Synephrine Hydrochloride Suppresses Esophageal Cancer Tumor

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Bioactive Constituents, Metabolites, and Functions

Synephrine hydrochloride suppresses esophageal cancer tumor growth and metastatic potential through inhibition of Galectin-3-AKT/ERK signaling Wen Wen Xu, Can-Can Zheng, Yunna Huang, Wen-You Chen, Qing-Sheng Yang, Jia-Yi Ren, Yue-Ming Wang, Qing-Yu He, Hua-Xin Liao, and Bin Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04020 • Publication Date (Web): 16 Aug 2018 Downloaded from http://pubs.acs.org on August 18, 2018

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Journal of Agricultural and Food Chemistry

1

Title: Synephrine hydrochloride suppresses esophageal cancer tumor growth and

2

metastatic potential through inhibition of Galectin-3-AKT/ERK signaling

3 4

Running title: Synephrine suppresses ESCC progression via AKT/ERK

5 6

Wen Wen Xu1,2,3,#, Can-Can Zheng4,#, Yun-Na Huang1,2,3, Wen-You Chen5,

7

Qing-Sheng Yang5, Jia-Yi Ren6, Yue-Ming Wang1,2,3, Qing-Yu He4, Hua-Xin Liao1,2,3,*,

8

Bin Li4,*

9 10

1

11

Guangzhou

12

Bioengineering Medicine, Guangzhou, China; 3National Engineering Research Center

13

of Genetic Medicine, Jinan University, Guangzhou, China; 4Key Laboratory of

14

Functional Protein Research of Guangdong Higher Education Institutes, Institute of

15

Life and Health Engineering, College of Life Science and Technology, Jinan

16

University, Guangzhou 510632, China; 5Department of Thoracic Surgery, First

17

Affiliated Hospital, Jinan University, Guangzhou 510632, China;

18

Traditional Chinese Medicine, Jinan University, Guangzhou 510632, China.

Institute of Biomedicine, College of Life Science and Technology, Jinan university, 510632,

China;

2

Guangdong

Provincial

Key

Laboratory

of

6

School of

19 20

Correspondence Author:

21

Dr. Bin Li, Key Laboratory of Functional Protein Research of Guangdong Higher

22

Education Institutes, Institute of Life and Health Engineering, College of Life Science

ACS Paragon Plus Environment


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23

and Technology, Jinan University, Guangzhou, China. Phone: (86)-20-85224372; Fax:

24

(86)-20-85224372; Email: [email protected].

25 26

Dr. Hua-Xin Liao, Institute of Biomedicine, Guangdong Provincial Key Laboratory of

27

Bioengineering Medicine, National Engineering Research Center of Genetic Medicine,

28

Jinan

29

(86)-20-85222062; Email: [email protected].

University,

Guangzhou,

China.

Phone:

(86)-20-85222062;

Fax:

30

ESCC,

esophageal

squamous

cell

carcinoma;

EMT,

31

Abbreviations:

32

epithelial-mesenchymal transition; , 5-FU, fluorouracil; FBS, fetal bovine serum;

33

p-AKT, phospho-AKT; p-ERK, phospho-ERK; qRT-PCR, quantitative real-time

34

polymerase chain reaction; IPA, ingenuity pathway analysis; PLGEM, power law

35

global error model; ALT, alanine transaminase; AST, aspartate transaminase; EGCG,

36

epigallocatechin-3-gallate.

37 38 39 40


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41

Abstract

42

A library consisting of 429 food-source compounds was used to screen the natural

43

products with anticancer property in esophageal squamous cell carcinoma (ESCC).

44

We demonstrated for the first time that synephrine, an active compound isolated from

45

leaves of citrus trees, markedly suppressed cell proliferation (Inhibition rate with 20

46

µM synephrine at day 5: 71.1 ± 5.8% and 75.7 ± 6.2% for KYSE30 and KYSE270,

47

respectively) and colony formation (Inhibition rate with 10 µM synephrine: 86.5 ± 5.9%

48

and 82.3 ± 4.5% for KYSE30 and KYSE270, respectively), as well as migration

49

(Inhibition rate with 10 µM synephrine: 76.9 ± 4.4% and 62.2 ± 5.8% for KYSE30

50

and KYSE270, respectively) and invasion abilities (Inhibition rate with 10 µM

51

synephrine: 73.3 ± 7.5% and 75.3 ± 3.4% for KYSE30 and KYSE270, respectively)

52

of ESCC cells in a dose-dependent manner, without significant toxic effect on normal

53

esophageal

54

bioinformatics analyses were performed to explore the synephrine-regulated proteins.

