Discovery of 2′-hydroxychalcones as autophagy inducer in A549 lung cancer cells

Article abstract of DOI:10.1039/c3ob42429d

A series of 2′-hydroxychalcone derivatives was synthesized and the effects of all the compounds on growth of A549 lung cancer cell were investigated. The results showed that all compounds had inhibitory effects on the growth of A549 lung cancer cells and compound 2-7 possessed the highest growth inhibitory effect and induced autophagy of A549 lung cancer cells.

Full text of DOI:10.1039/c3ob42429d

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Discovery of 2’-hydroxychalcones as autophagy
inducer in A549 lung cancer cells
Cite this: DOI: 10.1039/x0xx00000x
Fang-Wu Wanga,†, Sheng-Qing Wangb,†, Bao-Xiang Zhaob, *and Jun-Ying Miaoa, *
A series of 2’-hydroxychalcone derivatives was synthesized and the effects of all the
compounds on growth of A549 lung cancer cell were investigated. The results showed that all
Received 00th January 2013,
Accepted 00th January 2013
compounds had inhibitory effects on the growth of A549 lung cancer cells and compound 2-7
possessed the highest growth inhibitory effect and induced autophagy of A549 lung cancer
cells.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Current data suggest that depending on the context, either the
induction or the inhibition of autophagy can provide therapeutic
benefits to patients.10
Introduction
Though the mounting knowledge about autophagy, the
mechanisms through which the autophagic machinery regulates
these diverse processes are not entirely understood.11 Targeting
autophagy in cancer provides new opportunities for drug
development since more potent and specific inhibitors of
autophagy are needed. The role of autophagy and its regulation
in cancer cells continues to emerge and studies aim to define
optimal strategies to modulate autophagy for therapeutic
advantage.12 Therefore, the design and synthesis of modulators
of autophagy may provide novel therapeutic tools and may
ultimately lead to new therapeutic strategies in cancer.
By so far, a number of anticancer compounds have been
reported as autophagy inducers that target diverse signaling
pathways in autophagy.13-21 Autophagy is a highly complicated
system sensing and responding to the status of different types of
stress, which explains why there are multiple mechanisms of
autophagy induction, and thus, abundant targets for drug
discovery. The consequences of promoting autophagy in cancer
cells are incompletely recognized and may largely depend on
cellular context.12 However, compared to other cancer types,
autophagy inducers for treatment of lung cancer, esp. NSCLC,
is relatively rare in reports.22 And in most cases, those
autophagy inducers in lung cancer produce a combination
induction of apoptosis and autophagy.23-27 Currently, aside from
classic rapamycin and rapalogs,28, 29 novel autophagy-specific
compounds targeting for lung cancer therapy are in great need.
Chalcones, envisaged as open-chain flavonoids, show
various biological activity, such as potent anti-inflammatory
activity,30, 31 anticancer activity,32-39 lipid lowering agents that
act by modulating lipoprotein lipase,40 antiparasitic activity,41
cathepsin B and L inhibitory activity,42 cytoprotective activity43
and anti-diabetic activity.44 A few studies showed minor
Lung cancer is the leading cause of cancer-related death in the
world at present. About 75% of all lung malignancies are non-
small cell lung cancers (NSCLC). Despite advances in
prevention and treatment, NSCLC is often diagnosed at an
advanced stage and has a poor prognosis.
Current research suggests that standard chemotherapy and
radiotherapy regimens have been optimized to maximal
efficiency. Many studies have shown that small molecules can
act as oncogenes or tumor suppressors and become a promising
new treatment strategy.
Autophagy is an evolutionarily conserved pathway for
degradation of cytoplasmic proteins and organelles via
lysosome. Current research clearly shows that autophagy
fulfills numerous functions in vital biological processes.
