Structure-activity relationship of synthetic 2-phenylnaphthalenes with hydroxyl groups that inhibit proliferation and induce apoptosis of MCF-7 cancer cells

Article abstract of DOI:10.1371/journal.pone.0141184

In this study, six 2-phenylnaphthalenes with hydroxyl groups were synthesized in high yields by the demethylation of the corresponding methoxy-2-phenylnaphthalenes, and one 2-phenylnaphthalene with an amino group was obtained by hydrogenation. All of the

Full text of DOI:10.1371/journal.pone.0141184

RESEARCH ARTICLE
Structure-Activity Relationship of Synthetic
2-Phenylnaphthalenes with Hydroxyl
Groups that Inhibit Proliferation and Induce
Apoptosis of MCF-7 Cancer Cells
1
2
3,4
3,4
Chi-Fen Chang *, Ci-Yi Ke , Yang-Chang Wu , Ta-Hsien Chuang
*
1
Department of Anatomy, School of Medicine, China Medical University, Taichung, Taiwan, 2 Department
of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan, 3 School of
Pharmacy, China Medical University, Taichung, Taiwan, 4 Research Center for Chinese Herbal Medicine,
China Medical University, Taichung, Taiwan
*
cfchang@mail.cmu.edu.tw (CFC); thchuang@mail.cmu.edu.tw (THC)
Abstract
OPEN ACCESS
In this study, six 2-phenylnaphthalenes with hydroxyl groups were synthesized in high
yields by the demethylation of the corresponding methoxy-2-phenylnaphthalenes, and one
Citation: Chang C-F, Ke C-Y, Wu Y-C, Chuang T-H
(
2015) Structure-Activity Relationship of Synthetic
-Phenylnaphthalenes with Hydroxyl Groups that
2
-phenylnaphthalene with an amino group was obtained by hydrogenation. All of the 2-phe-
2
nylnaphthalene derivatives were evaluated for cytotoxicity, and the structure-activity rela-
tionship (SAR) against human breast cancer (MCF-7) cells was also determined. The SAR
results revealed that cytotoxicity was markedly promoted by the hydroxyl group at the C-7
position of the naphthalene ring. The introduction of hydroxyl groups at the C-6 position of
the naphthalene ring and the C-4' position of the phenyl ring fairly enhanced cytotoxicity,
but the introduction of a hydroxyl group at the C-3' position of the phenyl ring slightly
decreased cytotoxicity. Overall, 6,7-dihydroxy-2-(4'-hydroxyphenyl)naphthalene (PNAP-
Inhibit Proliferation and Induce Apoptosis of MCF-7
Cancer Cells. PLoS ONE 10(10): e0141184.
doi:10.1371/journal.pone.0141184
Editor: Yi-Hsien Hsieh, Institute of Biochemistry and
Biotechnology, TAIWAN
Received: July 13, 2015
Accepted: October 6, 2015
Published: October 22, 2015
6
h) exhibited the best cytotoxicity, with an IC50 value of 4.8 μM against the MCF-7 cell line,
and showed low toxicity toward normal human mammary epithelial cells (MCF-10A).
PNAP-6h led to cell arrest at the S phase, most likely due to increasing levels of p21 and
p27 and decreasing levels of cyclin D1, CDK4, cyclin E, and CDK2. In addition, PNAP-6h
Copyright: © 2015 Chang et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
decreased CDK1 and cyclin B1 expression, most likely leading to G /M arrest, and induced
2
morphological changes, such as nuclear shrinkage, nuclear fragmentation, and nuclear
hypercondensation, as observed by Hoechst 33342 staining. PNAP-6h induced apoptosis,
most likely by the promotion of Fas expression, increased PARP activity, caspase-7, cas-
pase-8, and caspase-9 expression, the Bax/Bcl-2 ratio, and the phosphorylation of p38, and
decreased the phosphorylation of ERK. This study provides the first demonstration of the
cytotoxicity of PNAPs against MCF-7 cells and elucidates the mechanism underlying
PNAP-induced cytotoxicity.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: The authors would like to thank the
Ministry of Science and Technology, Taiwan (MOST
103-2320-B-039-027-; MOST 103-2113-M-039-003-
and MOST 103-2738-M-039-001-), China Medical
University (CMU103-N-05), and in part, the grant
from the Chinese Medicine Research Center, China
Medical University (the Ministry of Education, the Aim
for the Top University Plan) for financial support. The
funders had no role in study design, data collection
PLOS ONE | DOI:10.1371/journal.pone.0141184 October 22, 2015
1 / 17

PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
and analysis, decision to publish, or preparation of
the manuscript.
Introduction
Breast cancer is the most common cause of cancer death in females; therefore, the search for a
new and effective anticancer agent is imperative. Naphthalene derivatives display potent anti-
arrhythmia, anti-tumor, and antioxidant activities [1]. Pharmacologically, 2-phenylnaphtha-
lenes (PNAPs) have similar spatial and conformational requirements to genistein (an isofla-
vone) and exhibit a large number of biological and biomedical effects [2]. Chemically,
Competing Interests: The authors have declared
that no competing interests exist.
1
-substituted naphthalene, not 2-substituted naphthalene, is obtained by the electrophilic aro-
matic substitution of naphthalene. To the best of our knowledge, studies on the relationship
between the structure and cytotoxicity of multi-substituted PNAPs are rare. In this study, we
synthesized unsubstituted PNAP-1, seven methoxy-PNAPs (PNAP-2−PNAP-8), six corre-
sponding hydroxy-PNAPs (PNAP-2h−PNAP-7h), and one amino-PNAP (PNAP-8h) and
investigated their anticancer structure-activity relationships and mechanisms of action in the
MCF-7 cell line.
