A Novel Diol-Derivative of Chalcone Produced by Bioconversion,
3-(2,3-dihydroxyphenyl)-1-phenylpropan-1-one, Activates Phosphorylation of
ERK and CREB in Cultured Rat Cortical Neurons
Md. Al Rahim1), Akira Nakajima1), Norihiko Misawa2), Kazutoshi Shindo3), Kyoko Adachi2),
Yoshikazu Shizuri2), Yasushi Ohizumi4, 5, 6) and Tohru Yamakuni1)*
1)Department
of Pharmacotherapy, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai
980-8578, Japan
2)Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan
3)Department of Food and Nutrition, Japan Women’s University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan
4)Yokohama College of Pharmacy, 601 Matano-cho, Tozuka-ku, Yokohama 245-0066, Japan
5)Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-11-404 Aoba, Aramaki,
Aoba-ku, Sendai 980-8579, Japan
6)Department of Pharmacokinetics and Pharmacodynamics, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada,
Suruga-ku, Shizuoka 422-8526, Japan
The pharmacological effects of chalcones and their derivatives on the nervous system are unknown. We have recently
reported several chalcone and flavonoid derivatives, including a novel diol derivative of chalcone, produced by a
bioconversion system using a recombinant Escherichia coli, that enables us to produce chemical compounds which are
naturally rare and usually difficult to chemically synthesize. In this study, we for the first time evaluated the ability of these
compounds to stimulate phosphorylation of ERK and CREB, by using cultured rat cortical neurons. Among ten compounds
tested, the novel diol-derivative of chalcone, 3-(2,3-dihydroxyphenyl)-1-phenylpropan-1-one, most potently increased
phosphorylation of ERK and CREB. Therefore, our current study provides a rationale for this compound to be studied further
in order to not only elucidate the underlying mechanisms but also evaluate its therapeutic potential in vivo.
Keywords: chalcone-diol / ERK / CREB / cortical neuronal culture
Introduction
A great number of compounds from natural resources
have so far attracted considerable attention as novel
leading compounds for drug development (Liu, 1993) as
well as useful pharmacological tools (Furukawa et al.,
1993; Ohizumi, 1997; Obara et al., 2002). In the course
of our survey of substances activating PKA/ERK signaling
in the CNS neurons in culture by examining the ability of
six flavonoids to stimulate the signaling, we have
successfully found nobiletin, a citrus polymethoxy flavone
with neurotrophic action (Nagase et al., 2005a, b), which
not only reverses learning impairment associated with
NMDA receptor antagonism by activation of ERK in mice
*Correspondence author: Tohru Yamakuni, Ph.D.
Department of Pharmacotherapy, Graduate School of Pharmaceutical Sciences,
Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan
Tel: +81-22-795-6852 Fax: +81-22-795-6850
E-mail: yamakuni@mail.pharm.tohoku.ac.jp
(Nakajima et al., 2007), but also rescues memory
impairment in AD model rats, possibly via stimulation of
PKA/CREB signaling (Matsuzaki et al., 2006).
In order to further search for new substances activating
phosphorylation of ERK and CREB in the CNS neurons,
we focused on chalcones and their derivatives, since these
compounds are precursors of flavonoids enriched in edible
plants, e.g. citrus fruits, and thus show a structural
similarity each other. Recently, we have developed a new
bioconversion system using a recombinant E. coli to
successfully produce derivative compounds that are novel
and/or rare in nature, from various chemicals, including
chalcones and flavonoids (Shindo et al., 2003, Shindo et
al., 2004; Misawa et al., 2005). In the current study, ten
compounds produced by this bioconversion system were
employed to examine whether these compounds modulate
the phosphorylation of ERK and CREB in cultured rat
cortical neurons. Here we provide the first evidence
that a novel diol derivative of chalcone, 3-(2,3dihydroxyphenyl) -1-phenylpropan-1-one,potently
stimulates the phosphorylation of ERK and CREB in
cultured cortical neurons.
Materials and methods
1. Preparations of the Tested Compounds
The bphA1(2072) gene is an evolved gene shuffled
between the biphenyl dioxygenase large (α subunit genes
derived from polychlorinated biphenyl (PCB)-degrading
soil bacteria Pseudomonas pseudoalcaligenes KF707 and
Burkholderia xenovorans LB400 (Misawa et al., 2002).
The tested compounds shown in Table 1 were prepared by
the co-cultivation of the corresponding substrates and E.
coli cells carrying bphA1(2072) along with the
bphA2A3A4 genes from P. pseudoalcaligenes KF707,
which encoded the small (β) subunit, ferredoxin and
ferredoxin reductase of biphenyl dioxygenase, and the
bphB gene coding for subsequent desaturase from strain
KF707, as described previously (Shindo et al., 2003;
Misawa et al., 2005). The bphC gene from strain KF707
was together used as needed (Shindo et al., 2004). As an
example, compound #3, a novel chalcone-diol was
prepared as follows: E. coli JM109 carrying plasmid
pBS2072B, which contained bphA1 (2072) - bphA2A3A4 bphB , was grown in an LB medium containing 150 μg/ml
of ampicillin (Ap) at 30°C with reciprocal shaking (175
rpm) for 6-7 h until the absorbance at OD 600 nm had
reached approximately 1. Eight milliliters of this culture
was inoculated into 100 ml of an M9 medium with 150
μg/ml
of
Ap,
1
mM
of
isopropyl
-D-thiogalactopyranoside, 0.4% (w/v) glucose, and 10
mg of trans-chalcone (Aldrich) in a Sakaguchi flask, and
co-cultivated at 30℃ with reciprocal shaking (175 rpm)
for 2 days. The culture medium was extracted with
ethylacetate, and the organic layer was further purified by
silica gel column chromatography (hexane-ethylacetate =
4:1) to give a purified chalcone-diol (5.9 mg), as described
previously (Shindo et al., 2003).