Autophagy has become one of the most attractive topics in
cancer research. Among the various biological functions of
autophagy identified so far, the link between autophagy and
cancer is often viewed as controversial, because of its
apparently contradictory roles, such as survival and cell death.1
Autophagy has dual roles in cancer, acting as both a tumor
suppressor by preventing the accumulation of damaged proteins
and organelles and as a mechanism of cell survival that can
promote the growth of established tumors.1-3 Autophagy can
mediate cell death in a process known as autophagic cell death
(ACD). ACD refers to cell death by autophagy (rather than cell
death with autophagy) in which the final cell death process is
mediated by autophagy rather than another cell death modality
such as apoptosis or necroptosis.4, 5 It is shown that some drugs,
such as cisplatin, are able to induce autophagy in cancer cells
and autophagy may be associated with drug resistance in
tumors.6 Furthermore, the effect of anticancer drugs on cancer
cells may be increased by regulating the level of autophagy.7-9
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structural difference could lead to important difference in hydroxyacetophenone with substituted benzaldehyde under
mechanistic effect for anticancer.45
stirring at room temperature in 41.8-93.2% yields. The
In an ongoing study in our laboratory on discovery of structures of compounds (1-9) were confirmed by H NMR, C
1
13
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1
DOI: 10.1039/C3OB42429D
modulators of apoptosis or autophagy as potential new NMR and HRMS spectral data. In the H NMR spectra of the
anticancer agents,46-55 herein, we have identified a series of compounds, the CH protons of the olefin resonated as a doublet
chalcone derivatives with anticancer activity primarily by at
δ 7.40-7.67 ppm and 7.84-7.96 ppm, respectively. The
inducing autophagic cell death in A549 cells (an NSCLC cell hydroxy proton signal appeared at the range of
line). Although a couple of studies have shown that chalcones ppm as a single peak.
could induce autophagy to some extent in different types of
δ = 12.63-12.93
tumor,24,25,45,56 the anticancer effects in NSCLC are rarely
presented or not so significant as other cancer types.25
Furthermore, the autophagy induced by these reported
chalcones are largely dependent on the mechanisms overlapped
and interrelated with apoptosis, accompanied by apoptosis or
necroptosis as additional effects on tumor suppression. While
our compounds 2-6, specifically triggered autophagy without a
concomitant remarkable apoptotic activity, indicating they
might play a more prominent role in apoptosis-resistant cancers
than those agents inducing both autophagy and apoptosis, since
the anticancer effect only comes from ACD. Nevertheless,
Scheme 1 The synthesis of compounds.
compound
cells, possessed the highest inhibitory effect. In this context, we
speculated the robust autophagic induction by compound was
prior to and facilitated the consequent activation of apoptosis
and thus caused much tumor cell death. Importantly, this series
of chalcone compounds showed inhibitory effect on tumor cells
equivalent or even greater than anticancer chalcones ever
reported.
7 inducing both autophagy and apoptosis in A549
Inhibitory effects of the compounds on the proliferation of A549
cells
7
To define the biological effects of compounds 1-9 on A549
cells, we first observed the morphological changes of the cells
treated with the compounds under a phase contrast microscope.
Exposure of A549 cells to compounds 2-7 at 40 μM for 24 or
48 h resulted in a distinct decrease of cell viability as well as
induced the changes of A549 cell morphology. Vacuolization
and shrinkage of cells were observed, and the morphological
changes were even more significant at 48 h for these cells (Fig.
1). In order to evaluate the inhibitory effects of these
compounds on the proliferation of A549 cells we carried out the
SRB assay with compounds 1-9 at 48 h. The data showed that a
decrease of cell viability was observed with most compounds in
Results and discussion
Synthesis of compounds 1-9
The synthetic route of the proposed compound 1-9 is shown in
Scheme 1. Starting materials 2-hydroxyacetophenone was
prepared according to literatures.57 The 2’-hydroxychalcone
derivatives 1-9 were obtained by the reaction of 2-
a dose-dependent manner (Fig. 2). Among them, compound
7
showed a most potent inhibitory effect, according to the IC50
Fig 1. Effects of the compounds on A549 cell morphology at 24 or 48 h. A549 cells were treated with compounds 2-7 (40 μM) or DMSO 0.1% (v/v) as a vehicle control.
