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

Article abstract of DOI:10.1021/jf2017594

Naringenin, a well-known naturally occurring flavonone, demonstrates cytotoxicity in a variety of human cancer cell lines; its inhibitory effects on tumor growth have spurred interest in its therapeutic application. In this study, naringenin was derivatiz

Full text of DOI:10.1021/jf2017594

ARTICLE
pubs.acs.org/JAFC
A Synthetic Naringenin Derivative, 5-Hydroxy-7,
40-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
||
||
Yesol Bak,†, Heejong Kim,†, Jeong-Woo Kang,† Dong Hun Lee,† Man Sub Kim,† Yun Sun Park,†
Jung-Hee Kim,† Kang-Yeoun Jung,§ Yoongho Lim,† Jintae Hong,# and Do-Young Yoon*,†
†Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul, Republic of Korea
§Department of Biochemical Engineering, College of Engineering, Gangneung-Wonju National University, Gangneung, Republic of
Korea
#College of Pharmacy and Medical Research Center, Chungbuk National University, Cheongju, Republic of Korea
ABSTRACT: Naringenin, a well-known naturally occurring flavonone, demonstrates cytotoxicity in a variety of human cancer cell
lines; its inhibitory effects on tumor growth have spurred interest in its therapeutic application. In this study, naringenin was
derivatized to produce more effective small-molecule inhibitors of cancer cell proliferation, and the anticancer effects of its derivative,
5-hydroxy-7,40-diacetyloxyflavanone-N-phenyl hydrazone (N101-43), in non-small-cell lung cancer (NSCLC) cell lines NCI-H460,
A549, and NCI-H1299 were investigated. Naringenin itself possesses no cytotoxicity against lung cancer cells. In contrast, N101-43
inhibits proliferation of both NCI-H460 and A549 cell lines; this capacity is lost in p53-lacking NCI-H1299 cells. N101-43 induces
apoptosis via sub-G1 cell-cycle arrest in NCI-H460 and via G0/G1 arrest in A549 cells. Expression of apoptosis and cell-cycle
regulatory factors is altered: Cyclins A and D1 and phospho-pRb are down-regulated, but expression of CDK inhibitors such as p21,
p27, and p53 is enhanced by N101-43 treatment; N101-43 also increases expression levels of the extrinsic death receptor Fas and its
binding partner FasL. Furthermore, N101-43 treatment diminishes levels of cell survival factors such as PI3K and p-Akt dose-
dependently, and N101-43 additionally induces cleavage of the pro-apoptotic factors caspase-3, caspase-8, and poly ADP-ribose
polymerase (PARP). Cumulatively, these investigations show that the naringenin derivative N101-43 induces apoptosis via up-
regulation of Fas/FasL expression, activation of caspase cascades, and inhibition of PI3K/Akt survival signaling pathways in NCI-
H460 and A549 cells. In conclusion, these data indicate that N101-43 may have potential as an anticancer agent in NSCLC.
KEYWORDS: naringenin derivative, apoptosis, lung cancer, flavonoid
’ INTRODUCTION
Triggering apoptosis in tumor cells can therefore be an effective
strategy in anticancer therapy.
Lung cancer is one of the most common cancers globally and
the major cause of cancer deaths, having surpassed breast cancer
as the leading cause of cancer mortality in women in 1987; by
2002 it was predicted to account for approximately 25% of all
female deaths from cancer.1 The high incidence of mortality is a
result of the difficulty of early diagnosis and lung tumors’ elevated
potential for local invasion as well as metastasis to distant tissues
and organs.2 The most predominant forms of lung cancer are
non-small-cell lung cancer (NSCLC), accounting for about 85%
of cases, and small-cell lung cancer (SCLC), accounting for 15%
of cases.3 In comparison to small-cell carcinomas, NSCLCs are
relatively insensitive to chemotherapy. Thus, there is urgent need
to improve clinical management of this serious disease.
Apoptosis, or programmed cell death, plays a pivotal role in
the normal development and pathology of a wide variety of
tissues. It can be easily distinguished from necrosis through its
diverse alterations to the cell, including chromatin condensation,
DNA fragmentation, cytoplasmic shrinkage, and the formation of
apoptotic bodies.4,5 However, most cancer cells do not undergo
apoptosis due to impairment of apoptotic signal transmission.6
Apoptosis can occur through two mechanisms, the extrinsic
death-receptor dependent pathway and a mitochondria-depen-
dent, or intrinsic, pathway.7,8 The extrinsic pathway is activated
by cell-surface ligand-gated death receptors such as the tumor
necrosis factor receptor superfamily. The association of specific
ligands with the extracellular domains of death receptors triggers
receptor trimerization: these ligands include CD95L/FasL and
Apo2L/TRAIL, which bind their respective cognate receptors
CD95/Fas and DR4/DR5.9,10 The Fas-associated death domain
(FADD) recruits caspase-8 to the Fas receptor to form the death-
inducing signaling complex (DISC).11 Oligomerization of cas-
pase-8 upon DISC formation triggers its activation concomitant
with that of downstream effectors such as caspase-3.10,12 Intrinsic
apoptosis is initiated by disparate extracellular and intracellular
death stimuli: in the mitochondrial apoptosis pathway, caspase
Received: May 10, 2011
Revised:
August 18, 2011
Accepted: August 30, 2011
Published: August 30, 2011
r
2011 American Chemical Society
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activation stems from permeabilization of the outer mitochon-
drial membrane by pro-apoptotic factors in the Bcl family and is
regulated by Bcl-2 and mitochondrial lipids. Mitochondrial
release of cytochrome c directly triggers caspase-3 activation
through formation of the cytochrome c/Apaf-1/caspase-9-con-
taining apoptosome complex. Caspase-3 then cleaves cellular
targets to promote programmed cell death.10,13
that regulate apoptosis. We demonstrate that N101-43 induces
apoptosis via up-regulation of death receptor expression, activa-
tion of the apoptotic caspase cascade, and inhibition of PI3K/Akt
cell survival signaling pathways in NCI-H460 and A549 cells.
