Neurotrophic Action of 5-Hydroxylated Polymethoxyflavones: 5

Subscriber access provided by Temple University Libraries

Article

Neurotrophic action of 5-hydroxylated polymethoxyflavones: 5demethylnobiletin and gardenin A stimulate neuritogenesis in PC12 cells Szu-Ping Chiu, Ming-Jiuan Wu, Pei-Yi Chen, Yi-Ru Ho, Mi-Hsueh Tai, Chi-Tang Ho, and Jui-Hung Yen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf4024678 • Publication Date (Web): 04 Sep 2013 Downloaded from http://pubs.acs.org on September 7, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

Journal of Agricultural and Food Chemistry

1

Neurotrophic action of 5-hydroxylated polymethoxyflavones: 5-demethylnobiletin and

2

gardenin A stimulate neuritogenesis in PC12 cells

3

Szu-Ping Chiu1, Ming-Jiuan Wu2, Pei-Yi Chen3,4, Yi-Ru Ho1, Mi-Hsueh Tai1, Chi-Tang Ho5,

4

Jui-Hung Yen1,3*

5 1

6

Department of Molecular Biology and Human Genetics, Tzu Chi University, Hualien 970, Taiwan

7 2

8

Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan

9 3

10 4

11

5

12

Institute of Medical Science, Tzu Chi University, Hualien 970, Taiwan

Center of Medical Genetics, Buddhist Tzu Chi General Hospital, Hualien 970, Taiwan

Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901-8520, USA

13 14 15

Running title: Neurotrophic action of 5-hydroxylated PMFs in PC12 cells

16

*

17

Corresponding authors. Tel: +886-3-856-5301 ext 2683, Fax: +886-3-856-1422

E-mail: [email protected]

18

1

ACS Paragon Plus Environment


Page 2 of 37

19

Abstract

20

Polymethoxyflavones (PMFs) exhibit a broad spectrum of biological properties, including

21

anti-cancer, anti-atherogenic, and neuroprotective effects. The aim of this study is to

22

investigate the neurotrophic effects of the 5-demethylnobiletin, a hydroxylated PMF found in

23

citrus plants, and gardenin A, a synthetic PMF analog, on neurite outgrowth and neuronal

24

differentiation in PC12 cells. The results of this study showed that 5-demethylnobiletin and

25

gardenin A (10-20 µM) potently induce neurite outgrowth in PC12 cells, accompanied by the

26

expression

27

growth-associated protein-43(GAP-43) and synaptophysin. We observed that the addition of

28

K252a, a TrKA antagonist, significantly inhibited NGF-induced neurite outgrowth in PC12

29

cells, but 5-demethylnobiletin- or gardenin A-induced neurite outgrowth was not affected.

30

Treatment with 5-demethylnobiletin and gardenin A markedly induced the phosphorylation of

31

both cyclic AMP response element-binding protein (CREB) and CRE-mediated transcription,

32

which was suppressed through the administration of the inhibitor 2-naphthol AS-E phosphate

33

(KG-501) or using CREB siRNA. Furthermore, our results showed that MAPK/ERK kinase

34

1/2 (MEK1/2), protein kinase A (PKA), and protein kinase C (PKC) inhibitors blocked the

35

CRE transcription activity and neurite outgrowth induced through 5-demethylnobiletin or

36

gardenin A. Consistently, increased ERK phosphorylation and PKA and PKC activities were

37

observed in PC12 cells treated with 5-demethylnobiletin or gardenin A. These results reveal

38

for the first time that 5-demethylnobiletin and gardenin A promote neuritogenesis through the

39

activation of MAPK/ERK-, PKC-, and PKA-dependent, but not TrkA-dependent, CREB

40

signaling pathways in PC12 cells.

of

neuronal

differentiation

and

synapse

formation

marker

proteins,

41 42

KEYWORDS: Polymethoxyflavones; 5-demethylnobiletin; gardenin A; PC12 cells; neurite

43

outgrowth

2


Page 3 of 37

44


INTRODUCTION

45

Neurotrophic factors, such as nerve growth factor (NGF), play critical roles in neuronal

46

development and survival and in the maintenance of synaptic connection and plasticity1. The

47

loss of neurotrophic support is a critical factor in the pathogenesis of neurodegenerative

48

disorders, such as Alzheimer’s disease and Parkinson’s disease2. Therefore, the administration

49

of neurotrophic agents to stimulate neurogenesis, neuritogenesis, neuronal differentiation and

50

synaptogenesis has been considered to be a potential therapeutic strategy for patients with

51

neurodegenerative diseases. Recently, it was shown that several phytochemicals, such as

52

polyphenolics and flavonoids, possess high neurotrophic potency and might penetrate the

53

blood-brain barrier (BBB), influencing numerous neuronal functions within the brain3. These

54

small molecules mimic the neurotrophic ability of NGF and might contribute to the

55

development of important prevention strategies or therapeutics to combat neurodegenerative

56

disorders.

