Repression of Human Hepatocellular Carcinoma Growth by

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Repression of human hepatocellular carcinoma growth by regulating Met/ EGFR/VEGFR-Akt/NF-#B pathways with theanine and its derivative DTBrC Guoying Zhang, Zheng Li, Xiaochun Wan, Ying Zhang, Rongqin Zhu, Zhenzhen Liu, Dexin Ji, Huarong Zhang, Fei Wu, Huihui Tian, Kun Liu, and Benhao Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02509 • Publication Date (Web): 28 Aug 2016 Downloaded from http://pubs.acs.org on August 30, 2016

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Page 1 of 43

Journal of Agricultural and Food Chemistry 1

Repression

of

human

hepatocellular

carcinoma

growth

by regulating

Met/EGFR/VEGFR-Akt/NF-κB pathways with theanine and its derivative DTBrC

Guoying Zhang†*, Zheng Li†, Xiaochun Wan‡*, Ying Zhang§, Rongqin Zhu†, Zhenzhen Liu†, Dexin Ji†, Huarong Zhang†, Fei Wu†, Huihui Tian†, Kun Liu†, Benhao Wu§ †

School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, 264005, P.R. China; ‡

Key Laboratory of Tea Biochemistry & Biotechnology, Ministry of Agriculture, Anhui

Agricultural University, Hefei, Anhui Province 230036, China;

§

Shandong Yingdong Yinghao Biotechnology Inc., No.101, Hangtianlu, Gaoxinqu,

Yantai, Shandong Province 264670, China Running title: Theanine and DTBrC inhibit human hepatocellular carcinoma growth *Address correspondence to these authors: Guoying Zhang and Xiaochun Wan *Guoying Zhang’s Email: [email protected]; Tel & Fax: +86-535-6884952 *Xiaochun Wan’s email: [email protected]

Disclosure of Potential Conflicts of Interest: The authors declare no potential conflicts of interest.

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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1

ABSTRACT

2

To explore the potential of theanine against cancer, we have studied the anticancer

3

activities

4

(R)-2-(6,8-dibromo-2-oxo-2H-chromene-3-carboxamido)-5-(ethyl-amino)-5–oxo-pent

5

anoic ethyl ester (DTBrC), in vitro, ex vivo, and in vivo models of human

6

hepatocellular carcinoma (HHC). Theanine and DTBrC displayed inhibitory effects on

7

the growth and migration of HHC cells in vitro, ex vivo, and in vivo. Theanine and

8

DTBrC significantly enhanced the repression of HHC cell growth in combination with

9

anticancer drug pirarubicin. Theanine and DTBrC completely suppressed HGF- and

10

EGF+HGF- induced migration with reduction of p53 tumor suppressor level and

11

enhanced the p53 protein expression in HHC cells. The Akt and NF-κB knockdown

12

greatly reduced the cancer cell migration with decrease in CD44 expression. DTBrC

13

and theanine significantly repressed the protein expressions in the Met/EGFR/

14

VEGFR-Akt/NF-κB pathways, which might be the mechanism for their biologic

15

effects.

16

Keywords: Theanine; DTBrC; Human hepatocellular carcinoma; Inhibition; Signaling

17

pathways

18

INTRODUCTION

19

Human hepatocellular carcinoma (HHC) is one of the most common cancers in the

20

world.1,

21

chances for cure in only small subsets of HCC patients in the early stage.

22

Nevertheless, a large majority of patients with advanced stage of HCC depend on

2

of

theanine

from

tea

and

its

semi-synthesized

derivative,

Surgical resection, local treatment, and liver transplantation may offer

2


Page 3 of 43


23

chemotherapy to control metastasis and recurrence. Unfortunately, HCC is inherently

24

resistant to chemotherapeutic agents, resulting in a dismal prognosis for HCC

25

patients. Metastasis and recurrence are the main causes of mortality in many HCC

26

patients. During the complicated process of metastasis, abnormal proliferation and

27

migration of cancer cells are the most important steps. The major mechanisms that

28

block the efficacy of chemotherapy, resulting in failure to inhibit metastasis and

29

recurrence in HCC include the resistance to apoptosis and defects of repressing

30

proliferation

31

Met/EGFR/VEGFR-Akt-nuclear factor-kappaB (NF-κB) signaling network in HHC.3-7

32

Therefore, it is necessary to explore novel drugs and/or food additives capable of

33

overcoming

34

EGFR/VEGFR-Akt-NF-κB pathways.

and/or

migration,

chemotherapeutic

which

resistance

are

of

due

HCC

to

cells

the

by

activation

targeting

of

Met/

35 36

Theanine (γ–glutamylethylamide) is a characteristic amino acid found in tea that can

37

be isolated from green tea and black tea, or synthesized by some chemical or

38

biochemical methods.8 Theanine has been widely used for many years as a safe food

39

additive without limitation to its dose. Some experimental results displayed the

40

anticancer activities of theanine9-12 Our previous study indicated that theanine and the

41

sera from the rats treated with theanine suppressed the invasion of rat hepatoma cells

42

in vitro and ex vivo and the rat hepatoma growth in vivo. 11-12 To explore the potential

43

of theanine against cancer and develop more effective and lower toxic compounds to

44

inhibit HHC progression, we have synthesized a novel theanine derivative, (R)-

3



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45

2-(6,8-dibromo-2-oxo-2H-chromene-3-carboxamido)- 5-(ethylamino)-5-oxo-pentanoic

46

ethyl ester (DTBrC) based on the structure of theanine and investigated effects of

47

theanine and DTBrC, a small molecule fluorescent compound, on cancer cell

48

migration,

49

receptors-mediated Akt-NF-κB signaling pathways in HHC cells.