Microscopic photographs (200×) were taken under an inverted phase contrast microscope (Nikon).
Fig 2. Effects of compounds 1-9 on A549 cell viability. A549 cells were treated with compounds 1-9 at the concentration of 10, 20 or 40 μM or treated with DMSO 0.1%
(v/v) (Control) for 48 h. Cell viability was analyzed by SRB assay and illustrated in the labelled column. Results are presented as mean ± SE; (n =3, *p < 0.05 vs control;
**p < 0.01 vs control).
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values (Table 1). From IC50 data, we found that the cytotoxic
potency was highly dependent on the substituents on the two
aryl rings. Comparing compounds with same Ar and R = Cl,
View Article Online
DOI: 10.1039/C3OB42429D
Me, H, respectively, the IC50 of compounds
reduce in sequence; in contrast, the IC50 of compounds
increase in sequence. However, for compounds and
IC50 has no such sequence. Comparing compounds with same R
and different Ar, the IC50 of compounds , and reduce in
sequence; IC50 of compounds , and are almost no changes,
and interestingly increase in
1
,
4
, and
, and
, the
7
3, 6
9
2
,
5
8
1
,
2
3
4
,
5
6
but the IC50 of compounds
sequence.
7
,
8
9
Table 1 IC50 of the compounds 1-9 and 5-FU for A549 cells at 48 h.
Apoptosis and necrosis assays of compound treated A549 cells
Apoptosis (programmed cell death-type I) and necrosis are
well-known mechanisms of cell death induced by anticancer
therapies. The apoptotic process involves a sequence of cell
shrinkage, increased cytoplasmic density, chromatin
condensation, and segregation into sharply circumscribed
masses abut the nuclear membrane which will produce
apoptotic bodies. DNA staining with certain dyes, such as
Hoechst 33258, allows the direct observation of the
condensation of the cell nucleus. After exposed to compounds
2-7 at 20, 40 μM for 48 h, A549 cells were stained by Hoechst
33258 to detect condensation of nucleus that appeared as
fluorescent blue. The results suggested that the compounds
induce apoptosis slightly in A549 at both concentrations, except
Fig 3. Apoptosis of A549 cells treated with compounds 2-7. (A) fluorescent
image of A549 cells stained by Hoechst 33258. (B) Fluorescence quantitative
statistics for apoptotic cells. Fluorescent micrographs of Hoechst 33258
staining (200×) were taken under a fluorescent microscope (Nikon). (n =3, *p <
0.05 vs control; **p < 0.01 vs control).
for compound
determinant factor for inhibiting growth of cells (Fig. 3).
However, compound significantly triggered apoptosis in
7, indicating that apoptosis seems hardly to be a
7
A549 cells at 48 h, and the apoptosis rate reached 25%. To
detect whether the compounds cause necrosis in A549 cells, we
measured the LDH activity in cell culture medium. As shown in
Fig. 4, there was no significant difference (p>0.05) in LDH
release between the control group and the cells treated with
compounds at the test range of concentration. To conclude, the
inhibitory effects of cell viability were not due to apoptosis or
necrosis in A549 cells.
Fig 4. Effects of compounds 2-7 on LDH activities of A549 cells. The culture
medium was collected as samples for LDH assay after 24 and 48 h treatment at
the concentration of 20 or 40 μM. (n =3, *p < 0.05 vs control; **p < 0.01 vs
control).
detected the autophagy activity in cells treated with
compounds.
Vacuolization in morphological change has suggested a sign of
autophagy activity. Since autophagy is a lysosomal degradation
pathway for the breakdown of intracellular proteins and
organelles, formation of acidic vesicles can be a viable marker
for autophagy. We first performed aridine orange (AO) staining
in A549 cells after treatment with compounds at 20 or 40 μM
for 48 h. According to acidic compartments stained as
fluorescent bright red, the cells showed elevated autophagic
Compounds induce autophagic cell death in A549 cells
Recently increasing evidence has demonstrated that autophagic
cell death can be induced as an alternative cell-death
mechanism when cells fail to undergo apoptosis. We therefore
Fig 5. Compounds 2-7 increased the acidic vesicle level in the cells at 48 h. Microscopic photographs (400×) were taken under a fluorescent microscope (Nikon).