’ MATERIALS AND METHODS
The phosphatidylinositol 3-kinase (PI3K) signaling pathway
plays pivotal roles in the signal transduction of survival in a wide
range of cell types including neurons.14,15 PI3K activates its
downstream effector protein Akt/protein kinase B (Akt/PKB)
by promoting its phosphorylation at residues serine 473
(Ser473) and threonine 308 (Thr308). Then activated Akt
phosphorylates a wide range of substrates, activating anti-apop-
totic (survival) factors and inactivating pro-apoptotic factors.15
Akt down-regulates the activities of glycogen synthase kinases
3α (GSK3α) and 3β (GSK3β) by phosphorylating the former at
residue serine 21 (Ser21) and the latter at residue serine 9
(Ser9).16 GSK3 is a highly evolutionary conserved intracellular
serineꢀthreonine kinase that exists as two isoforms, GSK3α and
GSK3β, ubiquitously expressed in mammalian tissues.17 Phos-
phorylation, and therefore inactivation of GSK3, can be catalyzed
by insulin, growth factors, and amino acids throughout the PI3K/
Akt, MAPK cascade, protein kinase C (PKC), or cAMP-depen-
dent protein kinase/protein kinase A.18ꢀ20 GSK3 regulates
diverse cell functions such as protein synthesis, cell proliferation,
cell differentiation, apoptosis, microtubule dynamics, and cell
motility.21
Plant extracts have begun to receive attention as therapeutic
agents in cancer treatment due to their capacity to inhibit tumor
growth, angiogenesis, and metastasis with minimal side effects.
Flavonoids are plant secondary metabolites often found in fruits,
vegetables, and plant-derived beverages or in dietary supple-
ments or herbal remedies.22 They comprise a diverse group of
plant natural products synthesized from phenylpropanoid and
acetate-derived precursors and play seminal roles in growth,
development, and host defense against microorganisms or
pests.23 In structure, they are low molecular weight polyphenols
with a characteristic 2-phenylbenzo-γ-pyrone structure harbor-
ing one or more hydroxyl groups. One of their most obvious
features is their ability to quench free radicals via formation of
resonance-stabilized phenoxyl radicals,24 and they can also
exhibit antifungal, anti-inflammatory, or antitumor effects.25
Additionally, they are safe for human consumption and, in some
cases, are suggested to be good chemopreventive agents.
Naringenin, a glycone of naringin, is abundant in citrus fruits,
grapes, and tomatoes.26 Naringenin has been discovered to
possess activity against uterine, blood, stomach, brain, and lung
cancer cell lines27ꢀ30 at doses that do not affect nontransformed
cells.31 Although many studies report these anticancer proper-
ties, their precise mechanisms remain unclear in NSCLC. It has
been known that naringenin inhibits NF-kB, which is a key
transcription factor involved in cancer growth.32 Therefore, to
find a novel derivative showing anticancer activity, we tried to
synthesize several naringenin derivatives. The cell viability test
for them revealed that several compounds exhibited at least a
20% decrease. Especially, N101-43 including an acetate group
and phenylhydrazine showed the best result. As a result, we
focused on the N101-43 compound. On the basis of naringenin’s
putative anticancer activity, we elucidated the effect of the newly
synthesized naringenin derivative N101-43 on the growth of
NSCLC cells and the intracellular signal transduction pathways
Reagents. CellTiter 96 AQueous One solution Cell Proliferation
Assay Reagent (MTS) was purchased from Promega (Madison, WI).
Hoechst stain solution and propidium iodide (PI) were purchased from
Sigma (St. Louis, MO). A pan-caspase inhibitor, Z-VAD-fmk, and a
PI3K inhibitor, LY294002, were from R&D Systems (Minneapolis,
MN) and Calbiochem (Darmstadt, Germany), respectively.