50

MATERIALS AND METHODS

51

Chemicals and antibodies

52

The primary antibodies to Bcl-2, Bax, caspase-3, PARP-1, cytochrome c, cyclic D1,

53

Akt, phosphor (p)Akt (Ser473), NF-κB (p-65), pNF-κB (p-65) (Ser536), Met, pMet

54

(Tyr1234/1235), EGFR, pEGFR (Tyr1068), VEGFR2, pVEGFR2 (Tyr1175), p53,CD44,

55

β-tubulin, GAPDH, EGF, and HGF were purchased from Cell Signaling Technology

56

Inc. (Beverley, MA, USA). The primary antibody to β-actin was purchased from Santa

57

Cruz Technology Inc. (Dallas, Texas, USA). Annexin V-FITC/PI Apoptosis Detection

58

Kit and Z-VAD-FMK (Z) were purchased from Beyotime, Haimen, China. Fibronectin

59

and Boyden chambers were obtained from BD Inc. (BD Biosciences, San Jose, CA,

60

USA）and Corning Inc. (Corning, NY, USA), respectively. Pirarubicin was purchased

61

from Yuhuangding Hospitai, Yantai, China. DMEM, RPMI 1640 medium, penicillin,

62

streptomycin, fetal

63

3-[4,5-Dimethylthiazol-2-yl]-

64

Ly294002 (Ly), Bay 11-7082 (Bay), L-theanine, and all other chemicals including

65

materials for the synthesis of DBrC and DTBrC were acquired from Sigma Chemical

66

Co. (St. Louis, MO, USA).

growth,

apoptosis,

bovine

and

serum

tumor

(FBS),

growth

as

well

trypsin/EDTA,

2,5-diphenyltetrazolium

bromide

as

the

related

propidium

iodide,

(MTT),

DMSO,

4


Page 5 of 43


67

Preparation of theanine derivative DTBrC

68

The scheme of synthesis of the theanine derivative, (R)- 2-(6,8-dibromo-

69

2-oxo-2H-chromene- 3- carboxamido)-5-(ethylamino)-5-oxo-pentanoic ethyl ester

70

(DTBrC) was shown in Fig. 1A. The procedure of DTBrC synthesis was conducted as

71

follows.

72

General

73

All the material we used were purchased from commercial suppliers and used without

74

further purification, unless otherwise noted. Solvents were distilled prior to use and

75

flash chromatography was performed using silica gel (200-300 mesh). 6,

76

8-dibromo-coumarin-3-carboxylic acid (3) was synthesized according to the literature

77

method.13 Reactions were routinely monitored by thin-layer chromatography on 0.25

78

mm silica gel plates (60 GF-254) and visualized with ultraviolet lamp (254 nm) or

79

Ninhydrin Spray (0.5% in butanol). Melting points were determined on an

80

electrothermal melting point apparatus, and the thermometer was uncorrected. 1H,

81

13c

NMR spectra were determined on a Bruker Avance III spectrometer using TMS as

82

an internal standard in DMSO-d6 or CDCl3 solutions, operating at a frequency of 500

83

MHz for proton and 125 MHz for carbon. Peak positions are given in parts per million

84

(δ) from tetramethylsilane, and coupling constant value (J) are given in Hz. ESI-MS

85

were determined on an API 4000 spectrometer. All chemicals were purchased from

86

Sigma Chemicals Ltd.

87

General procedure for the synthesis of ethyl L-theanine (2)

88

SOCl2 (0.055 ml, 0.76 mmol) was added slowly into a solution of L-Theanine （1）

5



Page 6 of 43 6

89

(87 mg, 0.5 mmol) in absolute methanol or ethanol (1 ml). The reaction mixture

90

was stirred at room temperature for about 2 hours, and the end of the reaction was

91

observed by TLC. The resulting mixture was concentrated under reduced pressure to

92

give crude product of (S)-ethyl 2-amino-5- (ethylamino)-5-oxopentanoate (2). The

93

crude product was used in the next step without further purification.