2. Cortical Neuronal Culture and Treatment
Primary culture of cortical neurons was prepared as
described previously with minor modifications (Nagase et
al., 2005b; Matsuzaki et al., 2006). In brief, under ether
anesthesia of an 18-day Sprague-Dawley (SD) rat (Japan,
SLC), embryos were immediately decapitated. The brain
was quickly removed, and cortex was collected in ice-cold
Dulbecco’s Phosphate Buffered Saline (DPBS) under
sterile conditions. The cortical neurons were dissociated
according to the standard methods with SUMITOMO
Nerve-Cell Culture System (SUMITOMO BAKELITE),
plated on 35-mm culture dish coated with poly-L-lysine
(Life Technologies) at a density of 1 x 10 6 cells/dish in
Neurobasal Medium without phenol red (Gibco Co., Ltd.)
containing B-27 supplement (Gibco Co., Ltd.), 500 M
L-glutamine and 0.005% penicillin-streptomycin, and
cultured at 37°C in a humidified atmosphere consisting of
5% CO2 and 95% air. At 4 days in vitro (D.I.V.), a half of
the medium was replaced by the medium containing a
mitotic inhibitor, cytosine arabinofuranoside (Ara-C), to
inhibit non-neuronal proliferation.
At 10-14 D.I.V., cultured cortical neurons were treated
with the compounds tested, including a novel
chalcone-diol, or vehicle (0.1% DMSO) in the Ara-C
containing medium without B-27 supplement and
L-glutamine at 100 μM concentrations for 30-min, and
thereafter subjected to Western blot analysis.
Table 1 List of compounds tested for the ability to
stimulate ERK/CREB phosphorylation
Sample
ID
Chemical Nomenclature
1
2-(2,3-Dihydroxyphenyl)-chroman-4-one
2
6-(4-Oxo-chroman-2-yl)-pyridine-2carboxylic acid 2-oxo-2-phenylethylester
3
3-(2,3-Dihydroxyphenyl)-1phenylpropan-1-one
4
3-Napthalen-1-yl-benzene-1,2-diol
5
3-Napthalen-2-yl-benzene-1,2-diol
6
3-Benzothiazole-2-yl-benzene-1,2-diol
7
3-Benzooxazol-2-yl-benzene-1,2-diol
8
3-Pyridin-2-yl-benzene-1,2-diol
9
3-(2,3-Dihydroxyphenyl)-indan-1-one
10
3-Imidazole-1-ylmethyl-benzene-1,2-diol
Cultured cortical neurons were treated with 100 μM of each of
the listed compound for 30 min and subjected to Western blot
analysis as described under Experimental Procedures.
3. Cell Extract Preparation and Western Blot Analysis
Cell extract preparation and Western blot analysis were
conducted, as described previously (Nagase et al., 2005b;
Matsuzaki et al., 2006). After treatment with tested
compounds, cells were lysed with 60 μl of lysis buffer (1
mM EDTA, 1% SDS, 10 mM NaF, 10 nM calyculin, 320
nM okadaic acid, 1 μM sodium orthovanadate, 1 mM
p-APMSF, 10 μg/ml pepstatin, 10 μg/ml antipain, 10 μg/ml
leupeptin,
10
μg/ml
chymostatin,
10
μg/ml
phosphoramidon, 10 mM HEPES, pH 7.5) per 35-mm dish.
Cell lysates were heated at 95°C for 5 min, sonicated and
centrifuged at 14,000 r.p.m. for 20 min at 4°C to obtain the
supernatant as cell extracts. Cell extracts were separated
by 12.5% SDS-PAGE and transferred onto PVDF
membrane.
The membranes were incubated with
anti-phospho-ERK (Thr202/Tyr204), anti-phospho-CREB
(Ser133), anti-total ERK antibodies (1:1000, Cell
Signaling Technology).
OH
OH
O
F i g . 1 . Chemica l st ruct ure of a novel
chalcone-diol, 3-(2,3-dihydroxyphenyl)-1-phenylpropan-1
-one.
Immunoreactive
proteins
were
visualized
by
chemiluminescence as described previously (Matsuzaki et
al., 2006). The band intensities were quantitatively
analyzed using SCION image software.
4. Statistical Analyses
Student’s t test was performed to measure the level of
significance. A level of p < 0.05 was considered to be
statistically significant.