Several studies have demonstrated that genistein and compounds with the phenyl-1-benzo-
pyran-4-one backbone inhibit the growth of cancer cells via cell cycle arrest and the induction
of apoptosis [3–5]. Cyclin-cyclin dependent kinase (CDK) complexes are the principal regula-
tors of the cell cycle, which is a very complex and tightly regulated process [6,7]. The G /S
1
phase transition is regulated by activation of the cyclin D1-CDK4/6 complex and the cyclin
E-CDK2 complex [8,9], whereas the G /M phase transition is regulated by activation of the
2
cyclin B1-CDK1 complex [10,11]. Deregulation of the cell cycle causes a lack of differentiation
and aberrant growth.
Apoptosis is an evolutionary process that leads to programmed cell death [12]. The process
of apoptosis includes morphological changes (e.g., cell shrinkage, membrane blebbing, chro-
matin condensation, and nuclear fragmentation) and biochemical changes (e.g., DNA break-
down, protein cleavage, and protein cross-linking) [13,14]. Many anticancer agents induce cell
death by activating caspases, which form part of a common apoptotic pathway [15,16]. Extrin-
sic and intrinsic pathways that regulate caspase-dependent apoptosis have been identified [17].
In the extrinsic pathway, Fas, a death receptor, leads to the formation of a death-inducing
FADD-caspase-8 signaling complex [18]. The intrinsic pathway is controlled by the Bcl-2 fam-
ily of proteins. First, the Bax/Bcl-2 ratio increases, and this increase is followed by the release of
cytochrome c, leading to the activation of caspase-9 and caspase-3 [19]. The MAPK pathway is
well known for its regulation of cell survival and apoptosis. The MAPK family is composed of
three major kinases: ERK, p38, and JNK [20]. ERK is preferentially activated by growth factors,
leading to cell growth and survival. p38 and JNK are preferentially activated by cytokine stimu-
lation and oxidative stress, resulting in cell differentiation and apoptosis [21,22]. It is unclear
whether the intrinsic/extrinsic pathway or the MAPK pathway is activated in PNAP-mediated
apoptosis. In this study, we assessed the cytotoxicity of a series of PNAPs against MCF-7 cells
and elucidated the mechanism underlying PNAP-induced cytotoxicity.
Materials and Methods
Chemistry
The approaches used to synthesize the fifteen PNAP derivatives are shown in Fig 1. 2-Phenyl-
naphthalene (PNAP-1) and seven methoxy-PNAPs (PNAP-2−PNAP-8) were synthesized
from commercially available phenylacetonitriles and benzaldehydes according to previously
reported methods [23]. Six corresponding hydroxy-PNAPs (PNAP-2h−PNAP-7h) were
obtained by the demethylation of PNAP-2−PNAP-7 in excellent yields (91% to 100%). One
amino-PNAP (PNAP-8h) was obtained by the hydrogenation of PNAP-8 at a yield of 86%.
PLOS ONE | DOI:10.1371/journal.pone.0141184 October 22, 2015
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PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
1
13
The H and C NMR spectra of PNAP-2h−PNAP-8h are available in the Supporting Infor-
mation (Figures A−G in S1 File). All of the PNAPs were dissolved in DMSO to a concentration
of 50 mM to prepare stock solutions, which were stored at -20°C.
General Procedure for the Preparation of hydroxy-PNAPs. BBr in CH Cl at a concen-
3
2
2
tration of 1 M (5 mL, 5 mmol) was slowly added to a solution of methoxy-PNAPs (PNAP-2
PNAP-7, 0.5 mmol) in CH Cl (20 mL) at 0°C. The mixture was allowed to warm to room
−
2
2
temperature and was stirred for 1 h. The resulting solution was poured into H O (50 mL). The
2
H O layer was extracted with EtOAc (3 × 50 mL), and the combined extracts were washed with
2
H O (3 × 50 mL), dried with anhydrous MgSO , and filtered. The filtrate was concentrated,
2
4
and the residue was purified by column chromatography over silica gel and eluted with EtOAc
to obtain hydroxy-PNAPs (PNAP-2h−PNAP-7h). The full spectral data of these compounds
are described as follows.
6
-Hydroxy-2-phenylnaphthalene (PNAP-2h). Yield 91%; white solid, mp 176–177°C (lit.
1
[
24], mp 169−170°C). H NMR (500 MHz, CDCl ) δ 5.12 (1H, br s), 7.12 (1H, dd, J = 8.8, 2.4
3
Hz), 7.17 (1H, s), 7.36 (2H, t, J = 7.4 Hz), 7.47 (1H, t, J = 7.4 Hz), 7.69–7.71 (3H, m), 7.75 (1H,
13
d, J = 8.8 Hz), 7.80 (1H, d, J = 8.8 Hz), 7.97 (1H, s); C NMR (125 MHz, CDCl ) δ 109.3,
3
1
1
18.2, 125.7, 126.3, 126.9, 127.1, 127.2 (2×C), 128.8 (2×C), 129.2, 130.2, 133.8, 136.4, 141.2,
-
1
53.5; IR (KBr) 3339, 3046, 1618, 1495, 1292, 1194 cm ; ESIMS m/z (rel int) 219 (100,
-
-
[
M-H] ); HRESIMS m/z calcd for C H O: 219.08044; found: 219.08036 [M-H] .