55

Western blot and qRT-PCR data indicated that synephrine may downregulate

56

Galectin-3 to inactivate AKT and ERK pathways. In addition, we found that the

57

sensitivity of ESCC to fluorouracil (5-FU) could be enhanced by synephrine.

58

Furthermore, in vivo experiments showed that synephrine had significant antitumor

59

effect on ESCC tumor xenografts in nude mice (Inhibition rate with 20 mg/kg

60

synephrine is 61.3 ± 20.5%) without observed side effects on animas. Taken together,

61

Synephrine, a food-source natural product, may be a potential therapeutic strategy in

62

ESCC.

epithelial

cells.

Mechanistically,

quantitative


proteomics

and


63 64

Keywords: Synephrine, natural product, esophageal cancer progression, Galectin-3,

65

AKT and ERK pathways

66


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Introduction

68

Esophageal cancer is the eighth most common cancer in the world and the sixth

69

leading cause of cancer death worldwide. Esophageal squamous cell carcinoma

70

(ESCC) is the predominant histological subtype and accounts for nearly 90% of the

71

esophageal cancer cases in China. Despite improvements in diagnosis and treatment

72

in recent years, 5-year survival rate of ESCC is still low. Limited treatment

73

efficiency and the severe side effects traditional chemotherapeutic drugs generally

74

cause, such as hepatotoxicity and renal damage, are the major obstacles in cancer

75

therapy. Therefore, development of new therapeutic agents with anticancer property

76

and low side effect is urgently needed.

77

There is an intense effort to develop natural products as anticancer agents

78

recently. Increasing natural active ingredients derived from medicinal herbs were

79

successfully applied to cancer therapy in clinic,1-3 among which paclitaxel, a

80

complex diterpene isolated from the bark of the Pacific yew tree, is one of the most

81

commonly used anticancer drugs.4 To identify novel compounds with anticancer

82

bioactivity, we used a library consisting of 429 natural products which have

83

reasonable aqueous solubility and membrane permeation profile. After literature

84

study and experimental screening, synephrine, a natural phenolic compound from the

85

leaves of citrus trees and various citrus juices,5 was found to exert the strongest

86

inhibitory effect on ESCC cell proliferation. Citrus contains various flavonoids and

87

alkaloids with multiple biological activities, and synephrine was isolated for the first

88

time from extracts of Citrus fruits. Purified synephrine is a sympathomimetic



89

α-adrenergic receptor agonist and displays potent vasoconstrictive effects on isolated

90

rat aorta.6, 7 Up to now, the function of synephrine in cancer treatment is largely

91

unknown.

92

The molecular mechanism how synephrine exerts its biological functions

93

including anticancer effect has not been reported but warrants investigation. In present

94

study, quantitative proteomics was performed to identify the differentially expressed

95

proteins in synephrine-treated ESCC cells, and bioinformatics analysis of the protein

96

alterations suggested that Galectin-3 and the AKT and ERK pathways have crucial

97

roles in the bioactivity of synephrine. Our previous studies reported that constitutively

98

activated PI3K/AKT signaling, which played an important role in esophageal tumor

99

growth and metastasis, is a potential target for ESCC treatment.8, 9 The ERK pathway

100

has also been documented to be an important regulator of tumorigenesis and

101

metastasis.10 Galectin-3, a member of the beta-galactosidase binding lectin family,

102

was reported to correlate with esophageal cancer progression and promote

103

tumorigenesis by activating AKT and ERK pathways.11-14

104

In this study, we examined the effect of synephrine on growth, chemosensitivity

105

and metastatic potential of esophageal cancer cells in vitro and in vivo, and

106

investigated whether Galectin-3-dependent regulation of AKT and ERK pathways has

107

a crucial role in the anticancer effects of synephrine.