Synthesis of 5-Hydroxy-7,40-diacetyloxyflavanone-N-ph-
enyl Hydrazone (N101-43). As shown in Figure 1A, N101-43 is a
newly synthesized naringenin derivative. Prior synthesis of 5,7,
40-trihydroxy flavanone N-phenyl hydrazone (named N101-3) was
required for N101-43 synthesis. Naringenin (3.0 g (11.02 mmol)) and
phenyl hydrazine (1.3 mL (13.22 mmol)) were dissolved in absolute
ethanol (20 mL), and glacial acetic acid (1.0 mL) added. The solution
was refluxed on an oil bath for 36 h at 110ꢀ120 °C with stirring until a
yellow precipitate formed. The precipitate was collected by filtration and
washed with water and ethanol. The crude product was purified by flash
column chromatography using a gradient solvent system with acetone
and chloroform to obtain the desired product, N101-3, as a brown solid
(3.18 g (8.78 mmol), 79.7% yield): 1H NMR δ 2.81 (dd, J = 12.09, 16.86
Hz, 1H), 3.27 (dd, J = 2.91, 17.01 Hz, 1H), 5.07 (dd, J = 2.55, 11.7 Hz,
1H), 5.90 (dd, J = 2.4, 20.16 Hz, 2H), 6.81 (d, J = 8.61 Hz, 2H), 6.95 (d,
J = 7.71 Hz, 2H), 7.24 (t, J = 7.88, 15.75 Hz, 2H), 7.34 (d, J = 8.61 Hz,
2H), 9.33 (s, 1H), 9.55 (s, 1H), 9.77 (s, 1H), 12.50 (s, 1H); 13C NMR δ
31.8, 75.9, 95.1, 96.5, 98.8, 112.0 (2C), 115.1 (2C), 119.2, 128.1 (2C),
129.3, 130.2 (2C), 144.9, 145.1, 157.5, 157.7, 159.2, 159.7; IR (cmꢀ1
)
3433, 2948, 2054, 1639, 1600, 1499, 1250, 1250, 1154, 1055, 1032,
1019; HRFABMS calcd for C21H18N2O4 (M + Na)+ 385.1164, found
385.1160. Next, to a solution of N101-3 (0.2 g (0.55 mmol)) in THF
(15 mL) was added Et3N (0.3 mL (2.21 mmol)) under argon gas. After
10 min of stirring at room temperature, acetyl chloride (0.1 mL (1.21
mmol)) was slowly added. After a further 10 min, when thin-layer
chromatography demonstrated complete disappearance of the starting
material, distilled water was used to quench the reaction. The crude
product was purified by flash column chromatography using a gradient
solvent system with acetone and chloroform to obtain the desired
product, N101-43, as a white solid (0.22 g (0.50 mmol), 91.3% yield):
1H NMR δ 2.31 (d, J = 13.2 Hz, 6H), 2.67 (dd, J = 12.45, 16.47 Hz, 1H),
3.15 (dd, J = 3.3, 16.5 Hz, 1H), 5.14 (dd, J = 2.94, 12.09 Hz, 1H), 6.32
(dd, J = 2.19, 19.41 Hz, 2H), 6.93ꢀ7.00 (m, 3H), 7.14ꢀ7.19 (m, 3H),
7.31 (dd, J = 7.68, 8.79 Hz, 2H), 7.50 (d, J = 8.79 Hz, 2H), 12.21 (s, 1H);
13C NMR δ 21.1, 21.2, 30.9, 76.0, 101.8, 103.7, 104.2, 113.0 (2C), 121.3,
122.1 (2C), 127.3 (2C), 140.0 (2C), 136.8, 143.1, 143.7, 150.8, 152.3,
157.0, 159.2, 169.2, 169.5; IR (cmꢀ1) 3334, 2922, 2850, 1751, 1601,
1499, 1368, 1190, 1123, 1021; HRFABMS calcd for C25H22N2O6 (M +
Na)+ 469.1376, found 469.1372.
Antibodies. Antibodies specific to PARP, caspase-3, caspase-8,
caspase-9, phospho-GSK3α/β (Ser-21/9), Bcl-2, Bcl-xL, and Bax, as
well as an α-mouse IgG horseradish peroxidase (HRP) conjugated
secondary antibody, were purchased from Cell Signaling Technology
(Beverly, MA). Antibodies specific to PI3K, phospho-Akt1/2/3
(Ser-473), Akt, GSK-3 α/β, p27, p21, NF-Kb p65, and GAPDH, as
well as an α-rabbit IgG HRP conjugated secondary antibody, were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibo-
dies specific to cyclin D1, cyclin A, and phospho-pRb were purchased
from BD Bioscience (San Diego, CA). Anti-p53 antibody was from
Oncogene (Cambridge, MA).
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Figure 1. Antiproliferative and apoptotic effects of naringenin and naringenin derivatives in human lung cancer cell lines. (A) Chemical structures of
naringenin and the naringenin derivatives 5,7,40-trihydroxyflavanone N-phenyl hydrazone (N101-3) and 5-hydroxy-7,40-diacetyloxyflavanone-N-phenyl
hydrazone (N101-43). (B) Cytotoxic effects of naringenin and naringenin derivatives on human lung cancer cell lines (NCI-H460, A549, NCI-H1299)
and keratinocytes (HaCaT). Cells were treated with naringenin or naringenin derivatives in complete medium for 24 h before assessment of cell viability
by MTS assay. Data are presented as the mean ( standard deviation (n = 3). (/)p < 0.05 and (//) p < 0.005 versus DMSO treated cells. (C) NCI-H460
or A549 cells cultured in different concentrations of N101-43 for 24 h observed by phase-contrast microscopy (40ꢁ) and photographed. (D) Nuclear
morphological changes observed by fluorescence microscopy (Hoechst staining, 100ꢁ).
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Cell Culture. We obtained the NCI-H460, A549, and NCI-H1299
non-small-cell lung cancer cell lines from the American Type Culture
Collection (ATCC; Rockville, MD). NCI-H460, A549, and NCI-H1299
cells were cultured in RPMI medium supplemented with heat-inacti-
vated 10% (v/v) fetal bovine serum (FBS; both from Hyclone Labora-
tories, Logan, UT) and incubated under humidified conditions at 5%
CO2 and 37 °C.