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DOI: 10.1039/C3OB42429D
Fig 6. Compounds 2-7 increased punctate LC3-II in A549 cells at 48 h. Immunohistochemistry image was taken under a Confocal Laser scanning microscopy (Zeiss).
activity after the treatment compared with the control group
Our results suggested that the compounds 2-7 induced
(Fig. 5). Nevertheless, the examination of autophagy should be excessive autophagy activity in A549 cells and led to
conducted by multiple methods because autophagosome autophagic cell death, which is another considerable
accumulation may not represent fully functional autophagy. It mechanism used as a therapeutic strategy to treat cancer.
has been widely accepted that LC3-II protein correlates with Induction of autophagic cell death may be an ideal approach in
the induction of autophagy and both the ratio of LC3-II to LC3- those cancers that are resistant to apoptosis by anticancer
I and the amount of LC3-II can be used to monitor therapies (e.g., chemotherapy, radiation). Moreover, those
autophagosome formation. Immunofluorescence of LC3-II is compounds also offered useful tools for studying the molecular
useful for in vitro and in vivo studies, and punctate LC3-II can mechanisms underlying the relation between autophagy and
be faithful evidence for autophagy. After treatment at 40 μM cancer or apoptosis. The cross-talking between autophagy and
compounds for 48 h, we used anti-LC3-II antibodies for apoptosis is complex and confusing, since they are linked by
immunohistochemistry experiment and observed apparent effector proteins (e.g., Bcl-2, Bcl-XL, ATG5, p53) and share
increase in punctate LC3-II, comparing to control group (Fig. common pathways (e.g., PI3K/Akt/mTOR, NFκB, ERK). It’s
6). We further examined the levels of LC3-II by immunoblot noteworthy that compound
7 performed the most significant
and the data showed both the enhancement of conversion of inhibitory effects on A549 cells through combination of
LC3-I to LC3-II and upregulation of LC3 levels (Fig. 7). All apoptosis and autophagy, even if other compounds had low
above data are consistent to demonstrate that the compounds level of activation for apoptosis. In this context, we speculated
can induce autophagic cell death in A549 cells and the that the induction of apoptosis by compound
7 was probably
inhibition of growth was mainly attributed to the autophagy triggered by autophagic activity, rather than the reverse. Even
induction.
though many studies have suggested the concurrence of the two
cell events under their overlapped mechanisms, there are also
several examples in which the induction of autophagy
facilitates the activation of apoptosis.58, 59
Experimental
General
Thin-layer chromatography (TLC) was conducted on silica gel
1
60 F254 plates (Merck KGaA). H NMR spectra were recorded
on a Bruker Avance 300 (300 MHz) spectrometer, and 13C
NMR spectra were recorded on a Bruker Avance 400 (100
MHz) spectrometer, using CDCl3 as solvent and
tetramethylsilane (TMS) as internal standard. HRMS spectra
were recorded on a Q-TOF6510 spectrograph (Agilent).
Melting points were determined on an XD-4 digital micro
melting point apparatus.
General procedure for the synthesis of compounds 1-9
To a stirred solution of substituted 2-hydroxyacetophenone (1.0
mmol) in ethanol (15 mL) was added 40% sodium hydroxide
solution (2.0 mL) in an ice-salt bath. After stirred 30 min, 2-
Fig 7. Western blot analysis of the protein level of LC3-II and GAPDH as a
normalization control (A) and quantitative statistics (B). A549 cells were treated
with compounds 2-7 at 40 μM for 48 h. (n =3, *p < 0.05 vs control; **p < 0.01
vs control).
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hydrazinylpyridine (1.2 mmol) was added and the reaction
mixture was stirred for 12 h at room temperature as shown in
Scheme 1. The progress of the reaction was monitored by TLC.