16 11
6
,7-Dihydroxy-2-phenylnaphthalene (PNAP-3h). Yield 98%; white solid, mp 205–
1
2
07°C. H NMR (500 MHz, DMSO-d ) δ 7.12 (1H, s), 7.20 (1H, s), 7.32 (1H, t, J = 7.5 Hz), 7.45
6
(
2H, t, J = 7.5 Hz), 7.50 (1H, d, J = 8.4 Hz), 7.65 (1H, d, J = 8.4 Hz), 7.72 (2H, d, J = 7.5 Hz),
Fig 1. Synthetic approaches for the production of PNAP-1–PNAP-8 and PNAP-2h–PNAP-8h. The eight 2-phenylnaphthalenes (PNAP-1–PNAP-8)
were easily obtained from phenylacetonitriles and benzaldehydes through six steps. Subsequently, the six hydroxy-PNAPs (PNAP-2h− PNAP-7h) were
obtained by demethylation of the corresponding methoxy-PNAPs (PNAP-2− PNAP-7). Unfortunately, attempts at the demethylation of PNAP-8 under the
same conditions led to complex mixtures. Another hydrophilic amino-PNAP (PNAP-8h) was produced by the reduction of nitro-PNAP (PNAP-8).
doi:10.1371/journal.pone.0141184.g001
PLOS ONE | DOI:10.1371/journal.pone.0141184 October 22, 2015
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PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
1
3
7
1
3
.86 (1H, s), 9.57 (2H, br s); C NMR (125 MHz, DMSO-d ) δ 109.5, 110.2, 112.2, 123.4,
6
26.4, 126.8 (2×C), 127.0, 128.3, 129.0 (2×C), 129.3, 134.7, 140.9, 147.3, 147.4; IR (KBr) 3495,
395, 1628, 1528, 1119 cm ; ESIMS m/z (rel int) 235 (100, [M-H] ); HRESIMS m/z calcd for
C H O : 235.0754; found: 235.0755 [M-H] .
-
1
-
-
1
6
11 2
2
-(4'-hydroxyphenyl)naphthalene (PNAP-4h). Yield 100%; white solid, mp 166–167°C
1
(
lit. [25], mp 167−169°C). H NMR (500 MHz, CDCl ) δ 4.89 (1H, br s), 6.95 (2H, d, J = 8.6
3
Hz), 7.44–7.50 (2H, m), 7.61 (2H, d, J = 8.6 Hz), 7.70 (1H, dd, J = 8.5, 1.7 Hz), 7.84–7.90 (3H,
13
m), 7.97 (1H, s); C NMR (125 MHz, CDCl ) δ 115.7 (2×C), 125.0, 125.4, 125.7, 126.2, 127.6,
3
1
1
28.0, 128.3, 128.7 (2×C), 132.3, 133.7, 133.9, 138.1, 155.1; IR (KBr) 3564, 3055, 1603, 1512,
-
1
-
180 cm ; ESIMS m/z (rel int) 219 (41, [M-H] ); HRESIMS m/z calcd for C H O: 219.08044;
16
11
-
found: 219.08043 [M-H] .
-hydroxy-2-(4'-hydroxyphenyl)naphthalene (PNAP-5h). Yield 93%; white solid, mp
1
6
1
27–128°C (lit. [26], mp 258−262°C). H NMR (500 MHz, DMSO-d ) δ 6.86 (2H, d, J = 8.4
6
Hz), 7.01 (1H, dd, J = 8.8, 2.0 Hz), 7.10 (1H, s), 7.56 (2H, d, J = 8.4 Hz), 7.63 (1H, dd, J = 8.6,
.6 Hz), 7.70 (1H, d, J = 8.6 Hz), 7.78 (1H, d, J = 8.8 Hz), 7.94 (1H, s), 9.51 (1H, br s), 9.70 (1H,
1
1
3
br s); C NMR (125 MHz, DMSO-d ) δ 108.6, 115.9 (2×C), 119.1, 124.1, 125.2, 126.7, 127.8
6
(
1
2
2×C), 128.3, 129.7, 131.2, 133.5, 134.6, 155.3, 157.0; IR (KBr) 3183, 3030, 1603, 1512, 1265,
-
1
-
204 cm ; ESIMS m/z (rel int) 235 (100, [M-H] ); HRESIMS m/z calcd for C H O :
16 11 2
-
35.0754; found: 235.0751 [M-H] .
,7-Dihydroxy-2-(4'-hydroxyphenyl)naphthalene (PNAP-6h). Yield 98%; white solid,
6
1
mp 280–282°C. H NMR (500 MHz, DMSO-d ) δ 6.84 (2H, d, J = 8.1 Hz), 7.08 (1H, s), 7.13
6
(
1H, s), 7.42 (1H, d, J = 8.4 Hz), 7.53 (2H, d, J = 8.1 Hz), 7.58 (1H, d, J = 8.4 Hz), 7.73 (1H, s),
13
9
1
1
2
.46 (3H, br s); C NMR (125 MHz, DMSO-d ) δ 109.5, 109.9, 115.8 (2×C), 122.0, 122.2,
6
26.2, 127.7, 127.8 (2×C), 129.3, 131.6, 134.8, 146.8, 147.3, 156.8; IR (KBr) 3495, 3341, 1597,
-
1
-
520, 1119 cm ; ESIMS m/z (rel int) 251 (100, [M-H] ); HRESIMS m/z calcd for C H O :
1
6
11 3
-
51.07027; found: 251.07032 [M-H] .
,7-Dihydroxy-2-(3',4'-dihydroxyphenyl)naphthalene (PNAP-7h). Yield 100%; white
6
1
solid, mp 230°C (decomp.). H NMR (500 MHz, DMSO-d ) δ 6.80 (1H, d, J = 8.2 Hz), 6.98
6
(
1H, dd, J = 8.2, 2.0 Hz), 7.08 (1H, s), 7.09 (1H, d, J = 2.0 Hz), 7.13 (1H, s), 7.37 (1H, d, J = 8.4
13
Hz), 7.57 (1H, d, J = 8.4 Hz), 7.68 (1H, s), 8.94 (2H, br s), 9.46 (2H, br s); C NMR (125 MHz,
DMSO-d ) δ 109.5, 110.0, 114.1, 116.2, 117.7, 122.0, 122.2, 126.2, 127.7, 129.3, 132.3, 135.1,
6
-
1
1
(
44.9, 145.7, 146.8, 147.3; IR (KBr) 3372, 3329, 1602, 1234, 1111 cm ; ESIMS m/z (rel int) 267
100, [M-H] ); HRESIMS m/z calcd for C H O : 267.0652; found: 267.0647 [M-H] .