Cell Viability Assays. Cell viability was quantified using an MTS
assay (Promega, Madison, WI). Approximately 0.7 ꢁ 104 cells were
seeded in each well of a 96-well plate containing 100 μL of RPMI
supplemented with 10% FBS, penicillin (100 μg/mL), and streptomycin
(0.25 μg/mL) for overnight growth. After 20 h, various concentrations
of N101-43 were added and incubation was continued for a further 24 h.
One hundred microliters of medium was removed and 20 μL of MTS
solution (2 mg/mL) added to cells for 30 minꢀ1 h at 37 °C. Optical
density was measured at 492 nm using an ELISA reader (Apollo LB
9110, Bertholo, Technologies GmbH, Germany).
siRNA Transfection. siRNA oligonucleotide of p53 and FasL and
scrambled siRNA were purchased from ST Pharm. Co., Ltd. (Seoul,
Korea). Sequences: p53 oligonucleotide, sense 50-CACUACAACUA-
CAUGUGUA-30 and antisense 50-UACACAUGUAGUUGUAGUG
(dTdT)-30;33 FasL siRNA, 50-CUGGGCUGUACUUUGUACATT-
30;34 scrambled siRNA, sense 50-CCUACGCCACCAAUUUCGU
(dTdT)-30 and antisense 50-ACGAAAUUGUGGCGUAGG (dTdT)-
30.33 Approximately, 6 ꢁ 104 cells were seeded in each well of a 6-well
plate containing 10% FBS incubated overnight to give 3ꢀ40% con-
fluence. Transfection was performed using a TransIT-TKO reagent
(Mirus, Milwaukee, WI) to give a final RNA concentration of 50 nM.
After 48 h, cells were treated with N101-43 for 24 h and harvested for
Western blot analysis.
Western Blot Analysis. N101-43-treated cells were scraped from
plate wells and collected by centrifugation (72g, 5 min, 4 °C). Cells were
then washed with ice-cold phosphate-buffered saline (PBS) and recen-
trifuged (1890g, 5 min, 4 °C). The cell pellets obtained were resus-
pended and lysed in buffer containing 20 mM Tris (pH 7.4), 200 mM
sodium chloride, 1 mM ethylenediaminetetraacetic acid (EDTA),
10 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 0.5%
NP-40, 0.05% sodium deoxycholic acid, and protease inhibitor cocktail,
and lysates were clarified by centrifugation at 17010g for 30 min at 4 °C.
Protein concentrations were measured by using Bradford assay
(Bio-Rad, Hercules, CA) with a UV spectrophotometer (Biochrom,
WPA, Biowave II, U.K.). Cell lysates were mixed with equal volumes of
5ꢁ SDS sample buffer, boiled for 5 min, and separated by 10ꢀ12%
sodium dodecyl sulfateꢀpolyacrylamide gel electrophoresis (SDS-
PAGE). After electrophoresis, proteins were transferred to polyvinyli-
dene difluoride (PVDF) membranes (Millipore, Billerica, MA). Mem-
branes were blocked in 5% nonfat dry milk dissolved in PBST (3.2 mM
Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, 0.05%
Tween-20) overnight at 4 °C. Primary antibodies specific to PARP,
caspase-3, caspase-8, caspase-9, Bcl-2, Bcl-xL, Bax, phospho-GSK3 (Ser
21/9), PI3K, phospho-Akt1/2/3 (Ser-473), Akt, GSK-3 α/β, p27, p21,
cyclin D1, cyclin A, phospho-pRb, and p53 were adsorbed to mem-
branes. HRP-conjugated α-rabbit or α-mouse IgG was used as a secon-
dary antibody. Immune-positive bands were visualized by using Westzol
plus Western blot detection system (iNtRON Biotechnology, Sung-
Nam, South Korea). All membranes were stripped, reblocked, and
reprobed with anti-GAPDH antibody as an internal control to confirm
equal protein loading.
coverslips were then washed with PBS a further three times, dried
completely, and mounted on microscope slides with mounting solution.
Slides were imaged with fluorescence microscopy.
Cell Cycle Analysis by Flow Cytometry. Approximately 1 ꢁ
105 cells were seeded per well of 6-well plates and incubated for 24 h
before treatment with various concentrations of N101-43 and a further
24 h of incubation. The cells were then washed, harvested, and fixed with
ice-cold 70% ethanol at ꢀ20 °C. After fixation, the cells were washed
with PBS and stained for 30 min with PBS containing 50 μg/mL PI and
100 μg/mL RNase A. The proportion of apoptotic cells was determined
by flow cytometry on a FACScalibur instrument and analyzed with
CellQuest software (BD Bioscience). The sub-G1 population indicated
apoptosis-associated chromatin degradation.
Flow Cytometry Analysis after Annexin V and PI Staining.
Apoptosis was detected by flow cytometry using an Annexin V-FITC
Apoptosis Detection Kit (BD Pharmigen). Briefly, 1.5 ꢁ 105 cells were
seeded per well of 6-well plates and incubated to adhere for overnight.