After completion, the reaction mixture was neutralized by
hydrochloric acid. The precipitates were filtered, washed with
water, and consequently recrystallized from ethanol to afford 1-
9.
7.42-7.46 (m, 3H, Ar-H), 7.67 (d, 1H,
7.66-7.68 (m, 3H, Ar-H), 7.92 (d, 1H,
J
J
= 15.3 Hz, -CH=CH-),
= 15.3 Hz, -CH=CH-),
View Article Online
12.63 (s, 1H, OH); 13C NMR (100 MDHOzI,: 1C0.D10C39l/3C):3OB42429D
δ
20.65,
118.36, 119.68, 120.16, 127.95, 128.71, 129.04, 129.40, 130.90,
134.65, 137.54, 145.23, 161.58, 193.59; HRMS: calcd for
[M+H]+ C16H15O2: 239.1072; found: 239.1067.
The spectroscopic data of compounds 1-9
(E)-1-(5-chloro-2-hydroxyphenyl)-3-phenylprop-2-en-1-one
Yellow solid; yield: 87.9%; mp: 103-106 ◦C [lit. 110-112 ◦C]60;
1H NMR (300 MHz, CDCl3):
6.99 (d, 1H, = 9.0 Hz, Ar-H),
(1)
(E)-3-(benzo[d][1,3]dioxol-5-yl)-1-(2-hydroxy-5-methylphenyl)
prop-2-en-1-one
118◦C [Lit. 111-112 ◦C]64; 1H NMR (300 MHz, CDCl3):
(s, 3H CH3), 6.05 (s, 2H CH2), 6.84 (d, 1H, = 9.3 Hz, Ar-H),
6.93 (d, 1H, = 8.4 Hz, Ar-H), 7.17 (dd, 1H, = 1.5, 8.1 Hz,
Ar-H), 7.21 (d, 1H, = 1.5 Hz Ar-H), 7.31 (dd, 1H, = 1.8, 8.4
Hz, Ar-H), 7.48 (d, 1H, = 15.3 Hz, -CH=CH-), 7.69 (dd, 1H,
= 1.5, 9.9 Hz, Ar-H), 7.84 (d, 1H, = 15.3 Hz, -CH=CH-),
12.70 (s, 1H, OH); 13C NMR (100 MHz, CDCl3):
20.60,
(5
). Pale yellow solid, yield 86.2%; mp 115-
δ
J
δ
2.36
7.43-7.49 (m, 4H, Ar-H), 7.63 (d, 1H,
7.69-7.73 (m, 2H, Ar-H), 7.88 (d, 1H,
J
J
= 15.6 Hz, -CH=CH-),
= 1.5 Hz, Ar-H), 7.96
J
J
= 15.6 Hz, -CH=CH-), 12.72 (s, 1H, OH); 13C NMR
J
J
(d, 1H,
(100 MHz, CDCl3):
J
J
δ
119.37, 120.26, 120.58, 123.54, 128.82,
J
J
128.88, 129.12, 131.30, 134.30, 136.20, 146.52, 162,08;
HRMS: calcd for [M+H]+ C15H12ClO2: 259.0526; found:
259.0528.
J
δ
101.76, 106.77, 108.68, 118.04, 118.28, 119.72, 125.64, 127.79,
129.12, 129.22, 137.25, 145.01, 148.47, 150.22, 161.50, 193.34;
HRMS: calcd for [M+H]+ C17H15O4: 283.0970; found:
283.0961.
(E)-3-(benzo[d][1,3]dioxol-5-yl)-1-(5-chloro-2-hydroxyphenyl)
prop-2-en-1-one
[lit. 145-146 ◦C]61; H NMR (300 MHz, CDCl3):
CH2), 6.88 (d, 1H, = 7.8 Hz, Ar-H), 6.98 (d, 1H,
Ar-H), 7.17-7.21 (m, 2H, Ar-H), 7.40 (d, 1H, = 15.6 Hz, -
CH=CH-), 7.41-7.45 (m, 1H, Ar-H) 7.85 (s, 1H Ar-H), 7.87 (d,
1H,
= 15.6 Hz, -CH=CH-), 12.80 (s, 1H, OH) ; 13C NMR (100
MHz, CDCl3): 101.88, 106.84, 108.82, 117.19, 120.21,
120.64, 123.46, 126.18, 128.69, 128.78, 136.01, 146.40, 148.58,
150.64, 162.02, 192.55; HRMS: calcd for [M+H]+ C16H12ClO4:
303.0424; found: 303.0419.
