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 (Nakajima et al., 2007), but also rescues memory A great number of compounds from natural resources impairment in AD model rats, possibly via stimulation of have so far attracted considerable attention as novel PKA/CREB signaling (Matsuzaki et al., 2006). leading compounds for drug development (Liu, 1993) as In order to further search for new substances activating well as useful pharmacological tools (Furukawa et al., phosphorylation of ERK and CREB in the CNS neurons, 1993; Ohizumi, 1997; Obara et al., 2002). In the course of we focused on chalcones and their derivatives, since these our survey of substances activating PKA/ERK signaling in compounds are precursors of flavonoids enriched in edible the CNS neurons in culture by examining the ability of six plants, e.g. citrus fruits, and thus show a structural flavonoids to stimulate the signaling, we have successfully similarity each other. Recently, we have developed a new found nobiletin, a citrus polymethoxy flavone with bioconversion system using a recombinant E. coli to neurotrophic action (Nagase et al., 2005a, b), which not successfully produce derivative compounds that are novel only reverses learning impairment associated with NMDA and/or rare in nature, from various chemicals, including receptor antagonism by activation of ERK in mice chalcones and flavonoids (Shindo et al., 2003, Shindo et al., 2004; Misawa et al., 2005). In the current study, ten *Correspondence author: Tohru Yamakuni, Ph.D. Department of Pharmacotherapy, Graduate School of Pharmaceutical Sciences, compounds produced by this bioconversion system were Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan Tel: +81-22-795-6852 Fax: +81-22-795-6850 employed to examine whether these compounds modulate E-mail: [email protected] the phosphorylation of ERK and CREB in cultured rat cortical neurons. Here we provide the first evidence sterile conditions. The cortical neurons were dissociated that a novel diol derivative of chalcone, 3-(2,3- according to the standard methods with SUMITOMO dihydroxyphenyl)-1-phenylpropan-1-one,potently Nerve-Cell Culture System (SUMITOMO BAKELITE), stimulates the phosphorylation of ERK and CREB in plated on 35-mm culture dish coated with poly-L-lysine cultured cortical neurons. (Life Technologies) at a density of 1 x 106 cells/dish in Neurobasal Medium without phenol red (Gibco Co., Ltd.) Materials and methods containing B-27 supplement (Gibco Co., Ltd.), 500 M L- 1. Preparations of the Tested Compounds glutamine and 0.005% penicillin-streptomycin, and The bphA1(2072) gene is an evolved gene shuffled cultured at 37°C in a humidified atmosphere consisting of between the biphenyl dioxygenase large (α subunit genes 5% CO2 and 95% air. At 4 days in vitro (D.I.V.), a half of derived from polychlorinated biphenyl (PCB)-degrading the medium was replaced by the medium containing a soil bacteria Pseudomonas pseudoalcaligenes KF707 and mitotic inhibitor, cytosine arabinofuranoside (Ara-C), to Burkholderia xenovorans LB400 (Misawa et al., 2002). inhibit non-neuronal proliferation. The tested compounds shown in Table 1 were prepared by At 10-14 D.I.V., cultured cortical neurons were treated the co-cultivation of the corresponding substrates and E. with the compounds tested, including a novel chalcone- coli cells carrying bphA1(2072) along with the diol, or vehicle (0.1% DMSO) in the Ara-C containing bphA2A3A4 genes from P. pseudoalcaligenes KF707, medium without B-27 supplement and L-glutamine at 100 which encoded the small (β) subunit, ferredoxin and μM concentrations for 30-min, and thereafter subjected to ferredoxin reductase of biphenyl dioxygenase, and the Western blot analysis. bphB gene coding for subsequent desaturase from strain KF707, as described previously (Shindo et al., 2003; Table 1 List of compounds tested for the ability to Misawa et al., 2005). The bphC gene from strain KF707 stimulate ERK/CREB phosphorylation was together used as needed (Shindo et al., 2004). As an Sample example, compound #3, a novel chalcone-diol was Chemical Nomenclature prepared as follows: E. coli JM109 carrying plasmid ID pBS2072B, which contained bphA1 (2072) - bphA2A3A4 - 1 2-(2,3-Dihydroxyphenyl)-chroman-4-one bphB , was grown in an LB medium containing 150 μg/ml 6-(4-Oxo-chroman-2-yl)-pyridine-2- 2 of ampicillin (Ap) at 30°C with reciprocal shaking (175 carboxylic acid 2-oxo-2-phenylethylester rpm) for 6-7 h until the absorbance at OD 600 nm had 3-(2,3-Dihydroxyphenyl)-1- 3 reached approximately 1. Eight milliliters of this culture phenylpropan-1-one was inoculated into 100 ml of an M9 medium with 150 4 3-Napthalen-1-yl-benzene-1,2-diol μ g/ m l o f A p , 1 m M o f i s o p r o p yl -D- thiogalactopyranoside, 0.4% (w/v) glucose, and 10 mg of 5 3-Napthalen-2-yl-benzene-1,2-diol trans-chalcone (Aldrich) in a Sakaguchi flask, and co- 6 3-Benzothiazole-2-yl-benzene-1,2-diol cultivated at 30℃ with reciprocal shaking (175 rpm) for 2 7 3-Benzooxazol-2-yl-benzene-1,2-diol days. The culture medium was extracted with ethylacetate, 8 3-Pyridin-2-yl-benzene-1,2-diol and the organic layer was further purified by silica gel column chromatography (hexane-ethylacetate = 4:1) to 9 3-(2,3-Dihydroxyphenyl)-indan-1-one give a purified chalcone-diol (5.9 mg), as described 10 3-Imidazole-1-ylmethyl-benzene-1,2-diol previously (Shindo et al., 2003). 2. Cortical Neuronal Culture and Treatment Cultured cortical neurons were treated with 100 μM of each of the listed compound for 30 min and subjected to Western blot Primary culture of cortical neurons was prepared as analysis as described under Experimental Procedures. described previously with minor modifications (Nagase et al., 2005b; Matsuzaki et al., 2006). In brief, under ether 3. Cell Extract Preparation and Western Blot Analysis anesthesia of an 18-day Sprague-Dawley (SD) rat (Japan, Cell extract preparation and Western blot analysis were SLC), embryos were immediately decapitated. The brain conducted, as described previously (Nagase et al., 2005b; was quickly removed, and cortex was collected in ice-cold Matsuzaki et al., 2006). After treatment with tested Dulbecco’s Phosphate Buffered Saline (DPBS) under compounds, cells were lysed with 60 μl of lysis buffer (1 mM EDTA, 1% SDS, 10 mM NaF, 10 nM calyculin, 320 and a possible downstream effector, CREB, the ten nM okadaic acid, 1 μM sodium orthovanadate, 1 mM p- compounds (listed in Table 1) were screened by assaying APMSF, 10 μg/ml pepstatin, 10 μg/ml antipain, 10 μg/ml the activity to stimulate the phosphorylation of ERK and leupeptin, 10 μg/ml chymostatin, 10 μg/ml CREB in cultured rat cortical neurons, using Western blot phosphoramidon, 10 mM HEPES, pH 7.5) per 35-mm analysis. Rat cortical neuronal cultures were treated with dish. Cell lysates were heated at 95°C for 5 min, sonicated the tested compounds at 100 μM for 30 min and subjected and centrifuged at 14,000 r.p.m. for 20 min at 4°C to to Western blot analysis. As shown in Fig. 2, it was obtain the supernatant as cell extracts. Cell extracts were observed that eight of the tested compounds failed to separated by 12.5% SDS-PAGE and transferred onto stimulate the phosphorylation of ERK and CREB, and PVDF membrane. The membranes were incubated with some of them rather depressed the phosphorylations. The anti-phospho-ERK (Thr202/Tyr204), anti-phospho-CREB compound #4, 3-napthalen-1-yl-benzene-1,2-diol, showed (Ser133), anti-total ERK antibodies (1:1000, Cell a little increase in the phosphorylation of CREB, yet it was Signaling Technology). not effective enough to be studied further. On the other hand, among the compounds tested, compound #3, a novel diol derivative of chalcone, 3-(2,3- OH dihydroxyphenyl)-1-phenylpropan-1-one (Fig. 1), only markedly increased the phosphorylation of ERK and its OH 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.