Cells were treated with various concentrations of N101-43 for 24 h,
harvested, and washed with PBS, and the cell pellets were resuspended in
annexin binding buffer. Cells were double-stained with annexin V-FITC
and PI following the manufacturer’s instruction. Early apoptosis is defined
by annexin V+/PIꢀ staining, and late apoptosis is defined by annexin
V+/PI+ staining as determined by FACS Calibur flow cytometry (Becton
Dickinson & Co., Franklin Lakes, NJ) and Cell Quest Pro software.
Reverse-Transcription Polymerase Chain Reaction (RT-
PCR) and Real-Time qPCR Analyses. Total RNA was isolated
using the Easy-BLUE Total RNA Extraction Kit (iNtRon Biotechnol-
ogy, Seoul, Korea) according to the manufacturer’s instructions. Reverse
transcription was performed using M-MuLV reverse transcriptase
(New England Biolabs, Beverly, MA). RT-PCR analysis was performed
using a PCR thermal cycler Dice instrument (TaKaRa, Otsu, Shiga,
Japan) with the following primer sets: FasL, 50-CAA GAT TGA CCC
CGG AAG TA-3 (forward), 50-GGC CTG TGT CTC CTT GTG AT-30
(reverse); GAPDH, 50-TGA TGA CAT CAA GAA GGT GGT-30
(forward), 50-TCC TTG GAG GCC ATG TAG GCC-30 (reverse).
GAPDH served as internal control. Real-time quantitative PCR was
performed with a relative quantification protocol using the Chromo 4
Real-Time PCR system and iQ SYBR Green Supermix (both from Bio-
Rad, Hercules, CA). All target genes were normalized to expression of
the housekeeping gene GAPDH. Each sample was run with the
following primer sets: FasL, 50-GCA GCC CTT GAA TTA CCC AT-
30 (forward), 50-CAG AGG TTG GAC AGG GAA GAA-30 (reverse);
Fas, 50-TGA AGG ACA TGG CTT AGA AGT G-30 (forward), 50-GGT
GCA AGG GTC ACA GTG TT-30 (reverse); GAPDH, 50-GGC TGC
TTT TAA CTC TGG TA-30 (forward), 50-TGG AAG ATG GTG ATG
GGA TT-30 (reverse). Fold changes in expression represent the ratio of
Fas or FasL expression in NCI-H460 and A549 cells treated with N101-
43 to untreated control.
Statistical Analysis. Data presented are the mean ( SD of results
from at least three independent experiments. Statistical significance was
assessed with Student’s t test. /, P < 0.05, or //, P < 0.005, were
considered to be statistically significant.
’ RESULTS
N101-43 Suppresses Cell Proliferation and Induces Apop-
totic Bodies in NSCLC. We treated non-small-cell lung cancer-
cell lines (NCI-H460, A549, NCI-H1299) and normal keratino-
cyte cells (HaCaT) with 10ꢀ40 μM naringenin or naringenin
derivatives (N101-3, N101-43) for 24 h and assessed cell viability
by MTS assay. Naringenin and N101-3 had little effect on
proliferation in any cell line. However, N101-43 significantly
reduced viability much more in NCI-H460 and A549 cells (both
p53 +/+) than in NCI-H1299 (p53 ꢀ/ꢀ) in a dose-dependent
Hoechst Staining. Apoptotic nuclear morphology was observed
through Hoechst staining. Cells were seeded on coverslips and then
cultured in a range of concentrations (0, 10, 25, and 40 μM) of N101-43
for 24 h. Coverslips were washed twice with PBS and fixed with 4%
paraformaldehyde for 1 h at room temperature. After three washes in
PBS, the cells were stained with Hoechst staining solution at 37 °C;
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manner (Figure 1B). In contrast, however, the viability of HaCaT
cells was very slightly decreased after culture with N101-43
(Figure 1B). These results suggest that N101-43 effectively
inhibits the growth of NSCLCs.
We wished to explore N101-43’s capacity for growth inhibi-
tion more fully. We found that N101-43 suppressed cell growth
in NCI-H460 and A549 cell lines, with a more pronounced effect
on the former (Figure 1B,C). To determine whether this effect
was apoptotic in nature, we cultured NCI-H460 and A549
cells with various concentrations of N101-43 for 24 h, then
stained cultures with Hoechst dye and monitored cellular
appearance by fluorescence microscopy. Nuclear condensation
Figure 2. Continued
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Figure 2. Effects of N101-43 on cell cycle progression in NCI-H460 and A549 cells. Cells were treated with the indicated concentrations of N101-43 for
24 h, fixed with 70% ethanol, and stained with PI; 10000 events were measured per experimental condition by flow cytometry. (A) Representative
examples of cell cycle analysis by flow cytometry with PI staining. (/) p < 0.05 versus DMSO treated cells. (B) Flow cytometry analyses of the cell cycle.
(C) Flow cytometry analysis after annexin V and PI staining. Apoptosis was detected by flow cytometry using an Annexin V-FITC Apoptosis Detection
Kit (BD Pharmigen). Cells were treated with various concentrations of N101-43 for 24 h, harvested, and washed with PBS, and the cell pellets were
resuspended in annexin binding buffer. Cells were double-stained with annexin V-FITC and propidium iodide (PI) following the manufacturer’s
instruction. (D) Expression levels of cell cycle regulators cyclins A and D1, p27, p21, p53, and phospho-pRb detected by Western blot analysis. GAPDH
was used as an internal control for sample loading. Densitometric quantification was performed using ImageJ densitometry. The intensity of the control
was normalized to 1, and the intensity of each band from N101-43-treated cells is compared to the control. (E) Effect of p53 knockdown on p53 and p21
expressions in NCI-H460 and A549 cells. p53 and p21 were detected by Western blot analysis.
and fragmentation, morphological features of apoptosis, mani-
fested themselves dose-dependently (Figure 1D). These results
indicate that N101-43 engendered apoptotic rather than necrotic
cell death in NCI-H460 and A549 cells.