(2). Yellow solid, yield 93.2%; mp 152-154 ◦C
1
δ
6.06 (s, 2H
J = 8.7 Hz,
(E)-1-(2-hydroxy-5-methylphenyl)-3-(4-methoxyphenyl)prop-2-
J
en-1-one
94-96 ◦C]65; H NMR (300 MHz, CDCl3):
3.87 (s, 3H OCH3), 6.92-6.97 (m, 3H, Ar-H), 7.30 (dd, 1H,
(6). Pale yellow solid, yield 59.4%; mp 99-102◦C [lit.
J
1
δ
2.36 (s, 3H CH3),
J
=
J
1.8, 8.4 Hz, Ar-H), 7.55 (d, 1H,
7.69 (m, 3H, Ar-H), 7.89 (d, 1H,
J
J
= 15.6 Hz, -CH=CH-), 7.63-
= 15.6 Hz, -CH=CH-), 12.73
20.63, 55.40,
δ
(s, 1H, OH); 13C NMR (100 MHz, CDCl3):
δ
114.50, 117.66, 118.28, 119.78, 126.58, 127.71, 129.29, 130.56,
137.21, 145.10, 161.52, 161.99, 193.52; HRMS: calcd for
[M+H]+ C17H17O3: 269.1178; found: 269.1178.
(E)-1-(5-chloro-2-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-
en-1-one
(3). Yellow solid, yield 84.8%; mp 112-114 ◦C [lit.
(E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-one
yellow solid, yield 55.7%; mp 94-96 ◦C [lit. 97.1-97.8 ◦C]66; 1H
NMR (300 MHz, CDCl3): 6.95 (t, 1H, = 7.5 Hz, Ar-H), 6.98
(dd, 1H, = 0.9, 8.4 Hz, Ar-H), 7.42-7.45 (m, 3H, Ar-H), 7.51
(t, 1H,
7.66-7.68 (m, 2H, Ar-H) 7.93 (dd, 1H
7.94 (d, 1H, C
= 15.6 Hz, -CH=CH-), 12.81 (s, 1H, OH); 13
(7). Pale
1
118 ◦C]62; H NMR (300 MHz, CDCl3):
δ
3.88 (s, 3H, OCH3),
δ
6.94-6.99 (m, 3H Ar-H), 7.43 (dd, 1H, J = 2.7, 8.7 Hz, Ar-H),
J
7.45 (d, 1H,
J
= 15.3 Hz, -CH=CH-), 7.65 (dd, 2H,
= 2.4 Hz, Ar-H), 7.93 (d, 1H,
Hz, -CH=CH-), 12.85 (s, 1H, OH); 13C NMR (100 MHz,
CDCl3): 55.48, 114.58, 116.75, 120.16, 120.70, 123.39,
J
= 2.7, 11.4
J
Hz, Ar-H), 7.86 (d, 1H
J
J
= 15.3
J
= 7.2 Hz, Ar-H), 7.67 (d, 1H,
J
J
= 15.6 Hz, -CH=CH-),
= 1.5, 8.1 Hz, Ar-H),
δ
J
127.04, 128.70, 130.83, 135.89, 146.40, 162.02, 162.31, 192.62;
HRMS: calcd for [M+H]+ C16H14ClO3: 289.0631; found:
289.0628.
NMR (100 MHz, CDCl3):
δ 118.65, 118.91, 120.03, 120.07,
128.72, 129.08, 129.72, 130.99, 134.58, 136.46, 145.49, 163.64,
193.74; HRMS: calcd for [M+H]+ C15H13O2: 225.0916; found:
225.0914.