57

The rat pheochromocytoma cell line PC12 is widely used as a cell culture model for

58

neuronal differentiation and survival4. PC12 cells exhibit the cell surface expression of the

59

NGF-specific receptor tyrosine kinase (TrkA). Upon exposure to NGF, PC12 cells exhibit

60

neurite outgrowth, neuronal differentiation and the formation of synaptic connections,

61

associated with the increased expression of neuronal-specific genes, such as growth

62

associated protein-43 (GAP-43), type III β-tubulin, and synaptophysin5. NGF induces rapid

63

TrkA dimerization, autophosphorylation, and the activation of the mitogen-activated protein

64

kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent signal pathway. It has

65

been reported that the NGF-mediated activation of MAPK/ERK induces phosphorylation of

66

the cAMP response element binding protein (CREB) at Ser133, which further enhances the

67

transcription activity of protein-coding genes or microRNA, associated with neuronal

68

differentiation, synapse formation, synaptic plasticity, and long-term memory6,7,8,9. In addition

69

to the MAPK/ERK-dependent pathway, several individual signal transduction cascades are 3



Page 4 of 37

70

also involved in the activation of CREB, including cAMP-dependent protein kinase A (PKA),

71

protein kinase C (PKC), calcium/calmodulin-dependent protein kinase (CaMK), and

72

phosphatidylinositol 3-kinase (PI3-K)/Akt10.

73

Polyphenolic compounds, such as flavonoids, derived from fruits or vegetables, exhibit

74

diverse pharmacological properties that stimulate neuritogenesis and neuroprotective

75

activities. Recent studies have shown that these naturally derived compounds, such as

76

curcuminoids11, epigallocatechin-3-gallate (EGCG)12, fisetin13 and luteolin14, interact with

77

critical diverse signaling molecules that regulate neurotrophic activity. Citrus fruits contain a

78

wide range of flavonoids. Polymethoxyflavones (PMFs) are present in the peels of citrus

79

fruits, such as sweet oranges (Citrus sinensis (L.) Osbeck) and mandarin oranges (Citrus

80

reticulate Blanco)15. The citrus PMFs display a broad spectrum of biological activities,

81

including

82

neuroprotective

83

(3',4',5,6,7,8-hexamethoxyflavone) and its metabolites have been reported to stimulate

84

neuritogenesis through MAPK/ERK- and CREB-dependent pathways in vitro and enhance or

85

improve impaired memory in animal models with neurological disorders21,22,23. Recently, we

86

also demonstrated that the hydroxylated PMF, 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone

87

(5-OH-HxMF), isolated from citrus peel extract, is a potent promoter of neurite outgrowth in

88

PC12 cells through cAMP/PKA/CREB pathways24.

89

anti-inflammatory,

anti-cancer,

properties16,17,18,19,20.

anti-angiogenesis,

The

well-studied

anti-atherogenic, citrus

PMF

and

nobiletin

In the present study, we selected two structurally related 5-hydroxylated PMFs,

90

5-demethylnobiletin

(5-hydroxy-6,7,8,3',4'-pentamethoxyflavone)

91

(5-hydroxy-6,7,8,3',4',5'- hexamethoxyflavone) (Figure 1), to examine their effects on

92

neuritogenesis in PC12 cells. 5-Demethylnobiletin can be isolated from the extract of aged

93

orange peels extract and derived from the auto-hydrolysis of nobiletin during long-term

94

storage15; gardenin A (GA) is a synthetic derivative of 5-hydroxylated PMFs25. The only

95

structural difference between 5-demethylnobiletin and gardenin A is on the 5'-position of the 4


and

gardenin

A

Page 5 of 37


96

B-ring moiety with -H and -OCH3, respectively. Recent studies have compared the

97

structure-relative activities of hydroxylated PMFs with their permethoxylated counterparts. It

98

has been reported that 5-hydroxylated PMFs, such as 5-demethylnobiletin, possess much

99

stronger anti-cancer properties compared with their PMFs counterparts, suggesting the critical

100

role of the hydroxyl group at the 5-position in the enhanced inhibitory effects on the growth

101

of cancer cells25. Thus, we investigated the effectiveness of the 5-hydroxylated PMFs,

102

5-demethylnobiletin and gardenin A, on the neurite outgrowth and neuronal differentiation in

103

PC12 cells.