108 109


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Materials and Methods

111

Chemicals

112

A natural product compound library and naturally extracted synephrine

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hydrochloride (synephrine HCl, CAS No.: 5985-28-4) were purchased from

114

TargetMol (Boston, MA, USA). The purity of all tested compounds is higher than

115

98%. Fluorouracil (5-FU) was purchased from Cayman Chemical (Ann Arbor, MI,

116

USA) and dissolved in dimethyl sulfoxide (DMSO).

117 118

Cell lines

119

The human ESCC cell lines KYSE30 and KYSE270 (obtained from DSMZ,

120

Braunschweig, Germany) were cultured in RPMI 1640 medium (Life Technologies,

121

Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Life

122

Technologies) at 37°C in 5% CO2.

123 124

Cell viability assay

125

Cells viability was determined with the Cell Counting Kit-8 (CCK-8; Dojindo

126

Molecular Technologies Inc., Rockville, MD, USA) according to the instructions, and

127

the results were quantified by measuring the absorbance of the solution at 450 nm.

128

129

Colony-formation assay

130

Colony-formation assay was performed as described previously.8 Cells were

131

seeded in 6-well plates at a density of 500 cells per well and cultured for 14 days.



132

After washing with PBS, the cells were fixed with 75% ethanol for 15 min and then

133

stained with crystal violet. The numbers of colonies were counted.

134 135

Soft Agar Assay

136

Soft agar assay was carried out to test the anchorage-independent growth ability

137

of cells.15 Briefly, the cells treated with synephrine or control were trypsinized and

138

suspended in RPMI 1640 medium containing 0.33% agar and 10% FBS, and then

139

layered on consolidated mixture of 0.6% agar and 10% FBS in RPMI 1640 medium.

140

Colonies were photographed 20 d later, and the ratio between the number of colonies

141

formed by treated cells and control cells was calculated.

142 143

In vitro cell migration and invasion assay

144

The uncoated Transwell chambers (8 µm pore size; BD Biosciences, Bedford,

145

MA, USA) were used to determine cell migration ability.16 Cells in serum-free

146

medium were seeded to the upper chamber, and the lower compartment was filled

147

with complete medium as a chemoattractant. After 24 h, the migrated cells adhering to

148

the lower surface of the chamber were fixed with methanol and stained with crystal

149

violet. Images of three different fields were captured from each membrane. The

150

migrated cells were quantified by submerging the chambers in 1% sodium dodecyl

151

sulfate buffer and measuring the absorbance of the solution at 570 nm. Similar

152

methods was performed to monitor invasive potential of ESCC cells with the use of

153

BD BioCoat matrigel coated invasion chambers (BD Biosciences).


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154 155

Western blot analysis

156

Details on preparation of whole cell lysates and Western blot were described

157

previously.17 The primary antibodies used include phospho-AKT (p-AKT), AKT,

158

phospho-ERK (p-ERK), and ERK from Cell Signaling Technology (Beverly, MA,

159

USA); vimentin and actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA);

160

E-cadherin from BD Biosciences; and Galectin-3 from Proteintech (Chicago, IL,

161

USA). The signals were visualized using Clarity Western ECL substrate (Bio-Rad,

162

Hercules, CA, USA) and detected by exposure to autoradiographic film.

163 164

Quantitative real-time polymerase chain reaction (qRT-PCR)

165

The qRT-PCR was conducted as described previously.18 In brief, total RNA was

166

extracted using TRIzol reagent (Life Technologies), and reverse transcription was

167

performed with PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, Dalian, China)

168

according to the manufacturer’s protocol. The mRNA expression levels of Galectin-3

169

and of GAPDH as internal control were detected using SYBR Green Supermix

170

(Bio-Rad). The sequences of primers were: 5’- ATGGCAGACAATTTTTCGCTCC-3’

171

(forward) and 5’- GCCTGTCCAGGATAAGCCC-3’ (reverse) for Galectin-3; 5’-

172

AGAAGGCTGGGGCTCATTTG-3’

173

AGGGGCCATCCACAGTCTTC-3’ (reverse) for GAPDH.