We next assayed NCI-H1299 (p53 ꢀ/ꢀ) lung cancer cells to
probe whether N101-43 might induce p21 and p53 indepen-
dently. As shown in Figure 2D (right panel), neither p53 nor p21
expression was detected in these cells. To compare drug response
in NSCLC (NCI H460 and A549) versus NSCLC with p53
knockdown, we transfected with p53 siRNA, and alternative
expression of p53 and p21 induced by N101-43 was examined by
Western blotting (Figure 2E). p21, a CDK inhibitor that induces
G1 arrest, was inhibited in NCI-H460 and A549 cells treated with
p53 siRNA (Figure 2E), indicating that N101-43-induced G1
arrest is p53-dependent. Therefore, it was assumed that p53-
dependent and/or other pathways can be involved in the
inhibiting effect of N101-43 on NSCLC cell growth. These
results show that the NSCLC tumor-killing effect of N101-43
is primarily mediated via the p53-dependent apoptotic pathway.
Taken together, these findings imply that N101-43 imposes cell
cycle arrest at sub-G1 phase and/or G0/G1 in NCI-H460 and
A549 cells via G1/S cyclin down-regulation and p53-dependent
p21 up-regulation.
N101-43 Induces Pro-apoptotic Signaling Pathways. Cas-
pases play crucial roles in apoptosis through their proteolysis of
specific targets. To establish more concretely N101-43’s manner
of inducing apoptosis, we assessed caspase activation as well as
expression of pro- and anti-apoptotic proteins in NCI-H460 and
A549 cells treated with N101-43. Treatment with N101-43
significantly induced cleavage of caspase-3 and -8 in either cell
line (Figure 3A). We further queried the effect of N101-43 on
PARP, determining that its processing likewise rose dose-depen-
dently (Figure 3A). A pan-caspase inhibitor, Z-VAD-fmk,
was used to confirm caspase activation. Z-VAD-fmk inhibited
N101-43 Induces Cell Cycle Arrest and Up-regulates Cell
Cycle Related Proteins. Flow cytometry analyses revealed
enhanced apoptosis in NCI-H460 cells treated with N101-43
for 24 h (Figure 2A), concomitant with a dose-dependent
increase from 7.81 to 28.45% in the proportion of the NCI-
H460 cell population at sub-G1 phase; there was no significant
alteration in the accumulation of cells in G0/G1 phase (from
48.86 to 45.18%) after 24 h of treatment (Figure 2B). A549 cells
demonstrated a similar response to the compound: the propor-
tion of cells at sub-G1 phase was increased from 8.16 to 15.39%
by treatment with high concentrations of N101-43 (p = 0.079),
and the percentage of cells at G0/G1 phase rose from 40 to 45ꢀ50%.
Our findings therefore suggested that N101-43 could arrest pro-
gression in the sub-G1 or G0/G1 cell cycle in NSCLCs. To confirm
apoptosis, flow cytometry was performed using an Annexin V-FITC
Apoptosis Detection Kit. N101-43 induced late apoptosis defined
by annexin V+/PI+ staining in NCI-H460 and A549 cell lines, with a
more pronounced effect on the former (Figure 2C).
To confirm our observation that N101-43 induced cell cycle
arrest, we performed immunoblot analyses on cell cycle regulated
proteins. As illustrated in Figure 2D, expression of cyclin D1, cyclin
A, and phospho-pRb was down-regulated in N101-43-treated NCI-
H460 and A549 cell lines. In contrast, expression of p21 and p53,
which tightly control entry into G1, was elevated in both cell lines;
p27 was up-regulated in N101-43-treated NCI-H460, whereas
N101-43 treatment slightly up-regulated p27 in A549 cells.
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Figure 3. Effects of N101-43 on expression of apoptotic factors in NCI-H460 and A549 cell lines. Cells were treated with different concentrations of
N101-43 for 24 h. (A) Processing of caspase-3, caspase-8, caspase-9, and PARP and (B) inhibitory effect of a pan-caspase inhibitor, Z-VAD-fmk, on
expression of caspase-3, caspase-8, caspase-9, and PARP detected by Western blot analyses. Z-VAD-fmk (100 μM) was pretreated for 2 h, and then
N101-43 (40 μM) was treated for 24 h into cells. (C) Expression levels of Bax, Bcl-2, and Bcl-xL detected by Western blot analyses. The intensity of the
control was normalized to 1, and the intensity of each band from N101-43-treated cells is compared to the control.
N101-43-induced cleavages of PARP and pro-casoase-8 in
both NSCLC cells, and the decreased levels of pro-caspase-3
and -9 were recovered in N101-43-treated NCI-H460 cells
(Figure 3B).