16 11 4
-
-
2-(4'-Aminophenyl)-6,7-dimethoxynaphthalene (PNAP-8h). PNAP-8 (77 mg, 0.25
mmol) was subjected to hydrogenation (50 psi) using 10% Pd/C as the catalyst in tetrahydrofu-
ran (THF, 10 mL) and EtOH (1 mL) at room temperature for 12 h. The reaction mixture was
filtered, and the filtrate was concentrated. The residue was purified by column chromatography
over silica gel and eluted with EtOAc to yield PNAP-8h. Yield 86%; pale yellow solid, mp 190–
1
1
91°C. H NMR (500 MHz, CDCl ) δ 3.74 (2H, br s), 4.01 (6H, s), 6.79 (2H, d, J = 8.1 Hz), 7.12
3
(
1H, s), 7.15 (1H, s), 7.52 (2H, d, J = 8.1 Hz), 7.56 (1H, d, J = 8.4 Hz), 7.71 (1H, d, J = 8.4 Hz),
1
3
7
1
2
.82 (1H, s); C NMR (125 MHz, CDCl ) δ 55.9 (2×C), 106.1, 106.5, 115.5 (2×C), 123.2, 123.6,
3
26.7, 127.8, 128.1 (2×C), 129.6, 131.8, 137.0, 145.6, 149.2, 149.7; IR (KBr) 3460, 3439, 3018,
-
1
+
995, 1624, 1504, 1244, 1130 cm ; ESIMS m/z (rel int) 280 (100, [M+H] ); HRESIMS m/z
+
calcd for C H NO : 251.07027; found: 251.07032 [M+H] .
1
8
17
2
Cell culture
MCF-7 cells were obtained from the laboratory of Dr. Yang-Chang Wu (School of Pharmacy,
China Medical University, Taiwan), and MCF-10A cells were obtained from the laboratory of
PLOS ONE | DOI:10.1371/journal.pone.0141184 October 22, 2015
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PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
Dr. Wei-Chien Huang (Graduate Institute of Cancer Biology, China Medical University, Tai-
wan). MCF-7 cells were maintained in DMEM/F12 medium containing 10% fetal bovine
serum and 1% penicillin-streptomycin. MCF-10A cells were maintained in DMEM/F12
medium containing 1.05 mM CaCl , 100 mg/ml cholera toxin, 5% horse serum, 10 μg/ml insu-
2
lin, 500 ng/ml hydrocortisone and 1% penicillin-streptomycin. These cells were cultured in cell
culture incubators, which were set to temperatures of 37°C and supplemented with 5% CO2.
Cell viability assay
3
MCF-7 cells were plated at a density of 5×10 cells per well in 96-well plates, incubated over-
night and then treated with different concentrations (0, 0.5, 1, 2.5, 5, 10, 25, and 50 μM) of fif-
teen PNAPs (PNAP-1−PNAP-8 and PNAP-2h−PNAP-8h). In addition, MCF-10A cells were
3
plated at a density of 5×10 cells per well in 96-well plates, incubated overnight and then
treated with different concentrations (0, 1, 2.5, 5, 10, 25, 50, and 100 μM) of three PNAPs
(PNAP-3h, -6h, and -7h). After 48 h of treatment, 10 μl of MTT solution (5 mg/ml in PBS)
was added to each well, and the cells were incubated for an additional 4 h at 37°C. The medium
was then completely removed, and 50 μl of DMSO was added to solubilize the MTT formazan
crystals. The absorbance at a wavelength of 550 nm was then measured using a MQX200R
microplate reader (BioTek, VT, USA).
Cell cycle analysis
5
MCF-7 cells were seeded at a density of 3×10 cells per well in six-well plates, incubated over-
night and then treated with PNAP-1, -3h, and -6h at three different concentrations (5, 10, and
2
7
5 μM). After 48 h of treatment, the cells were trypsinized, washed once with PBS, and fixed in
0% ethanol for 1 h at -20°C. The fixed cells were washed with ice-cold PBS, suspended in 0.5
ml of PBS containing 0.2 mg/ml RNase and 0.1% Triton X-100 for 30 min at room tempera-
ture, and stained with 20 μg/ml propidium iodide. The stained cells were analyzed using a
FACScan flow cytometer (Becton Dickinson, CA, USA). The fluorescence emitted from the
propidium iodide-DNA complex was estimated from a minimum of 10,000 cells per sample
and analyzed using the Cell Quest Alias software (BD Biosciences, USA).
Western blot analysis
5
MCF-7 cells were seeded at a density of 3×10 cells per well in six-well plates, incubated over-
night and then treated with PNAP-1, -3h, and -6h at three different concentrations (5, 10, and
2
5 μM) for 48 h. The cells were washed with PBS and lysed in ice-cold RIPA buffer for 30 min.
The supernatant was collected and centrifuged at 15,000 rpm and 4°C for 30 min. The protein
concentration was measured through a Bio-Rad assay using a MQX200R microplate reader
(
BioTek, VT, USA). The cell lysates were separated by 10% SDS-PAGE and electrophoretically
transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA,
USA). After several washes, the membranes were blocked with 5% skim milk in TBST (Tris-
buffered saline containing 0.1% Tween-20) for 1 h at room temperature and incubated over-
night at 4°C with different primary antibodies against cyclin D1, CDK4, cyclin E, CDK2, p21,
p27, cyclin B1, CDK1, cleaved caspase-7, -8, -9, PARP, Bcl-2, Bcl-xl, Bax, Bid, Fas, p-p38, p38,
p-JNK, JNK, p-ERK, or ERK (1:1000 dilution). The membranes were then washed three times
with TBST and probed with horseradish peroxidase (HRP)-conjugated secondary antibody
(
1:2000) for 1 h at room temperature. After three washes in TBST, the bound antibody was
visualized using the ECL Western Blotting Reagent (PerkinElmer, Boston, MA, USA), and the
chemiluminescence was detected using Fuji Medical X-ray film (Tokyo, Japan).