104

MATERIALS AND METHODS

105

Chemicals

106

5-Demethylnobiletin (5-hydroxy-6,7,8,3',4'-pentamethoxyflavone) was purified as

107

previously described26. Gardenin A (GA), RPMI-1640 medium, poly-L-lysine, dimethyl

108

sulfoxide (DMSO), forskolin, KN-62, H-89, 2-naphthol AS-E phosphate (KG-501),

109

Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS) and

110

other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), unless

111

otherwise indicated. U0126, PD98059 and LY294002 were purchased from Promega

112

(Madison, WI, USA). Bisindolylmaleimide I (BIM) was purchased from Cayman Chemical

113

(Ann Arbor, MI, USA). TrkA antagonist K252a was purchased from Enzo Life Sciences (Ann

114

Arbor, MI, USA). The mouse 7S nerve growth factor (NGF) was purchased from Millipore

115

(Billerica, MA, USA).

116

Cell culture

117

The PC12 cell line was obtained from the Bioresource Collection and Research Center

118

(Hsinchu, Taiwan). The cells were maintained in RPMI-1640 medium containing 2 mM

119

glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES and 1 mM sodium

120

pyruvate, supplemented with 10% heat-inactivated horse serum (Invitrogen, Carlsbad, CA,

121

USA) and 5% fetal bovine serum (FBS)(Biological Industries, Kibbutz Haemek, Israel) in a 5



122

5% CO2 incubator at 37 °C.

123

Cell viability analysis

124

The cell viability was measured through the mitochondrial-dependent reduction of

125

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to purple formazan.

126

The cells were incubated with MTT solution (1 mg/ml final concentration) for 4 h at 37 °C.

127

The formazan crystals were dissolved in DMSO, and the extent of the reduction of MTT was

128

measured as the absorbance at 550 nm.

129

Analysis of neurite outgrowth in PC12 cells

130

The quantification of neurite outgrowth in PC12 cells was performed as previously

131

described27. Briefly, the cells (2 x 105/ml) were seeded onto poly-L-lysine-coated 6-well

132

plates with normal serum medium. After 24 h, the cells were changed to low serum (1% horse

133

serum and 0.5% FBS) medium and treated with vehicle (0.1% DMSO) or the indicated

134

compounds for 48 h. Changes in the morphology of PC12 cells were observed and

135

photographed under an inverted microscope (Olympus IX71), and subsequently, the

136

neurite-positive cells were counted. The neurite-bearing cells were analyzed from at least ten

137

randomly selected microscopic fields with an average of 100 cells per field. The number of

138

differentiated cells in the field was visually examined, and cells showing at least one neurite,

139

with a length equal to the cell body diameter, were counted. The data are expressed as a

140

percentage of the total number of cells in the field. Each experiment was conducted in

141

triplicate.

142

RT quantitative PCR (RT-Q-PCR) analysis of GAP-43

143

PC12 cells (1 x 106/ml) were seeded onto poly-L-lysine-coated 6-well plates in normal

144

serum medium for 24 h. The cells were subsequently shifted to low serum medium for 24 h

145

prior to exposure to vehicle (0.1% DMSO) or the indicated compounds for 48 h. Total cellular

146

RNA was isolated using the Total RNA Mini Kit (Geneaid, Taipei, Taiwan). The reverse

147

transcription of 2 µg of RNA was performed using the High Capacity cDNA Reverse 6


Page 6 of 37

Page 7 of 37


148

Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR

149

was performed with 2 µL of cDNA in a 25-µL reaction containing 200 nM primers [GAP-43,

150

5'-CTAAGGAAAGTGCCCGACAG-3'

151

5'-GCAGGAGAGACAGGGTTCAG-3'

152

5'-CCTCTGAACCCTAAGGCCAA-3' (forward) and 5'-AGCCTGGATGGCTACGTACA-3'

153

(reverse)] and Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, Burlington, CA).