(forward)

174

175

Mass spectrometry and bioinformatics analyses


and

5’-


176

Preparation of cell lysates, trypsin digestion and desalination were performed,

177

and the peptides were analyzed with an Orbitrp Fusion Lumos mass spectrometer

178

(Thermo Fisher Scientific, Waltham, MA, USA).19 The raw data were searched using

179

Proteome Discoverer (Thermo Fisher Scientific) and Spectronaut (Omicsolution Co.

180

Ltd., Shanghai, China) software. A fold difference of ≥ 1.5 was defined as

181

differentially expressed. Ingenuity pathway analysis (IPA) software (Ingenuity

182

Systems, Redwood City, CA, USA) was used for pathway analysis (9).

183 184

Tumor xenograft experiment

185

Female BALB/c nude mice aged 6-8 weeks were maintained under standard

186

conditions and cared for according to the institutional guidelines for animal care.

187

Tumor xenografts were established by subcutaneously injecting the KYSE270 cells in

188

equal volume of PBS and Matrigel (BD Biosciences) into the flanks of nude mice.20

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The mice were randomly divided into treatment and control groups when the tumor

190

xenografts reached ~5 mm diameter. The mice in treatment groups received oral

191

gavage of synephrine thrice a week at doses of 10 mg/ kg and 20 mg/ kg, respectively,

192

whereas the control group received the vehicle only. The tumor volumes were

193

measured every three days and tumor volumes were calculated with the equation V =

194

(length x width2)/2. At the end of the experiment, tumors, as well as liver, lungs, and

195

kidneys, were harvested for Western blot and histologic analyses. All the animal

196

experiments were approved by the Ethics Committee for Animal Experiments of Jinan

197

University (No. 10365).


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198 199

Statistical analysis

200

All in vitro experiments were repeated at least three times. The data were expressed as

201

the mean ± SD and compared using ANOVA followed by Scheffe test. P values < 0.05

202

were deemed significant.



203

Results

204

Screening of anticancer compounds from a food-source natural product library

205

To identify the food-source compounds with anticancer property, a compound

206

library consisting of 429 food-source natural products was used for initial screening

207

by literature study. Totally 55 compounds, which have not been reported to exert any

208

effect in cancer cells, were selected for subsequent functional identification of

209

potential anticancer agents (Figure 1A). KYSE270 cells were treated with the 55

210

compounds individually at a concentration of 10 µM for 72 h, and the cell viability

211

was determined. As shown in Figure1 B, synephrine, a natural phenolic compound

212

from the leaves of citrus trees and various citrus juices,5 was identified as one of the

213

most effective compounds in inhibiting ESCC cell proliferation, suggesting that this

214

food-source natural produce could be a novel anticancer agent.

215 216

Synephrine reduces ESCC cell proliferation

217

To test the effect of synephrine on proliferation of ESCC cells, KYSE30 and

218

KYSE270 cells were exposed to increasing concentrations of synephrine for up to 5

219

days and cell viability was determined by CCK-8 assay. The results showed that

220

synephrine inhibited proliferation of ESCC cells in a dose-dependent manner (Figure

221

2A). By using colony-formation assay, we found that synephrine treatment

222

significantly repressed the anchorage-dependent growth in both cell lines (Figure 2B).

223

Moreover, soft agar assay was established to evaluate the anchorage-independent

224

growth ability of cells, and a decrease in number of colonies formed was observed in


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the ESCC cells treated with synephrine (Figure 2C). In addition to our results showed

226

that synephrine had no significant cytotoxic effect on immortalized normal

227

esophageal epithelial cells (Figure 2D), these data indicated that synephrine may

228

exert anticancer bioactivity in ESCC cells without side effect.