PLOS ONE | DOI:10.1371/journal.pone.0141184 October 22, 2015
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PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
Table 1. Effect of 2-Phenylnaphthalenes PNAP-1− PNAP-8, Their Demethylation Derivatives PNAP-2h− PNAP-7h, and Amino PNAP-8h on the Viabil-
ity of MCF-7 Cells.
a
Compound
Viability (% of control)
0
μM
0.5 μM
1.0 μM
2.5 μM
5.0 μM
10.0 μM
25.0 μM
50.0 μM
PNAP-1
PNAP-2
PNAP-3
PNAP-4
PNAP-5
PNAP-6
PNAP-7
PNAP-8
PNAP-2h
PNAP-3h
PNAP-4h
PNAP-5h
PNAP-6h
PNAP-7h
PNAP-8h
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
97.3 ± 2.6
97.5 ± 3.4
98.6 ± 0.7
96.2 ± 3.8
94.7 ± 1.6
95.2 ± 3.4
90.4 ± 2.5
89.5 ± 1.8
104.9 ± 0.9
98.6 ± 1.5
102.8 ± 1.9
100.5 ± 1.2
101.8 ± 1.8
97.1 ± 1.7
99.9 ± 0.8
94.6 ± 1.2
97.4 ± 2.4
97.1 ± 0.7
97.0 ± 2.2
94.8 ± 1.9
94.7 ± 2.2
89.8 ± 1.1
88.5 ± 3.8
102.5 ± 0.9
97.4 ± 4.2
100.3 ± 4.5
101.1 ± 2.6
96.3 ± 1.1
98.6 ± 3.2
98.9 ± 2.4
97.0 ± 1.7
100.7 ± 3.0
96.7 ± 1.3
100.4 ± 2.8
96.5 ± 2.2
76.7 ± 4.8
88.1 ± 1.8
78.6 ± 1.9
102.3 ± 1.5
94.8 ± 2.0
102.0 ± 8.2
100.6 ± 1.7
72.3 ± 3.1*
97.5 ± 1.0
101.1 ± 1.9
91.9 ± 1.7
101.6 ± 1.9
95.8 ± 1.4
102.7 ± 2.2
93.2 ± 0.8
50.2 ± 0.7
68.0 ± 3.6
66.5 ± 0.5
102.1 ± 0.8
86.8 ± 2.0
103.7 ± 3.6
97.7 ± 0.8
48.4 ± 3.9**
99.6 ± 0.6
89.3 ± 5.4
96.1 ± 2.2
83.1 ± 4.9
88.9 ± 0.5
96.8 ± 2.8
89.5 ± 1.0
49.2 ± 1.1
50.7 ± 1.6
52.1 ± 1.0
97.2 ± 1.9
77.6 ± 1.1**
102.3 ± 2.2
77.3 ± 5.8*
40.9 ± 3.9**
85.0 ± 3.6
93.2 ± 2.1
92.6 ± 2.3
56.5 ± 2.1
52.9 ± 0.6
61.7 ± 2.4
77.3 ± 1.8
48.3 ± 2.2
48.3 ± 0.7
47.0 ± 1.2
82.7 ± 4.4
32.9 ± 1.3**
95.5 ± 3.8
70.2 ± 2.7*
18.2 ± 3.5**
55.3 ± 0.9**
85.2 ± 5.8
86.1 ± 0.4
59.0 ± 2.0
51.8 ± 0.9
56.0 ± 1.5
60.4 ± 2.1
48.9 ± 1.5
47.6 ± 0.7
48.0 ± 0.8
60.0 ± 3.2**
18.7 ± 2.5**
78.0 ± 4.9
56.2 ± 2.2*
12.4 ± 2.0**
40.3 ± 2.4**
75.3 ± 0.9*
The MCF-7 cell line was treated with PNAP-1− PNAP-8, and PNAP-2h− PNAP-8h at eight concentrations and then incubated at 37°C for 48 h. The cell
viability was examined via the MTT assay.
a
The number of viable cells remaining after PNAP treatment is expressed as a percentage of the vehicle control, which was arbitrarily assigned a value of
100.0%. The results are presented as the means ± SEM from three independent assays.
*
P<0.05 and **P<0.01 compared with the control.
doi:10.1371/journal.pone.0141184.t001
Cell morphology studies
5
The cells were plated in 12-well plates at a density of 1×10 cells/well, incubated overnight and
then treated with PNAP-1, -3h, and -6h at three different concentrations (5, 10, and 25 μM)
for 48 h. The cells were washed once with PBS, and a subset of cells was stained with Hoechst
3
3342 (10 μg/ml for 15 min). The morphological changes were then observed by light micros-
copy at 100× magnification and fluorescence microscopy at 200× magnification.
Statistical analysis
All of the data are expressed as the means ± SEM from three replicate experiments. The differ-
ences among the groups were compared by analysis of variance (ANOVA) and post-hoc Tukey
Honestly Significant Difference (HSD) test using the SPSS 12.0 software for Windows (USA).
P values less than 0.05 were considered statistically significant.