154

The amplification was conducted using an ABI Prism 7300 Real-Time PCR System. The

155

following PCR conditions were used: 94 °C for 4 min, followed by 40 cycles at 94 °C for 1

156

min, 58 °C for 1 min, and 72 °C for 1 min. The ∆∆Ct method was used for the analysis of

157

GAP-43 mRNA expression, estimated in triplicate samples and normalized to β-actin

158

expression level.

159

Western blot analysis of GAP-43, synaptophysin, CREB, ERK1/2 and β-actin proteins

(forward)

and

(reverse);

β-actin,

160

PC12 cells (1 x 106/ml ) were seeded onto poly-L-lysine-coated 100-mm dishes in normal

161

serum medium for 24 h and subsequently shifted to low serum medium for 24 h prior to

162

exposure to vehicle (0.1% DMSO) or the indicated compounds for the indicated periods. The

163

cells were harvested using RIPA buffer (Thermo Fisher Scientific, Inc., Rockford, IL)

164

according to the manufacturer’s instructions. The cell lysate (20 µg) was separated on 10%

165

SDS-PAGE, and subsequently the proteins were transferred onto a PVDF membrane

166

(PerkinElmer, Boston, MA, USA) at 25 V overnight at 4 °C. The membranes were blocked at

167

4 °C in PBST blocking buffer (1% non-fat dried milk in PBS containing 0.1% Tween-20) for

168

24 h. The blots were incubated with each of the following antibodies at a 1:1000 dilution:

169

anti-GAP-43, anti-synaptophysin (Millipore, Billerica, MA, USA), anti-phospho-CREB

170

(Ser-133), anti-CREB, anti-ERK1/2, anti-phospho-ERK1/2 (Cell Signaling Technology, Inc.)

171

and Monoclonal anti-β-actin (1:8000) (Sigma-Aldrich). The blots were incubated with the

172

horseradish

173

Biotechnology, Santa Cruz, CA) for 1 h and the proteins of interest were detected using

peroxidase-conjugated

secondary

antibodies

7


(1:10,000)

(Santa

Cruz


174

Western LightningTM Chemiluminescence Reagent Plus (PerkinElmer, Boston, MA, USA)

175

according to the manufacturer’s instructions. The chemiluminescence signal was visualized

176

using Amersham HyperfilmTM ECL (GE Healthcare, Buckinghamshire, UK).

177

Immunofluorescence staining of GAP-43 proteins

178

PC12 cells were seeded onto poly-L-lysine-coated coverslips in normal serum medium for

179

24 h, and subsequently shifted to low serum medium prior to exposure to the indicated

180

reagents. After 48 h of incubation, the PC12 cells were fixed with 3.7% formaldehyde in

181

phosphate-buffered saline (PBS) for 15 min, permeabilized with 0.1% Triton X-100, and

182

soaked in blocking buffer (PBS containing 1% BSA) for 60 min at room temperature.

183

Immunostaining was performed through incubation with the anti-GAP-43 antibody, washing,

184

and subsequent incubation with the secondary Alexa Fluor 488-conjugated goat anti-mouse

185

IgG antibody (Invitrogen, Carlsbad, CA, USA) for 60 min. Subsequently, the cells were

186

washed four times in PBS and incubated for 2 min with 4’,6-diamidino-2-phenylindole (DAPI)

187

for nuclear staining. The coverslips were again washed, drained, and mounted with

188

Fluoromount G (Andes import). The cells were observed and photographed on an inverted

189

fluorescence microscope (Olympus IX71).

190

Analysis of cyclic AMP response element (CRE)-mediated transcription activity

191

PC12 cells (2 x 105/well) were seeded onto poly-L-lysine-coated 24-wells plate in DMEM

192

containing 10% horse serum and 5% FBS medium for 24 h. For transient transfection, cells

193

were co-transfected with the pCRE-Luc Cis-reporter plasmid (Stratagene, La Jolla, CA, USA)

194

and Renilla luciferase vector (Promega) using Lipofectamine 2000 reagent (Invitrogen).