(E)-1-(2-hydroxy-5-methylphenyl)-3-phenylprop-2-en-1-one
Pale yellow solid, yield 41.8%; mp 117-119 ◦C [lit. 105-106
◦C]63; 1H NMR (300 MHz, CDCl3):
2.36 (s, 3H CH3), 6.95 (d,
1H, = 8.7 Hz Ar-H), 6.88 (dd, 1H, = 1.8, 8.4 Hz, Ar-H),
(4)
(E)-3-(benzo[d][1,3]dioxol-5-yl)-1-(2-hydroxyphenyl)prop-2-
en-1-one
[Lit. 137-138 ◦C]64; 1H NMR (300 MHz, CDCl3):
CH2), 6.86 (d, 1H, = 8.1 Hz, Ar-H), 6.94 (t, 1H,
(8
). Pale yellow solid, yield 64.9%; mp 143-145 ◦C
6.05 (s, 2H
= 7.5 Hz,
δ
δ
J
J
J
J
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DNA in normal cells. A minimum of 500 cells were counted,
and each experiment was performed in triplicate.
View Article Online
Ar-H), 7.02 (dd, 1H,
J
= 0.9, 8.4 Hz, Ar-H), 7.15-7.19 (m, 2H,
= 15.3 Hz, -CH=CH-), 7.49-7.50 (m, 1H,
= 15.3 Hz, -CH=CH-), 7.91 (dd, 1H,
Ar-H), 7.48 (d, 1H,
Ar-H), 7.85 (d, 1H,
J
J
J =
DOI: 10.1039/C3OB42429D
LDH assay
1.5, 8.1 Hz,Ar-H), 12.89 (s, 1H, OH); 13C NMR (100 MHz,
Cell culture medium was collected after 48 h treatment with
0.1% DMSO (as control) or the compound. LDH assay was
performed using Lactate Dehydrogenase (LDH) kit (Nanjing
Jiancheng Co., China) according to the manufacturer’s
instructions.
CDCl3):
δ 101.80, 106.71, 108.74, 117.86, 118.58, 118.82,
120.04, 125.82, 129.01, 129.54, 136.24, 145.32, 148.49, 150.32,
163.56, 193.49; HRMS: calcd for [M+H]+ C16H13O4: 269.0814;
found: 269.0818.
Acridine orange (AO) staining
(E)-1-(2-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one
Discard the medium after treatment and gently rinse the cells
with PBS. The cells were stained with acridine orange dye (0.1
mg/mL) for 1 min. Subsequently, the cells were gently washed
twice with PBS, and were then observed under a fluorescence
microscope (Nikon). In acridine orange-stained cells, the
cytoplasm and nucleolus fluoresce bright green, whereas acidic
compartments fluoresce bright red.
(9). Pale yellow solid, yield 77.0%; mp 93-95 ◦C [lit. 95-
1
96◦C]67; H NMR (300 MHz, CDCl3):
δ
3.87 (s, 3H, OCH3),
6.91-6.97 (m, 3H, Ar-H), 7.02 (dd, 1H,
J = 0.9, 8.4 Hz, Ar-H),
7.49 (t, 1H,
CH=CH-), 7.64 (dd, 2H,
1H Ar-H), 7.91 (d, 1H,
OH); 13C NMR (100 MHz, CDCl3):
J
= 7.8 Hz, Ar-H), 7.54 (d, 1H,
= 1.8, 6.9 Hz, Ar-H), 7.91-7.94 (m,
= 15.3 Hz, -CH=CH-), 12.93 (s, 1H,
55.45, 114.53, 117.52,
J = 15.3 Hz, -
J
J
δ
Immunohistochemistry assay for LC3-II
118.58, 118.80, 120.12, 127.31, 129.58, 130.59, 136.17, 145.38,
162.05, 163.57, 193.66; HRMS: calcd for [M+H]+ C16H15O3:
255.1021; found: 255.1018.