229 230

Synephrine inhibits migration and invasion in ESCC cells

231

Cell motility is one of the malignant features of ESCC.21 The effects of

232

synephrine on migration of ESCC cells were studied by using chamber migration

233

assay, and the results showed that treatment with synephrine at 5 µM or 10 µM,

234

which had no inhibitory effect on cell proliferation of ESCC cells within 24 h,

235

significantly repressed the cells from migrating into the lower surface of chambers

236

(Figure 3A). Moreover, matrigel-coated chamber invasion assay was performed

237

and we found that synephrine markedly decreased the invasive potential of ESCC

238

cells (Figure 3B). To investigate whether the effect of synephrine on ESCC cell

239

motility was associated with epithelial-mesenchymal transition (EMT), expression

240

levels of representative epithelial (E-cadherin) and mesenchymal (Vimentin)

241

markers were detected by Western blot. The results showed that treatment of

242

synephrine induce the upregulation of E-cadherin and downregulation of Vimentin

243

(Figure 3C). A dose-dependent increase in E-cadherin and decrease in Vimentin

244

expression levels upon synephrine treatment indicated that the inhibitory effects of

245

synephrine on migration and invasion in esophageal cancer cells were due to

246

reversal of EMT.



247 248

Synephrine enhances the sensitivity of ESCC cells to 5-FU

249

Resistance to chemotherapy is one of the reasons for poor prognosis of ESCC.

250

We next examined whether synphrine has synergistic effects with 5-FU, one of the

251

commonly used chemotherapeutic drugs, in ESCC cells. KYSE30 and KYSE270 cells

252

were exposed to low-dose synephrine, low-dose 5-FU alone, or synephrine in

253

combination with 5-FU, and then cell viability and colony-formation assays were

254

performed. The results showed that whereas low-dose synephrine or 5-FU had no or

255

modest anti-proliferation effect, a combination of low-dose synephrine and low-dose

256

5-FU exerted significantly synergistic effect on suppressing cell growth (Figure 4A)

257

and colony formation (Figure 4B). In addition to the Western blot data showing that

258

significantly increased expression level of cleaved caspase-3 was observed in the

259

ESCC cells treated with the combination of 5-FU and synephrine, compared with

260

the cells treated with 5-FU or synephrine alone, our results demonstrated that

261

synephrine can sensitize ESCC cells to 5-FU treatment by inducing apoptosis.

262 263

Quantitative proteomics identifies the deregulation of AKT and ERK

264

pathways induced by synephrine

265

To investigate the molecular mechanisms involved in the anticancer effects of

266

synephrine, proteomic alterations in triplicate samples from the ESCC cells treated

267

with 10 µM synephrine for 48 h were quantified by quantitative proteomics

268

(Figure 5A). A total of 3394 proteins with quantitative information were identified,


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269

among which 2611 proteins were all detected in the triplicate experiments. A

270

statistical test was performed by using the power law global error model (PLGEM)

271

algorithm to analyze protein abundance, with a slope of 0.764 and an adjusted r2 of

272

0.975 (Pearson r = 0.725) (Figure 5B). As shown in Figure 5C, the residuals

273

distributed evenly and were independent from the rank of mean abundances. The

274

quantile-quantile (Q-Q) plot presented that the data had a fitted normal distribution

275

of the residual standard deviations between the modeled and the actual values

276

(Figure 5D). To explore the action mechanisms of synephrine in cancer, the 108

277

differentially expressed proteins (fold change ≥ 1.5) were uploaded to IPA

278

software for characterizing the canonical pathways involved in the anticancer

279

effects of synephrine. As shown in Figure 5E, AKT and ERK signaling pathways,

280

which have important roles in proliferation, survival and motility of cancer cells,

281

were most likely the potential pathways responsible for the action mechanisms of

282

synephrine in ESCC.

283 284

Synephrine inhibits AKT and ERK pathways through downregulation of

285

Galectin-3

286

According to the proteomic data above, we proposed that synphrine might

287

regulate AKT and ERK signaling. Expression levels of p-AKT, AKT, p-ERK and

288

ERK in synphrine-treated ESCC cells were detected by Western blot, and the

289

results showed upregulation of p-AKT and p-ERK expressions in a dose-dependent

290

manner (Figure 6A). As shown in Figure 5E, a cluster of synephrine-regulated



291

proteins, which suggested the involvement of AKT and ERK pathways, strongly

292

pointed to a hub protein, Galectin-3, an upstream activator of AKT and ERK

293

signaling. The data from Western blot and qRT-PCR confirmed our hypothesis that

294

synephrine reduced expression of Galectin-3 (Figure 6A and B), which inhibited

295

AKT and ERK pathways to exert anticancer effects in ESCC cells.