195

Twenty-four hours after transfection, the cells were changed to low-serum medium and

196

treated with vehicle (0.1% DMSO) or indicated compounds for 8 h. Luciferase activities were

197

determined by the Dual-Luciferase Reporter Assay System Kit (Promega) according to the

198

manufacturer’s instructions. The intensities of the luciferase reactions measured in the lysates

199

of the transfectants were normalized to their activity of Renilla luciferase, which was used as 8


Page 8 of 37

Page 9 of 37


200

an internal control.

201

Transfection of small interference RNA (siRNA) oligonucleotides

202

PC12 cells were seeded onto poly-L-lysine-coated 6-well plates in normal serum medium.

203

After 24 h, the cells were shifted to serum-free OPTI-MEM medium, followed by transfection

204

with 2'-OMe modified siRNA, as a negative control, or rat-specific CREB siRNA duplexes

205

[5'-GGAGUCUGUGGAUAGUGUA-3' (forward) and 5'-UACACUAUCCACAGACUCC-3'

206

(reverse)] (GeneDireX Inc., Las Vegas, NV, USA) at a final concentration of 150 pmol using

207

Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After 24 h, the

208

cells were changed to low-serum medium and treated with vehicle (0.1% DMSO),

209

5-demethylnobiletin or gardenin A for further analysis.

210

Analysis of protein kinase C (PKC) and protein kinase A (PKA) activity

211

Nonradioactive PKC and PKA activity assay kits (Enzo Life Sciences) were used to

212

measure the PKC and PKA activity in the samples. PC12 cells (1 x 106/ml ) were seeded onto

213

poly-L-lysine-coated 100 mm dishes in normal serum medium for 24 h, and subsequently

214

shifted to low serum medium for 24 h prior to exposure to the indicated compounds for the

215

indicated periods. The cellular proteins were harvested using RIPA buffer, and the protein

216

kinase activity was measured according to the manufacturer’s instructions. Briefly, the PKC

217

or PKA substrate microtiter plates were soaked in kinase assay dilution buffer at room

218

temperature. The cell lysates (50 ng) or standard (10 ng) were subsequently added, followed

219

by the addition of ATP to initiate the reaction. After incubation at 30 °C for 90 min, the

220

phosphospecific substrate antibody was added, and the reaction was incubated at room

221

temperature for 1 h. The HRP-conjugated secondary anti-rabbit IgG was subsequently added

222

to each well, and the reaction was incubated for an additional 30 min. The TMB substrate

223

solution was added to each well, and the reaction was further incubated for 30 min. Finally,

224

the stop solution was added, and the 96-well plate was read at 450 nm in a microplate reader.

225

Statistical Analysis 9



226

All experiments were repeated at least three times. All values are expressed as the mean ±

227

SD. The results were analyzed using One-way ANOVA with Dunnet’s post-hoc test and a p

228

value of < 0.05 was considered statistically significant.

229

RESULTS

230

The effect of 5-demethylnobiletin (5-demethyl NOB) and gardenin A (GA) on cell viability

231

and neurite outgrowth in PC12 cells

232

PC12 cells were cultured in a low-serum media system (1% horse serum and 0.5% FBS)

233

in the presence of the indicated concentration of compound for 48 h. The cell viability was

234

analyzed using an MTT assay, and the values were expressed as percentage of vehicle-treated

235

group (negative control). As shown in Figure 2A, NGF supported cell growth on PC12 cells

236

under low-serum culture conditions. 5-Demethyl NOB and GA also showed cell growth

237

supporting effect at higher concentrations (10-20 µM) and did not exert detectable

238

cytotoxicity on PC12 cells after 48 h incubation in low serum medium. To investigate whether

239

5-demethyl NOB and GA induce neurite outgrowth in PC12 cells, the cells maintained in

240

low-serum medium were treated with vehicle (0.1% DMSO), NGF (50 ng/ml), 5-demethyl

241

NOB or GA (2-20 µM). After 48 h, the neurite-bearing cells were photographed under an

242

inverted microscope (Figure 2B), and the number of neurite-bearing cells was counted. The

243

percentage of neurite-bearing cells reached 49.1± 2.2% and 53.5± 2.5% for 10 µM and 20 µM

244

5-demethyl NOB, respectively, and 34.4 ± 2.3% and 36.6 ± 1.5% for 10 µM and 20 µM

245

gardenin A, respectively (p

Neurotrophic Action of 5-Hydroxylated Polymethoxyflavones: 5

Recommend Documents