The mitochondrial, or intrinsic, apoptosis pathway comprises
pro- and anti-apoptotic members including Bcl-2 family
proteins.35 We investigated N101-43’s capacity to evoke intrinsic
apoptosis by Western blotting. As shown in Figure 3C, levels of
pro-apoptotic Bax were unchanged by N101-43 treatment,
whereas the levels of pro-survival Bcl-2 and Bcl-xL were markedly
reduced, in NCI-H460 cells. In A549, in contrast, no such
alterations in protein expression occurred subsequent to N101-
43 treatment, but caspase-3 was activated. These results suggest
that extrinsic signaling appears to predominate in A549 cells,
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Figure 4. Effects of N101-43 on Fas/FasL expression in NCI-H460 and A549 cells. (A) FasL gene induction in NCI-H460 and A549 cells treated with
N101-43. FasL expression was analyzed by RT-PCR; the PCR products depicted were amplified for 38 cycles. (B) Expression levels of Fas and FasL
mRNA measured by real-time quantitative PCR. GAPDH was used as an internal control. (C) Effect of FasL knockdown on expressions of PARP and
caspases in NCI-H460 and A549 cells. Expressions of PARP and caspases were detected by Western blot analysis.
whereas intrinsic and extrinsic apoptotic signaling likely con-
tribute to N101-43’s cytotoxicity in NCI-H460 cells.
To investigate the extrinsic signaling, RT-PCR and real-time
PCR analyses were performed to investigate modulation of Fas
expression: Fas and FasL transcription were increased dose-
dependently by N101-43 in both NCI-H460 and A549 cells, with
Fas mRNA substantially increased in NCI-H460 cells (Figure 4B,
left panel) and FasL mRNA levels rising in A549 cells (Figure 4A,B).
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Figure 5. Inhibitory effects of N101-43 on PI3K/AKT cell survival factors and IkB phosphorylation in NCI-H460 and A549 cell lines. Cells were treated
with different concentrations of N101-43 for 24 h. (A) IkB phosphorylation and (B) expression levels of PI3K, phospho-Akt, Akt, phospho-GSK3α/β,
and GSK3 α/β detected by Western blot analyses. The intensity of the control was normalized to 1, and the intensity of each band from N101-43-treated
cells is compared to the control. (C, D) Effects of PI3K inhibitor LY294002 on N101-43-induced FasL and p53 expression and p53 detected by RT-PCR
(C) and Western blot analyses, respectively. LY294002 (1 μM) was pretreated for 1 h, and then N101-43 (40 μM) was treated for 24 h into cells.
However, no significant changes in FADD levels were observed
(data not shown). These results suggest that extrinsic apoptotic
signaling mediated via enhanced FasL expression appears to pre-
dominate in A549 cells. To compare drug response in NSCLC
(NCI H460 and A549) versus NSCLC with p53 knockdown,
we transfected with FasL siRNA, and alternative expression of
PARP and caspases induced by N101-43 was examined by
Western blotting (Figure 4C). The expression levels of precursor
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caspase-8 and -9 and PARP were enhanced in N101-43-treated
cells by FasL knockdown (Figure 4C), assuming that N101-43-
induced cleavages of PARP and caspases are primarily FasL-
dependent. Collectively, these results show that the NSCLC
tumor-killing effect of N101-43 is primarily mediated via the
FasL-dependent apoptotic pathway.
We observed cell cycle arrest when cells were cultured with
N101-43. Characterization of this effect demonstrated that ex-
pression levels of cell cycle regulators were modulated by N101-43
treatment; arrest appeared to occur at sub-G1 or G0/G1 phase.
Apoptosis is a cell suicide mechanism frequently dysregulated
in oncogenesis. Caspase cascades operate in both intrinsic and
extrinsic apoptosis. Intrinsic (mitochondrial) programmed cell
death often involves an increase in mitochondrial membrane
permeability, coupled to release of cytochrome c, whereas the
extrinsic (death receptor) pathway involves oligomerization of
death receptors and recruitment of Fas-associated death domain
protein (FADD); activation of caspase-8 occurs downstream of
these signals and stimulates cleavage of other caspases such as
caspase-3 and PARP.41 To characterize the apoptotic mechan-
isms induced by N101-43, we assessed expression of pro- and
anti-apoptotic proteins, finding that cleavage of caspase-3, -8, and
-9 and PARP was promoted by N101-43 treatment.
The PI3K/Akt signaling pathway is influential in the regula-
tion of cell survival, as activation of anti-apoptotic downstream
effectors,42 phosphorylation-dependent inhibition of pro-apop-
totic signals of such as BAD protein, caspase-9, and the family of
Forkhead transcription factors.43ꢀ45 Recent works revealed that
Akt inhibition results in the up-regulation of FasL expression in
vascular smooth muscle cells (VSMC) and HeLa cells.46,47
Down-regulation of Akt leads to decrease in phosphorylation
of the endogenous Forkhead and its location to the nucleus. It
impairs the induction of FasL promoter.45 In addition, Akt
promotes cell survival via activation of NF-kB.48 NF-kB en-
hances cell survival and participates significantly in cancer cells’
acquisition of resistance to chemical or radiation therapy.36 As
such, NF-kB is an important target for lung cancer treatment and
chemoprevention. It has been reported that p53 binds directly to
a promoter of PIK3CA, which encodes the catalytic subunit of
PI3K, and inhibits its activity, whereas inactivation of p53 and
subsequent up-regulation of PIK3CA may contribute to the
pathophysiology of cancer.49 Our results demonstrated that
N101-43 induced p53 expression and inhibited NF-kB as well
as PI3K/Akt signaling in lung cancer cells. These data suggest that
the major pathways of apoptosis induced by treatment with N101-
43 are NF-kB and PI3K/Akt dependent. Because we need more
compounds and their biological data to obtain the relationships
between the different structures and their biological activities, the
current data are not enough for us to predict a structureꢀactivity
relationship (SAR). This remains for further study.