296 297

Synephrine suppresses growth of human ESCC xenografts in nude mice

298

without side effect

299

Nude mice bearing human ESCC tumor xenografts were used to evaluate the

300

therapeutic potential of synephrine in vivo. As shown in Figure 7A, oral

301

administration of synephrine (10 mg/kg and 20 mg/kg) caused a significant

302

dose-dependent suppression of tumor volume, with decreases of 58.2% and 71.6%

303

in the group receiving 10 mg/kg and 20 mg/kg, respectively, as compared with the

304

vehicle-treated mice. Western blot analysis of tumor xenografts showed that same

305

with the in vitro data (Figure 6A), marked downregulation of Galectin-3 and

306

inactivation of AKT and ERK pathways, as indicated by the decreased expression

307

levels of p-AKT and p-ERK, were observed in synephrine-treated tumors compared

308

with those in the control group (Figure 7B). There was no significant difference

309

between the treated and control groups in terms of morphology of the vital organs,

310

including lungs, liver and kidneys (Figure 7C). Moreover, the data showed that

311

synephrine treatment did not exert any overt change in serum alanine transaminase

312

(ALT) and aspartate transaminase (AST) levels of nude mice (Figure 7D), suggesting


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313

that synephrine had no toxic effect on animals. Collectively, these results

314

demonstrated the treatment efficiency and safety of synephrine as an anticancer

315

agent.

316 317



318

Discussion

319

Despite the advancement in chemotherapy in recent years, the treatment

320

outcome is always hampered by adverse side effects, such as nephrotoxicity, nausea,

321

hair loss, skin irritation, anemia, infertility, and presence of treatment-resistant

322

cancer cells.22 Therefore, there is an urgent need to invent novel nontoxic

323

therapeutic strategy that have anticancer effects on ESCC cells or sensitize them to

324

chemotherapeutic drugs. The naturally occurring compound present in human diets

325

is a good choice, especially due to absence of toxicity and low price. Over 60% of

326

the current anticancer drugs were derived directly from natural products, or

327

developed from the unique molecules through chemical modification, such as the

328

drugs commonly used in clinic

329

doxorubicin, and docetaxel. In addition, many natural products, such as

330

resveratrol,23-25 epigallocatechin-3-gallate (EGCG),26 curcumin,27-29 vitamin C,30, 31

331

ginger,32 mushroom dietary fiber,33, 34 have been documented to exert antioxidant,

332

anti-inflammatory, and antitumor activities. Screening of the natural products with

333

excellent treatment efficiency is a time- and cost-consuming, but meaningful task.

334

In this study, we combined literature study with cell viability assay to screen the

335

food-source compounds with anticancer property from a compound library

336

consisting of 429 natural products, which will benefit the identification of novel

337

therapeutic option without side effect for cancer treatment and warrants further

338

investigation.

339

including cyclophosphamide, paclitaxel,

Here, we provided the first evidence that synephrine can inhibit tumorigenicity.


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340

Although synephrine was found to inhibit VEGF-induced angiogenesis and

341

stimulate glucose uptake through activation of AMPK signaling pathway,35,

342

according to our knowledge, the bioactivity of synephrine in cancer has not been

343

reported. A recent review proposed that synephrine shares the structure similar with

344

catecholamines epinephrine, both of which contain hydroxyl groups at both meta

345

and para positions of the benzene ring. The author also suggested that it is the

346

hydroxyl group in the meta position of the ring that primarily promotes adrenergic

347

receptor binding and the subsequent effects.37 In another study, Kim and colleagues

348

found that R-(–)-β-O-methylsynephrine (OMe-Syn), a compound sharing the

349

similar structure with synephrine, was able to inhibit VEGF-induced angiogenesis

350

in vitro and in vivo.35 In present study, our results demonstrated that synephrine

351

significantly inhibited cell proliferation and colony formation in vitro (Figure 2),

352

and suppressed growth of ESCC tumor xenografts in vivo (Figure 7). Moreover,

353

synephrine did not exert observed toxic effect on normal esophageal epithelial cells or

354

the vital organs of animals. Metastasis is the leading reason for poor prognosis of

