases and PARP and on the expression of mitochondrial apoptotic proteins in A549 cells. Thecells were treated with different concentrations of DEDHF-7-p for 48 h. (A) Processing of caspases-3/-8/-9 and PARP. (B) Expression levels of Bax andBcl-2. and cytochrome c release were detected by Western blot analysis. GAPDH was used as an internal control. The intensity of the control wasnormalized to 1, and the intensity of each band from DEDHF-7-p treated cells was compared to that of the control. Densitometric quantification wasperformed using ImageJ densitometry. (C) The effect of DEDHF-7-p on Fas expression in A549 cells. Total RNA was extracted. Fas and GAPDHmRNA were analyzed by RT-PCR using specific primers.

Fig. 4 Inhibition of the PI3K/Akt pathway in DEDHF-7-p-treated A549cells. A549 cells were treated with the indicated concentrations ofDEDHF-7-p for 48 h. The expression levels of PI3K and Akt weredetected by Western blot analysis.

82 J Korean Soc Appl Biol Chem (2012) 55, 75−82

References

Bak Y, Kim H, Kang JW, Lee DH, Kim MS, Park YS, Kim JH, Jung KY,

Lim Y, Hong J, and Yoon DY (2011) A Synthetic Naringenin Derivative,

5-Hydroxy-7,4'-diacetyloxyflavanone-N-phenyl Hydrazone (N101-43),

Induces Apoptosis through Up-regulation of Fas/FasL Expression and

Inhibition of PI3K/Akt Signaling Pathways in Non-Small-Cell Lung

Cancer Cells. J Agric Food Chem 59, 10286–10297.

Bellacosa A, Kumar CC, Di Cristofano A, and Testa JR (2005) Activation of

AKT kinases in cancer: Implications for therapeutic targeting. Adv

Cancer Res 94, 29–86.

Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA,

Mao X, Nunez G, and Thompson CB (1993) bcl-x, a bcl-2-related gene

that functions as a dominant regulator of apoptotic cell death. Cell 74,

597–608.

Bussink J, van der Kogel AJ, and Kaanders JH (2008) Activation of the PI3-

K/AKT pathway and implications for radioresistance mechanisms in

head and neck cancer. Lancet Oncol 9, 288–296.

Chen D, Chen MS, Cui QC, Yang H, and Dou QP (2007) Structure-

proteasome-inhibitory activity relationships of dietary flavonoids in

human cancer cells. Front Biosci 12, 1935–1945.

Chen Y, Yang L, and Lee TJ (2000) Oroxylin A inhibition of

lipopolysaccharide-induced iNOS and COX-2 gene expression via

suppression of nuclear factor-kappaB activation. Biochem Pharmacol 59,

1445–1457.

Clarke PR and Allan LA (2009) Cell-cycle control in the face of damage—a

matter of life or death. Trends Cell Biol 19, 89–98.

Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, and Greenberg ME

(1997) Akt phosphorylation of BAD couples survival signals to the cell-

intrinsic death machinery. Cell 91, 231–241.

Francis AR, Shetty TK, and Bhattacharya RK (1989) Modulating effect of

plant flavonoids on the mutagenicity of N-methyl-N'-nitro-N-

nitrosoguanidine. Carcinogenesis 10, 1953–1955.

Franke TF, Yang SI, Chan TO, Datta K, Kazlauskas A, Morrison DK, Kaplan

DR, and Tsichlis PN (1995) The protein kinase encoded by the Akt

proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-

kinase. Cell 81, 727–736.

Jin CY, Park C, Hwang HJ, Kim GY, Choi BT, Kim WJ, and Choi YH (2011)

Naringenin up-regulates the expression of death receptor 5 and enhances

TRAIL-induced apoptosis in human lung cancer A549 cells. Molecular

Nutrition & Food Research 55, 300–309.

Kandaswami C, Lee LT, Lee PP, Hwang JJ, Ke FC, Huang YT, and Lee MT

(2005) The antitumor activities of flavonoids. In Vivo 19, 895–909.

Krzakowski M (2001) New agents within the preoperative chemotherapy of

non-small cell lung cancer. Lung Cancer 34 Suppl 2, S159–163.

Kulik G, Klippel A, and Weber MJ (1997) Antiapoptotic signalling by the

insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and

Akt. Mol Cell Biol 17, 1595–1606.

Lee ER, Kang YJ, Choi HY, Kang GH, Kim JH, Kim BW, Han YS, Nah SY,

Paik HD, Park YS, and Cho SG (2007) Induction of apoptotic cell death

by synthetic naringenin derivatives in human lung epithelial carcinoma

A549 cells. Biol Pharm Bull 30, 2394–2398.

Lee ER, Kang YJ, Kim HJ, Choi HY, Kang GH, Kim JH, Kim BW, Jeong

HS, Park YS, and Cho SG (2008) Regulation of apoptosis by modified

naringenin derivatives in human colorectal carcinoma RKO cells. J Cell

Biochem 104, 259–273.

Luo J, Manning BD, and Cantley LC (2003) Targeting the PI3K-Akt pathway

in human cancer: rationale and promise. Cancer Cell 4, 257–262.

Morikawa K, Nonaka M, Mochizuki H, Handa K, Hanada H, and Hirota K

(2008) Naringenin and hesperetin induce growth arrest, apoptosis, and

cytoplasmic fat deposit in human preadipocytes. J Agric Food Chem 56,

11030–11037.

Nunez G, Benedict MA, Hu Y, and Inohara N (1998) Caspases: The proteases

of the apoptotic pathway. Oncogene 17, 3237–3245.

Page C, Lin HJ, Jin Y, Castle VP, Nunez G, Huang M, and Lin J (2000)

Overexpression of Akt/AKT can modulate chemotherapy-induced

apoptosis. Anticancer Res 20, 407–416.

Park JH, Jin CY, Lee BK, Kim GY, Choi YH, and Jeong YK (2008)

Naringenin induces apoptosis through downregulation of Akt and

caspase-3 activation in human leukemia THP-1 cells. Food Chem

Toxicol 46, 3684–3690.

Sabarinathan D, Mahalakshmi P, and Vanisree AJ (2010) Naringenin promote

apoptosis in cerebrally implanted C6 glioma cells. Mol Cell Biochem

345, 215–222.

Sherr CJ (1996) Cancer cell cycles. Science 274, 1672–1677.

So FV, Guthrie N, Chambers AF, and Carroll KK (1997) Inhibition of

proliferation of estrogen receptor-positive MCF-7 human breast cancer

cells by flavonoids in the presence and absence of excess estrogen.

Cancer Lett 112, 127–133.

Szejtli J and Szente L (2005) Elimination of bitter, disgusting tastes of drugs

and foods by cyclodextrins. Eur J Pharm Biopharm 61, 115–125.

Totta P, Acconcia F, Leone S, Cardillo I, and Marino M (2004) Mechanisms

of naringenin-induced apoptotic cascade in cancer cells: Involvement of

estrogen receptor alpha and beta signalling. IUBMB Life 56, 491–499.

Wolf G, Elez R, Doermer A, Holtrich U, Ackermann H, Stutte HJ,

Altmannsberger HM, Rubsamen-Waigmann H, and Strebhardt K (1997)

Prognostic significance of polo-like kinase (PLK) expression in non-

small cell lung cancer. Oncogene 14, 543–549.