Results and Discussion
Effect of PNAPs on MCF-7 cell line viability and study of their structure-
activity relationship
To investigate the effects of the 2-phenylnaphthalenes PNAP-1−PNAP-8, their demethylation
derivatives PNAP-2h−PNAP-7h, and amino-PNAP (PNAP-8h) on cell survival, MCF-7 cells
were treated with different doses of each compound (0, 0.5, 1, 2.5, 5, 10, 25, and 50 μM) for
4
8 h. The rates of viability were determined via the MTT assay, and the results are shown in
Table 1. First, PNAP-1 showed no significant activity in MCF-7 cells, and PNAP-2−PNAP-8,
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PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
which contain methoxy groups, caused precipitation in the cell media at concentrations higher
than 5 μM (Table 1). Therefore, the more hydrophilic PNAP derivatives PNAP-2h−PNAP-8h
were designed and synthesized. Indeed, no precipitation was observed for PNAP-2h−PNAP-
8
h, most likely due to the formation of intermolecular hydrogen bonds between water and the
hydroxyl (or amino) groups on the 2-phenylnaphthalenes. Three of the derivatives (PNAP-3h,
6h, and -7h) exhibited significant dose-dependent toxicity against MCF-7 cells, with IC50 val-
-
ues of 17.9, 4.8, and 31.8 μM, respectively (Table 1), whereas the IC50 values obtained against
MCF-10A cells were 71.0, 50.9, and 55.1 μM, respectively. The results indicate that PNAP-3h
and -6h possess enhanced and selective cytotoxicity against MCF-7 cells and show low toxicity
toward MCF-10A cells.
The above-described results revealed that PNAP-5h, which has hydroxyl groups at the C-6
position of the naphthalene ring and at the C-4' position of the phenyl ring, exhibited better
cytotoxicity than PNAP-1, -2h, and -4h in our tests. This phenomenon shows that the intro-
duction of hydroxyl groups at the C-6 position of the naphthalene ring and the C-4' position of
the phenyl ring enhances cytotoxicity. Intriguingly, the two 2-phenylnaphthalenes PNAP-3h
and -6h, which contain a hydroxyl group at the C-7 position of the naphthalene ring, exhibited
better cytotoxic activities against the MCF-7 cell line (compare PNAP-3h to -2h and compare
PNAP-6h to -5h). These results suggest that cytotoxicity is markedly promoted by the presence
of a hydroxyl group at the C-7 position of the naphthalene ring. Moreover, it should be noted
that PNAP-6h exhibited the best activity against the MCF-7 cell line (IC50 = 4.8 μM). This
result also indicated that the introduction of a hydroxyl group at the C-4' position of the phenyl
ring would enhance the activities against the MCF-7 cell line. In contrast, the presence of a
hydroxyl group at the C-3' position of the phenyl ring in PNAP-7h may markedly diminish
cytotoxicity (compare PNAP-6h and -7h). In addition, the comparison of the cytotoxic activi-
ties of PNAP-6h and PNAP-8h suggests that the presence of a hydroxyl group, which serves as
a H-bond donor, on the naphthalene ring yields better activities than the presence of a methoxy
group, which serves as a H-bond acceptor. Based on the cell viability and SAR results, we fur-
ther investigated the potential reasons for the decrease in cell viability induced by PNAP-3h
and -6h and elucidated the underlying mechanism.
Effect of PNAPs on the morphological characteristics of MCF-7 cells
First, the effect of PNAP-1, -3h and -6h on cellular morphology was observed by phase-con-
trast microscopy. MCF-7 cells treated with either a vehicle control (0.05% DMSO) or 5 to
2
2
5 μM PNAP-1 showed dense cell populations under a phase-contrast microscope (Fig 2A–
D). The MCF-7 cells were regular in shape and size, with eccentric nuclei and a relatively
small amount of cytoplasm. The cell density and structure of cells treated for 48 h with 5 to
5 μM PNAP-3h and -6h showed obvious changes (Fig 2E–2J). The cells became rounded and
2
shrunken and lost contact with adjacent cells. In addition, the apoptotic cells no longer adhered
to the substrate, causing them to detach from the culture plates and float in the culture
medium. Thus, PNAP-3h and -6h led to a decrease in the viable MCF-7 cell numbers in a con-
centration-dependent manner, as shown in Table 1.
We also observed the nuclear morphology of MCF-7 cells subjected to Hoechst 33342 fluo-
rescent staining after treatment for 48 h with three concentrations (5, 10, and 25 μM) of
PNAP-1, -3h, and -6h (Fig 2L–2T). The nuclei of the vehicle control cells displayed an intact
oval shape and were exhibited a weak blue fluorescence (Fig 2K). However, the cells treated
with PNAP-3h (25 μM) and PNAP-6h (5, 10, and 25 μM) exhibited typical apoptotic features,
such as nuclear shrinkage, nuclear fragmentation, and nuclear hypercondensation (Fig 2Q–
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PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
Fig 2. Effect of PNAP-1, -3h, and -6h on MCF-7 morphology. MCF-7 cells were treated with PNAP-1, -3h, and -6h at three concentrations (5, 10, and
25 μM) for 48 h, and then, (A–J) the morphology was observed by light microscopy (100×). The cells were stained with Hoechst 33342, and then, (K–T) the
morphology was observed by fluorescence microscopy (200×). (A/K) control; (B/L) 5 μM PNAP-1; (C/M) 10 μM PNAP-1; (D/N) 25 μM PNAP-1; (E/O) 5 μM
PNAP-3h; (F/P) 10 μM PNAP-3h; (G/Q) 25 μM PNAP-3h; (H/R) 5 μM PNAP-6h; (I/S) 10 μM PNAP-6h; and (J/T) 25 μM PNAP-6h.
doi:10.1371/journal.pone.0141184.g002
2
T). Based on these microscopic observations, we propose that the observed cytotoxicity may
be partially mediated through PNAP-3h- and -6h-induced apoptosis.