Results
A novel chalcone-diol most potently stimulates the
phosphorylation of ERK and CREB among the
bioconverted compounds tested in cultured rat cortical
neurons. The effects of chalcones and the derivative
compounds are still unidentified in the nervous system.
To examine the effects of the bioconverted derivatives of
chalcone and flavonoid on the phosphorylation of ERK
and a possible downstream effector, CREB, the ten
compounds (listed in Table 1) were screened by assaying
the activity to stimulate the phosphorylation of ERK and
CREB in cultured rat cortical neurons, using Western blot
analysis. Rat cortical neuronal cultures were treated with
the tested compounds at 100 μM for 30 min and subjected
to Western blot analysis. As shown in Fig. 2, it was
observed that eight of the tested compounds failed to
stimulate the phosphorylation of ERK and CREB, and
some of them rather depressed the phosphorylations. The
compound #4, 3-napthalen-1-yl-benzene-1,2-diol, showed
a little increase in the phosphorylation of CREB, yet it was
not effective enough to be studied further. On the other
hand, among the compounds tested, compound #3, a
no ve l d io l d e r i vat i ve o f c h alco n e, 3 -(2 ,3 dihydroxyphenyl)-1-phenylpropan-1-one (Fig. 1), only
markedly increased the phosphorylation of ERK and its
downstream target, CREB, in cultured rat cortical neurons
(Fig. 2). The remarkable stimulatory effects of the
compound on the phosphorylation of the proteins
prompted us to study in more detail the pharmacological
characteristics of this novel chalcone-diol in cultured
cortical neurons. Currently, detail mechanistic study with
this compound is under investigation.
Discussion
Earlier studies have reported that chalcones and their
derivatives have beneficial pharmacological actions,
including anti-inflammatory (Hsieh et al., 2000; Won et al.,
2005), anti-pyretic (De Leon et al., 2003), anti-invasive
(Mukherjee et al., 2001) and anti-proliferative (Lin et. al.,
2002) activities. However, the effects of the chalcone and
their derivatives on the CNS neurons have been so far
unidentified. Thus, to our knowledge, this study is the first
to demonstrate that the chalcone derivative activates the
phosphorylation of ERK and CREB in cultured cortical
neurons. The activation of ERK is required to induce
nuclear translocation and the subsequent activation of
RSK2, which in turn phosphorylates CREB to stimulate
CRE-dependent transcription (Impey et al., 1998). Our
present results evidently showed that the chalcone-diol
enhanced the phosphorylation of not only ERK, but also
the phosphorylation of the downstream effector, CREB,
indicating a stimulatory effect of the compound on
ERK/CREB signaling in the cortical neurons. Very
recently, we also found that chalcone-diol stimulated
CRE-mediated transcription in both cultured cortical and
hippocampal neurons (data not shown).
A
Compounds #
C 1
2 3 4 5 6 7 8 9 10
P-ERK
P-CREB
Total ERK
B
**
P-ERK
(Fold of control)
5
4
3
2
1
0
C
1
2
3
4
5
6
7
8
9
10
Compounds #
Acknowledgements
The authors thank to Toshihiko Otomatsu of KNC
Laboratories Co. for large-scale preparation of
chalcone-diol. This work was supported in part by a
grant-in-aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science, and Technology of
Japan, and the grant from Takeda Science Foundation.
***
P-CREB
(Fold of control)
5
4
3
2
1
0
C
1
The chemical structure of chalcones consists of two
aromatic rings joined by a three-carbon α,-unsaturated
carbonyl system (Achanta et al., 2006). However, the
chalcone-diol generated by our bioconversion system is
characterized by the fact that it contains a catechol moiety
in its structure, which is attached to the second aromatic
ring by a saturated carbonyl system instead of an
unsaturated one. Thus, based on the structural difference
between this compound and the other chalcones reported
so far, it may be interpreted that the presence of a saturated
carbonyl system in between two aromatic rings is the
unique structural feature of this chalcone-diol.
In summary, this pharmacological approach combined
with bioconversion led us to successfully identify
a novel chalcone-diol as the agent, which
act i va te s phosphorylation of ERK and CREB in the
cultured cortical neurons. These results of the current study
would add new dimension to and attract greater attention
in the research interest of the chalcone compounds, and
further pharmacological studies would give new insights
into better understanding of the molecular mechanism of
this novel chalcone-diol.
2
3
4
5
6
7
8
9
10
Compounds #
Fig. 2. A novel chalcone-diol activates phosphorylation
of ERK and its downstream target, CREB, in cultured
rat cortical neurons.
Shown are representative results from at least three independent
experiments. Neurons plated at a density of 1 x 106 cells/35-mm
dish were cultured for 10-14 days. Cells were then stimulated
with ten different compounds at 100 μM for 30 min. (A) Western
blot analyses were performed using anti-phospho-ERK and
anti-phospho-CREB antibodies. Blots were then stripped and
reprobed with anti-total ERK antibody to verify that equal
amount of protein was electrophoresed in each lane. (B)
Relative densitometric expressions of the phosphorylated ERK
and CREB were normalized to the total ERK. Values are means ±
S.E.M. (n = 3). C: control.
**p < 0.01, ***p < 0.001 vs control.
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