After treatment, A549 cells were washed three times with 0.1
M PBS. Following fixation (15 min in 4% paraformaldehyde),
cells were washed three times in 0.1 M PBS and blocked with
sheep serum (1:30 dilution) in 0.1 M PBS for 20 min at room
temperature. Then cells were incubated with rabbit anti-human
LC3b antibody (1:100 dilution) in a humid chamber overnight
at 4 . Then cells were washed three times with 0.1 M PBS and
incubated for 30 min at 37 with the secondary antibody,
FITC-conjugated goat anti-rabbit IgG (1:200 dilution). Finally
cells were washed three times with 0.1 M PBS, and were
observed by a confocal laser scanning microscope (Zeiss,
Germany).
Cell culture
A549 cells were cultured in RPMI-1640 medium (Gibco)
supplemented with 10% calf bovine serum (HyClone), at 37 °C
in a humidified incubator with 5% CO2. For the experimental
study, cells were grown to 80−90% confluence, harvested with
0.05% trypsin (Sangon Biotech) in phosphate-buffered saline
(PBS), plated at the desired cell concentration. All the cells
were seeded at a density of 6250/cm2 into plates or appropriate
dishes 24 h before adding the compound.
Western blot analysis
Cells were washed twice with ice cold PBS and were then lysed
in protein lysis buffer (0.5% SDS in 25 mM Tris–HCl, pH 7.5,
4 mM EDTA, 100 mM NaCl, 1 mM PMSF, 10 μg/mL
leupeptin and 10 μg/mL soybean trypsin inhibitor). The protein
concentration of the cells was determined by the Bradford
method. After adding loading buffer and boiling, equal amount
of protein was loaded onto a 15% SDS polyacrylamide gel.
Cell viability assay
Cells were seeded in 96-well plates. After 24 h, cells were
treated with 0.1% DMSO (as control) or the compounds at
indicated concentrations for specified time durations. Cell
viability was evaluated by sulforhodamine B (SRB) assay.
Briefly, pour off the medium and fix cells by adding 100 μL of
cold 10% trichloroacetic acid (TCA) and incubate for 1 h at 4 .
Discard the supernatant and then wash the plates five times
with deionized water. Add 50μL of 0.4% (W/V) SRB solution
in 1% acetic acid to each well and shake 5 min on titer plate
shaker. Wash the plates five times with 1% acetic acid and
subsequently add 100 μL of 10 mM unbuffered Tris base (pH
10.5) to dissolve the bound dye. Mix 5 min on a microtiter plate
shaker and read optical densities at the wavelength of 540 nm
using Infinite 200 pro multimode Plate reader (Tecan).
After
electrophoresis,
the
resolved
protein
was
electrophoretically transferred to a Polyvinylidene Fluoride
(PVDF) membrane (Millipore, MA, USA). The membrane was
blocked with 5% non-fat milk in PBST (PBS containing 0.05%
Tween 20) for 1 h at room temperature. Subsequently the
membrane was probed with Rabbit anti-human LC3b antibody
(1:1000), or anti-GAPDH mouse monoclonal antibody (1:1000)
overnight at 4 , and was then washed twice with PBST, each
time for 5 min. The membrane was subsequently incubated
with HRP-conjugated goat anti-rabbit IgG (1:5000) or
polyclonal goat anti-mouse immunoglobulins/HRP (1:5000) for
1 h at room temperature and then washed three times with
PBST. Then the membrane was incubated with HRP substrate
for 5 min and the fluorescence signal were detected with X-ray
films. Intensity of the protein bands was quantified using
Quantity-One software (Bio-Rad).
Hoechst 33258 staining
The living A549 cells were stained with 10 mg/mL of Hoechst
33258 in the medium for 15 min at 37 . Subsequently, the
cells were gently washed twice with PBS, and were then
observed under a fluorescence microscope (Nikon). The
condensed DNA of apoptotic cells was identified by intense
local staining in the nucleus, in contrast to diffused staining of
Statistical analysis
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry
Page 8 of 9
ARTICLE
Journal Name
Data were presented as means ± SE from at least three
independent experiments and analyzed by Student’s t-test.
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