Effect of PNAPs on MCF-7 cell cycle phase distribution
Because cell proliferation is regulated by the cell cycle, the effect of PNAP-1, -3h and -6h on
cell cycle progression in the MCF-7 cell line was examined by flow cytometry and western blot
analyses. The results of the flow cytometric analysis indicated that treatment with PNAP-6h
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PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
for 48 h increased the sub-G population (Fig 3A). The sub-G peak, which consisted of cells
1
1
with reduced DNA content, represented the presence of apoptotic cells [27,28]. In addition, the
PNAP-6h-induced cell arrest at the G /M phase obtained at lower concentrations (5 and
2
1
0 μM) was accompanied by a decrease in the percentage of cells found at the Go/G1 phase.
PNAP-5h and -6h caused S phase arrest at a higher concentration (25 μM), led to a lower num-
ber of replicating cells and suppressed cell proliferation. However, the PNAP-1-treated cells
showed no significant change in the cell cycle distribution compared with the vehicle control
cells.
Based on these observations of the cell cycle distribution of MCF-7 cells, we analyzed the
changes in the abundance of cell cycle regulatory proteins. Cyclin-CDK complexes are the
principal regulators of cell cycle progression [7], and the activity of cyclin/CDK complexes are
negatively regulated by CDK inhibitors, such as p21 and p27 [29]. PNAP-3h (at 25 μM) and
PNAP-6h led to cell arrest at the S phase, most likely due to increased levels of p21 and p27
and reduced levels of cyclin D1, CDK4, cyclin E, and CDK2, as determined through the west-
ern blot analysis (Fig 3B and 3C). In addition, PNAP-6h induced an inhibition of CDK1 kinase
activity and a decrease in cyclin B1 expression, most likely leading to G /M arrest. Therefore,
2
we propose that the inhibition of cell viability by PNAP-3h and -6h may be partially mediated
through cell cycle arrest and apoptosis.
PNAPs activate the caspase pathway and increase the expression of
relative apoptotic proteins
Based on the above results, we found that PNAPs can prevent proliferation, induce apoptosis,
and decrease cell viability in the MCF-7 cell line. It is well known that numerous anticancer
drugs mediate apoptosis by activating caspases [16]. To demonstrate that the PNAP-induced
apoptosis in MCF-7 cells occurs via caspase activation, we investigated the expression of key
regulatory proteins associated with the caspase pathways in MCF-7 cells upon exposure to 5 to
2
5 μM PNAP-1, -3h, and -6h. Despite the lack of caspase-3 in MCF-7 cells, caspase-7, an apo-
ptosis executioner, may function as a replacement [30]. Caspase-7 is capable of cleaving spe-
cific tetrapeptide substrates (e.g., PARP), and PARP cleavage is a major hallmark of apoptosis
[
-
31]. The exposure of MCF-7 cells to three concentrations (5, 10, and 25 μM) of PNAP-3h and
6h for 48 h significantly increased the levels of cleaved caspase-7 and PARP and increased the
activity of cleaved caspase-7 and PARP in a dose-dependent manner (Fig 4A). Furthermore,
apoptosis could be stimulated via the extrinsic and intrinsic pathways. Caspase-8 is generally
considered an apoptosis activator in the extrinsic pathway, and caspase-9 plays an important
role in the intrinsic pathway [32]. These data show that the expression of cleaved caspase-8
and caspase-9 in MCF-7 cells increased after treatment with PNAP-3h and -6h in a dose-
dependent manner. Furthermore, cleaved caspase-8 and caspase-9 were partially inhibited by
the caspase-8-specific inhibitor Z-IETD-FMK and the caspase-9-specific inhibitor
Z-LEHD-FMK. Moreover, the two inhibitors also partially inhibited caspase-7 cleavage in
PNAP derivative-induced apoptosis (Fig 5A and 5B) and partially reversed the PNAP-6-
induced decrease in cell viability, as observed by microscopy and the MTT assay (Fig 5C).
These results suggest that the caspase-7-, caspase-8-, and caspase-9-mediated pathways may be
partially involved in the regulation of PNAP-induced apoptosis.
The activation of caspase-8 is the first step in the cascade of apoptotic events induced by Fas
stimulation [33], and the activation of caspase-9 is regulated by Bcl-2 family members [34–36].
However, the treatment of MCF-7 cells with 25 μM PNAP derivatives exerted a modest effect
on the expression of Bcl-xl, Bax, and Bid proteins at three time points (12, 24, and 48 h) (Fig
4
C and 4D). In contrast, the expression of Bcl-2 protein was significantly decreased and the
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PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
Fig 3. Effect of PNAP-1, -3h, and -6h on MCF-7 cell cycle progression. MCF-7 cells were treated with PNAP-1, -3h, and -6h at three concentrations (5,
10, and 25 μM) for 48 h, and then, (A) the cells were fixed and stained with propidium iodide to analyze the DNA content by flow cytometry. (B, C) Cell cycle-
associated proteins (cyclin D1, CDK4, cyclin E, CDK2, p21, p27, cyclin B1, and CDK1) were detected and analyzed by western blot. The expression was
quantified with the computerized Gel-Pro Analyzer image analysis system. The data are expressed as the means ± SEM from three independent assays.
*
P<0.05 and **P<0.01 are significantly different from the control.
doi:10.1371/journal.pone.0141184.g003
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PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
Fig 4. Effect of PNAP-1, -3h, and -6h on the expression of apoptosis-related proteins. (A) MCF-7 cells were treated with PNAP-1, -3h, and -6h at three
concentrations (5, 10, and 25 μM) for 48 h, and the expression of cleaved caspase-7, -8, -9 and PARP was determined by western blotting. (C) MCF-7 cells
were treated with 25 μM PNAP-1, -3h, and -6h at three concentrations (5, 10, and 25 μM) for 48 h, and the Bcl-2, Bcl-xl, Bax, Bax/Bcl-2, Bid, and Fas levels
were then detected by western blot. (B, D) The expression was quantified using a computerized Gel-Pro Analyzer image analysis system. The data are
expressed as the means ± SEM from three independent assays. *P<0.05 and **P<0.01 are significantly different from the control.