As a major cell survival signal, nuclear factor-kB (NF-kB) par-
ticipates in multiple steps in cancer cells’ resistance to chemical
and radiation therapies. Recent studies with animal models and
cell culture systems have established links between NF-kB and
lung carcinogenesis, highlighting its significance as a target in
lung cancer treatment and chemoprevention.36 We therefore
probed the possibility that N101-43 might inhibit NF-kB in
NCI-H460 and A549 cell lines. Indeed, N101-43 inhibited the
phosphorylation of IkB, an upstream mediator of NF-kB func-
tion, and thus p65 localization into nuclear was also inhibited in
cells activated by N101-43 (Figure 5A), supporting the involve-
ment of NF-kB inactivation in N101-43-induced apoptosis. Our
findings therefore suggested that N101-43 initially stimulates the
activation of Fas and FasL, in the extrinsic apoptosis module,
with the consequence of downstream activation of caspase-3 and
-8 and PARP through inhibition of NF-kB in lung cancer
cell lines.
It is known that PI3K and its downstream substrate Akt also
have a hand in apoptosis.37,38 We examined the expression of
these proteins, identifying dose-dependent reductions in PI3K
and phospho-Akt in both NCI-H460 and A549 cell lines.
phospho-GSK α/β also diminished upon N101-43 stimulation
of NCI-H460 cells; nevertheless, no significant change in total
GSK α/β protein levels was observed, indicating that N101-43
does not affect overall GSK α/β protein stability. We examined
whether or not PI3K inhibition can enhance FasL and p53
expression in NCI-H460 and A549 cell lines, where N101-43
suppressed cell growth in NCI-H460 and A549 cell lines, with a
more pronounced effect on the former (Figure 1B,C). PI3K
inhibitor LY294002 also showed a more pronounced effect on
N101-43-induced FasL and p53 expression in NCI-H460 as in
Figure 5D. These results indicate that N101-43 appears to
promote programmed cell death through the PI3K/Akt pathway
in lung cancer cell lines.
’ DISCUSSION
Plant extracts demonstrate significant potential as anticancer
therapeutic agents due to their ability to inhibit tumor growth,
angiogenesis, and metastasis with few side effects.39 Much of this
activity appears to stem from flavonoids, which are principal
components of many such extracts and demonstrate the capacity
to inactivate carcinogens, inhibit angiogenesis, and halt cell
proliferation or promote apoptosis.40 Naringenin is a flavonoid
abundant in citrus fruits, especially their peels and rinds, and has
been suggested to exhibit antiproliferative effects against human
tumor cells. Here we endeavored to investigate the effects of the
naringenin derivative N101-43 on lung cancer cell development.
We first investigated the antiproliferative effects of N101-43
on the NSCLC cell lines NCI-H460 (p53 +/+), A549 (p53 +/+),
and NCI-H1299 (p53 ꢀ/ꢀ). N101-43 was cytotoxic to NCI-
H460 and A549 cells and less so to NCI-H1299; HaCaT
keratinocyte control cells were not affected by the compound.
N101-43 therefore appeared to reduce cancer cell growth with
minimal collateral damage.
In summary, we demonstrate that the naringenin derivative
N101-43 induces apoptosis through the Fas (extrinsic) death
receptor, accompanied by processing of caspase-8, -3, and -9 and
PARP and inhibition of PI3K/Akt signaling, in NCI-460 and
A549 cells. N101-43 therefore displays promise as a pro-apopto-
tic factor that may benefit NSCLC therapy.
’ AUTHOR INFORMATION
Corresponding Author
*Postal address: Department of Bioscience and Biotechnology,
Bio/Molecular Informatics Center, Konkuk University, Hwayang-
dong 1, Gwangjin-gu, Seoul 143-701, Republic of Korea. Fax: +82-
2-444-4218. Phone: +82-2-450-4119. E-mail: ydy4218@konkuk.
ac.kr.
Author Contributions
These authors contributed equally to this work.
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Funding Sources
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This research was supported by a Korea Research Foundation
(KRF) grant funded by the Korean government (MEST 2010-
0019306, 2009-0072028). D.-Y.Y. is partially supported by the
Priority Research Centers Program (2009-0093824) and the
National R&D Program for Cancer Control, Ministry for Health,
Welfare, and Family aAffairs, Republic of Korea (0920080).
’ ACKNOWLEDGMENT
The synthetic naringenin derivative N101-43 was the
generous gift of Professor K. Y. Jung, Department of Bio-
chemical Engineering, Gangneung-Wonju National University
(Gangneung, Korea).
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NSCLC, non-small-cell lung cancer; DMSO, dimethyl sulfoxide;
N101-3, 5,7,40-trihydroxy flavanone N-phenyl hydrazone; N101-
43, 5-hydroxy-7,40-diacetyloxyflavanone-N-phenyl hydrazone-
PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered
saline; PI, propidium iodide; PVDF, polyvinylidene difluoride;
PI3K, phosphatidylinositol 3-kinase; SDS-PAGE, sodium dode-
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