355

cancer patients, and enhanced migration, invasion and EMT are phenotypes

356

associated with metastasis. EMT is characterized by loss of epithelial cell markers

357

such as E-cadherin, and increased expression of mesenchymal markers, such as

358

N-cadherin, vimentin and fibronectin.38 Our data indicated that treatment with

359

synephrine reduced the abilities of ESCC cells to migrate and invade, accompanied

360

with EMT reversal, indicated by increased E-cadherin and decreased vimentin

361

expression (Figure 3). Like other cancer types, resistance of tumor cells to


36


362

chemotherapy leads to treatment failure of ESCC. In addition to testing the effects

363

of synephrine as single agent in cancer treatment, we also examined the efficiency

364

of the combination with 5-FU in ESCC cells. Our results showed that synephrine

365

may potentiate the anticancer effect of 5-FU, thus supporting the implication of

366

synephrine in postoperative adjuvant therapy.

367

The action mechanism of synephrine in cancer cells was uncovered in present

368

study. With rapid development of mass spectrometry and bioinformatics analysis,

369

quantitative proteomics has been commonly used to dissect the molecular

370

mechanisms of anticancer drugs more accurately and systematically. IPA analysis of

371

the differentially expressed proteins in synephrine-treated ESCC cells suggested the

372

important role of AKT and ERK pathways in the bioactivity of synephrine, which

373

was confirmed by our Western blot data (Figures 5 and 6). Further analysis

374

indicated that Galectin-3, a well-known oncogene which promotes cancer

375

progression, may mediate the anticancer effects of synephrine in ESCC. Since the

376

significance of AKT and ERK signaling and their potential as therapeutic targets for

377

cancer treatment have been well documented in many types of human cancer, our

378

findings that synephrine inactivated these pathways to exert anticancer effects in

379

vitro and in vivo highlighted the therapeutic potential of synephrine in ESCC and

380

other cancer types. Besides PI3K/AKT and ERK signaling, other pathways may be

381

involved in the action mechanisms of synephrine. In the past decades, the

382

relationship between obesity and cancer has gained extensive attention.39 For

383

example, a meta-analysis of 221 datasets (282,137 incident cases) suggested a


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384

strong association between excess body-mass index (BMI) and various cancer

385

incidences including esophageal cancer.40 Since synephrine was originally

386

described as a potential product for treatment of overweight and obesity,41 and the

387

emerging evidence indicated that AMPK not only regulates fatty acid metabolism

388

and obesity,42 but also is a possible metabolic tumor suppressor and target for

389

cancer prevention and treatment,43 whether AMPK plays an important role in the

390

anticancer bioactivity of synephrine in cancer cells warrants further investigation.

391

Taken together, we demonstrate for the first time that synephrine is highly

392

effective in suppressing growth and motility of cancer cells, and sensitizing ESCC

393

cells to 5-FU treatment. More importantly, synephrine was found to exhibit strong

394

antitumor activity in mice without obvious side effect. These data shed light on the

395

mechanism of antitumor action of synephrine and suggest the potential implication

396

of synephrine in the management of ESCC.

397 398



399

Acknowledgement

400

This work was supported by National Natural Science Foundation of China (Project

401

81672953, 81773085), Guangdong Innovative and Entrepreneurial Research Team

402

Program (2013Y113), Guangzhou Science and Technology Project (201707010260),

403

and the Fundamental Research Funds for the Central Universities (21616322,

404

21617434).

405 406

Author contribution

407

B.L., W.W.X., and H.X.L. designed the research and wrote the manuscript; W.W.X.,

408

C.C.Z., Y.N.H., and J.Y.R. performed the experiments and contributed to the data

409

interpretation; W.Y.C., Q.S.Y., Y.M.W., and Q.Y.H. revised the manuscript for

410

important intellectual content and provided technical and/or material support.

411 412

Conflict of interest statement

413

The authors declare no conflict of interest.

414


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578

Figure Legends

579

Figure 1. Identification of synephrine as a novel cancer therapeutic agent. (A)

580

Diagram showing the procedure to screen natural products with anticancer activity

581

from a food-source compound library. (B) KSYE270 cells were treated with the 55

582

compounds (10 µM) individually for 72 h and cell viability was determined. (C)

583

Chemical structure of synephrine, a compound isolated from leaves of citrus trees.