doi:10.1371/journal.pone.0141184.g004
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PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
Fig 5. Effect of PNAP-6h on the expression of caspase-7, -8, and -9 proteins. MCF-7 cells were incubated in the presence or absence of (A) the
caspase-8 inhibitor z-IETD-FMK or (B) the caspase-9 inhibitor z-LEHD-FMK for 1.5 h before the addition of PNAP-6h. Western blot analyses were conducted
with antibodies to caspase-7, -8, and -9, and actin. (C) MCF-7 cells were treated with PNAP-6h in the presence or absence of caspase-8 inhibitor, or
caspase-9 inhibitor, and the morphology was then observed by light microscopy.
doi:10.1371/journal.pone.0141184.g005
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PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
ratio of Bax/Bcl-2 was increased in a time-dependent manner (12, 24, and 48 h). We also found
that PNAP-3h and -6h significantly induced the expression of Fas protein. Taken together,
these findings suggest that the treatment of MCF-7 cells with PNAP-3h and -6h leads to an
Fig 6. Expression of MAPKs in PNAP-induced apoptosis. (A) MCF-7 cells were treated with 25 μM PNAP-1, -3h, and -6h at three time points (12, 24, and
4
8 h). (B) MCF-7 cells were treated with PNAP-1, -3h, and -6h at three concentrations (5, 10, and 25 μM) for 48 h. ERK, p38, and JNK, as well as their
respective phosphorylated forms, were then analyzed by western blot. The data are expressed as the means ± SEM from three independent assays.
P<0.05 and **P<0.01 are significantly different from the control.
*
doi:10.1371/journal.pone.0141184.g006
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PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
Fig 7. Proposed model of PNAP-6h-mediated MCF-7 cell cycle arrest and apoptosis. PNAP-6h causes cell cycle arrest at the S and G2/M phases.
PNAP-6h modulates MCF-7 apoptosis via activation of the intrinsic/extrinsic pathways and the p38/ERK pathway.
doi:10.1371/journal.pone.0141184.g007
increase in the Bax/Bcl-2 ratio and Fas levels, possibly inducing the activation of caspase-8
and -9.
PNAPs induce p38 and ERK activation
The MAPK family plays a central role in the signaling pathways of cell proliferation, differenti-
ation, and apoptosis [37]. Therefore, the involvement of the MAPK pathway in the PNAP
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PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
derivative-mediated apoptosis of MCF-7 cells was examined. The expression levels of MAPK
(
(
ERK, p38, and JNK) proteins during PNAP derivative-induced apoptosis at three time points
12, 24, and 48 h) and at different PNAP concentrations (5, 10, and 25 μM) are shown in Fig
6
A and 6B, respectively. p38 and JNK kinases are mainly activated by extracellular stress,
resulting in cell differentiation and apoptosis [38]. In this study, we found that PNAP-3h and
6h treatment but not PNAP-1 treatment significantly increased the p38 phosphorylation lev-
-
els in MCF-7 cells in a time- and dose-dependent manner; however, the total amount of p38
protein did not change. This result shows an increase in the phosphorylation status of p38 and
indicates that PNAP-3h and -6h increases p38 activity (Fig 6A and 6B). In contrast to the
increase in p38 activity, no elevation in the phosphorylation of JNK or in the total JNK levels
was observed after treatment with PNAP-1, -3h, and -6h. In addition, PNAP-6h decreased the
expression of p-ERK in a dose-dependent manner (Fig 6B) because ERK is generally activated
by mitogenic and proliferative stimuli and is involved in cellular proliferation and survival
[39]. These results indicate that p38 and ERK are also associated with PNAP-3h and -6h-
induced apoptosis of MCF-7 cells.
Conclusion
In summary, a series of hydrophilic PNAP derivatives was designed and synthesized. PNAP-
6
h, a 6,7-dihydroxy-2-phenylnaphthalene with a para-hydroxyl group on the phenyl ring,
exhibited the strongest cytotoxicity in our tests. In addition, our SAR studies revealed that
-hydroxy-2-phenylnaphthalene was the active core. Moreover, PNAP-6h treatment resulted
7
in changes in cellular morphology and induced cell cycle arrest and apoptosis in a dose-depen-
dent manner. We proposed a model for PNAP-6-mediated MCF-7 cell cycle arrest and apo-
ptosis, as shown in Fig 7. PNAP-6h induced S phase arrest via the promotion of p21 and p27
and the inhibition of cyclin D1, CDK4, cyclin E, and CDK2. In addition, G /M phase arrest
2
was mediated by reductions in the expression of cyclin B1 and CDK1. PNAP-6h induced apo-
ptosis in MCF-7 cells not only via the intrinsic and extrinsic pathways but also through the p38
and ERK pathways.
Supporting Information
1
13
1
13
S1 File. The H and C NMR spectra of PNAP-2h−PNAP-8h. H and C NMR spectra of
1
13
1
13
PNAP-2h (Figure A). H and C NMR spectra of PNAP-3h (Figure B). H and C NMR
1
13
1
spectra of PNAP-4h (Figure C). H and C NMR spectra of PNAP-5h (Figure D). H and
1
3
1
13
C NMR spectra of PNAP-6h (Figure E). H and C NMR spectra of PNAP-7h (Figure F).
1
13
H and C NMR spectra of PNAP-8h (Figure G).
(
PDF)
Acknowledgments
We would like to acknowledge Wei-Yu Chang for providing the starting materials. We also
thank Dr. Wei-Chien Huang for kindly providing MCF-10A cells.
Author Contributions
Conceived and designed the experiments: CFC THC. Performed the experiments: CFC CYK.
Analyzed the data: CFC THC. Contributed reagents/materials/analysis tools: CFC YCW THC.
Wrote the paper: CFC THC.
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PNAPs Induce Apoptosis and Inhibit Proliferation of MCF-7
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