O Discussion Earlier studies have reported that chalcones and their F i g . 1 . Chemical structure of a novel chalcone- derivatives have beneficial pharmacological actions, diol, 3-(2,3-dihydroxyphenyl)-1-phenylpropan-1 including anti-inflammatory (Hsieh et al., 2000; Won et al., -one. 2005), anti-pyretic (De Leon et al., 2003), anti-invasive (Mukherjee et al., 2001) and anti-proliferative (Lin et. al., Immunoreactive proteins were visualized by 2002) activities. However, the effects of the chalcone and chemiluminescence as described previously (Matsuzaki et their derivatives on the CNS neurons have been so far al., 2006). The band intensities were quantitatively unidentified. Thus, to our knowledge, this study is the first analyzed using SCION image software. to demonstrate that the chalcone derivative activates the 4. Statistical Analyses phosphorylation of ERK and CREB in cultured cortical Student’s t test was performed to measure the level of neurons. The activation of ERK is required to induce significance. A level of p < 0.05 was considered to be nuclear translocation and the subsequent activation of statistically significant. RSK2, which in turn phosphorylates CREB to stimulate CRE-dependent transcription (Impey et al., 1998). Our Results present results evidently showed that the chalcone-diol A novel chalcone-diol most potently stimulates the enhanced the phosphorylation of not only ERK, but also phosphorylation of ERK and CREB among the the phosphorylation of the downstream effector, CREB, bioconverted compounds tested in cultured rat cortical indicating a stimulatory effect of the compound on neurons. The effects of chalcones and the derivative ERK/CREB signaling in the cortical neurons. Very compounds are still unidentified in the nervous system. To recently, we also found that chalcone-diol stimulated CRE- examine the effects of the bioconverted derivatives of mediated transcription in both cultured cortical and chalcone and flavonoid on the phosphorylation of ERK hippocampal neurons (data not shown). A Compounds # The chemical structure of chalcones consists of two aromatic rings joined by a three-carbon α,-unsaturated C 1 2 3 4 5 6 7 8 9 10 carbonyl system (Achanta et al., 2006). However, the chalcone-diol generated by our bioconversion system is P-ERK characterized by the fact that it contains a catechol moiety in its structure, which is attached to the second aromatic P-CREB ring by a saturated carbonyl system instead of an unsaturated one. Thus, based on the structural difference Total ERK 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