584 585

Figure 2. Effects of synephrine on ESCC cell proliferation. (A) KYSE30 and

586

KYSE270 cells were exposed to indicated concentrations of synephrine for up to 5 d,

587

and the cell viability was determined by CCK-8 assay. (B, C) Colony-formation and

588

soft agar assays showed that synephrine inhibited both anchorage-dependent (B) and

589

anchorage-independent growth (C) of ESCC cells in a dose-dependent manner. (D)

590

Synephrine did not exert toxic effect on normal esophageal epithelial cells SHEE.

591

Bars, SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with control cells.

592 593

Figure 3. Effects of Synephrine on ESCC cell motility. (A, B) Treatment with

594

indicated concentrations of synephrine for 24 h inhibited the migration (A) and

595

invasion (B) potential of KYSE30 and KYSE270 cells, as determined by chamber

596

migration and invasion assay. (C) Western blot analysis of protein expressions of

597

E-cadherin and vimentin in KYSE30 and KYSE270 cells treated with synephrine

598

for 24 h. Bars, SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with control

599

cells.



Page 32 of 41

600 601

Figure 4. Effects of synephrine on 5-FU chemosensitivity of ESCC cells. (A)

602

KYSE30 and KYSE270 cells were treated with synephrine (5 µM), 5-FU (2.5 µM)

603

alone, or the combination for the indicated time points, and cell viability was

604

measured by CCK-8 assay. (B) Colony formation assay showed that exposure to

605

synephrine enhanced the sensitivity of ESCC cells to chemotherapeutic drug 5-FU. (C)

606

ESCC cells were treated with synephrine, 5-FU alone, or the combination for 72 h,

607

and Western blot was performed to detect the expression levels of caspase-3 and

608

cleaved caspase-3. Bars, SD; **, P < 0.01; ***, P < 0.001 compared with control

609

cells.

610 611

Figure 5. Identification of synephrine-regulated proteins and signaling pathways by

612

quantitative proteomics and bioinformatics analysis. (A) Experimental scheme of the

613

identification of synephrine-regulated proteins. (B) PLGEM model using a

614

regression fitting analysis with a contour plot; black circles showed a good fitting

615

of the data by the PLGEM model. (C) Residual distribution along with the rank of

616

mean abundances. (D) A Q-Q plot was used to detect a normal distribution of the

617

residual standard deviations between the modeled and the determined data.

618

Ingenuity Pathway Analysis (IPA) suggested dysregulation of AKT and ERK

619

signaling pathways in synephrine-treated ESCC cells.

(E)

620 621

Figure 6. Synephrine downregulates Galectin-3 to inactivate AKT and ERK


Page 33 of 41


622

signaling in ESCC cells. (A, B) KYSE30 and KYSE270 cells were treated with

623

indicated concentrations of synephrine for 48 h. Cell lysates were collected for

624

Western blot analysis of p-AKT, AKT, p-ERK, ERK, Galectin-3, and actin (A), and

625

qRT-PCR was performed to compare Galectin-3 expression in the synephrine-treated

626

cells and control cells (B). Bars, SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001

627

compared with control cells.

628 629

Figure 7. Effects of synephrine on growth of human ESCC tumor xenografts in

630

nude mice. Nude mice bearing KYSE270-derived xenografts were orally

631

administrated with synephrine (10 mg/kg or 20 mg/kg) or vehicle thrice a week (n =

632

6 per group). (A) Tumor curves showed that synephrine significantly suppressed

633

growth of tumor xenografts. (B) Western blot analysis of p-AKT, AKT, p-ERK,

634

ERK and Galectin-3 expressions in synephrine- and vehicle-treated tumors. (C)

635

Hematoxylin and eosin (H&E) staining of lung, liver and kidney specimens

636

collected from mice of the treatment and control groups. (D) Comparison of serum

637

ALT and AST level between synephrine-treated and control groups. Bars, SD; **, P

638

< 0.01; ***, P < 0.001 compared with control group.




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Synephrine Hydrochloride Suppresses Esophageal Cancer Tumor

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