B ** unique structural feature of this chalcone-diol. 5 In summary, this pharmacological approach combined

) with bioconversion led us to successfully identify l 4 o r

t a novel chalcone-diol as the agent, which

n K o 3 activates phosphorylation of ERK and CREB in the c R

f E - o cultured cortical neurons. These results of the current study

P

d 2 l

o would add new dimension to and attract greater attention F ( 1 in the research interest of the chalcone compounds, and further pharmacological studies would give new insights 0 C 1 2 3 4 5 6 7 8 9 10 into better understanding of the molecular mechanism of this novel chalcone-diol. Compounds #

Acknowledgements *** The authors thank to Toshihiko Otomatsu of KNC 5 Laboratories Co. for large-scale preparation of chalcone- ) l 4 diol. This work was supported in part by a grant-in-aid for o r t Scientific Research from the Ministry of Education, n B

o 3 E c

Culture, Sports, Science, and Technology of Japan, and the R f C o - 2 grant from Takeda Science Foundation. d P l o F ( 1 References 0 Achanta, G., Modzelewska, A., Feng, L., Khan, S.R. and 8 C 1 2 3 4 5 6 7 9 10 Huang, P. (2006): A boronic- chalcone derivative Compounds # exhibits potent anticancer activity through inhibition of Fig. 2. A novel chalcone-diol activates phosphorylation the proteasome. Mol. Pharmacol., 70, 426-433. of ERK and its downstream target, CREB, in cultured De Leon, E.J., Alcaraz, M.J., Dominguez, J.N., Charris, J. rat cortical neurons. a n d T e r e n c i o , M . C . ( 2 0 0 3 ) : 1-(2,3,4- trimethoxyphenyl)-3-(3-(2-chloroquinolinyl))-2-propen- Shown are representative results from at least three independent 1-one, a chalcone derivative with analgesic, anti- experiments. Neurons plated at a density of 1 x 106 cells/35-mm dish were cultured for 10-14 days. 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