94

General procedure for the synthesis of theanine derivative DTBrC (4)

95

The 6, 8-dibromo-coumarin-3-carboxylic acid (3 / DBrC) (1 mmol) was added to a

96

solution of the crude L-theanine ester obtained before (ca. 0.5 mmol) in dry CH2Cl2

97

(20 ml). DIPEA (0.61 mmol) and EDCI (0.2 mol) were added to the mixture

98

subsequently. The reaction mixture was stirred for about 1 hour at room temperature,

99

and the end of the reaction was observed by TLC. Water (20 ml) was added. After a

100

few minutes stirring, the resulting mixture was extracted with EtOAc (3×10 ml). The

101

organic phase was washed with brine (10 ml), dried over anhydrous MgSO4, filtered

102

and evaporated. The residue was purified by flash chromatography (16.7%

103

acetone/n-hexane as eluant) on silica gel. The product was recrystallized from a

104

mixture of acetone and n-hexane, affording the theanine derivative, (R)-2-(6,

105

8-dibromo-2-oxo-2H-benzopyran-3-carboxamido)-5-(ethylamino)-5-oxo-pentanoic

106

ethyl ester (4 / DTBrC). A light yellow solid (Yield = 75.8%). m.p.: 134-136℃. 1H-NMR

107

δ: 1.12 (t, J = 7.3 Hz, 3H, NHCH2CH3), 1.28 (t, J = 7.2 Hz, 3H, OCH2CH3), 2.17-2.28

108

(m, 4H, CH2CH2), 3.22-3.29 (m, 2H, NHCH2CH3), 4.18-4.26 (m, 3H),

109

NH), 7.38 (d, J = 2.3 Hz, 1H, coumarin-5H), 7.71 (d, J = 2.3 Hz, 1H, coumarin-7H),

110

8.32 (s, coumarin-4H), 14.12 (br s, 1H, NH); 12.95 (s, 1H, NH).

13

5.75 (br s, 1H,

C-NMR (100MHz,

6


Page 7 of 43


111

CDCl3) δ: 14.07 (CH3), 14.71 (CH3), 28.99 (CH2), 31.65 (CH2), 34.34 (CH2), 61.66

112

(CH), 69.16 (CH2), 109.80 (C), 112.09 (CH), 119.82 (C), 133.14 (CH), 137.96 (CH),

113

157.48 (C), 165.63 (C), 170.20 (C), 171.04 (C). ESI-MS (m/z): 528.5 [M-H]-.

114

Detection of in vitro and in vivo fluorescent signals

115

DTBrC (80 mg/kg, i.p.) or DMSO as a control was injected into female C57/BL6 mice.

116

After 3 h, the fluorescent imaging in vivo and in vitro (DTBrC at 8 mg/ml in a tube) was

117

recorded under 530 nanometer excitation and 600 nanometer emission. The images

118

were obtained on a Kodak Image Station 4000 Multi-Modal Imaging System

119

(IS4000MM) equipped with an X-ray unit and on a Kodak Image Station 2000

120

(Carestream Health, Rochester, NY, USA.).

121

Preparation of sera from theanine-, DBrC- or DTBrC-fed rats

122

The rat sera were prepared according to our published methods with slight

123

modifications.11, 14, 15 Briefly, seven female SD rats in each treatment (age range, 6

124

week; from Tumor Research Institute, Chinese Academy of Sciences, Beijing, China)

125

were treated in accordance with guidelines established by the Animal Care and Use

126

Committee at Yantai University. DTBrC, DBrC or theanine were orally administered to

127

the rats at a dose of 80 mg/ml/kg body weight. The blood was then collected at 0, 0.5,

128

1, 3, 5, and 8 h from the rats (fasted for 16 h) after oral administration of DTBrC, DBrC

129

or theanine. The collected rat blood was left to clot for 3 h at room temperature and

130

centrifuged twice at 2500x g at 4ºC for 35 minutes. The sera were sterilized by

131

filtration and then heated at 56 ºC for 35 minutes. The prepared rat sera were

132

allocated, and stored at -80°C for ex vivo growth assay.

7



Page 8 of 43 8

133

Cell culture and MTT cell viability assays

134

The cell lines of HHC SMMC7721 and HepG2 were purchased from the Chinese

135

Academy of Sciences and the American Type Culture Collection, respectively. The

136

cell lines of SMMC7721 and HepG2 were maintained in DMEM and RPMI 1640

137

medium, respectively, containing 10% heat-inactivated fetal bovine serum (FBS),

138

glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 µg/ml). The cells were

139

incubated at 37℃ in a humidified incubator with 95% air/5% CO2 atmosphere. The

140

rate of cell growth (viability) was determined using MTT assay according to our

141

published methods with slight modification.14-16 Cells were seeded in 96-well plates

142

(Becton Dickinson, NJ, U.S.A.) at 2x103 per well and incubated overnight. The cells in

143

control group were treated with vehicle [0.1% of DMSO (v/v)].The cells were

144

incubated in DMEM or RPMI 1640 medium supplemented with 10% FBS containing

145

different concentrations of theanine, DBrC or DTBrC (16 to 250 µM), or in

146

combination of existing anticancer drug pirarubicin (12.5 to 25 nM), or 10% rat sera

147

(in the case of ex vivo assay) obtained at different time points after theanine, DBrC or

148

DTBrC were orally intubated the rats. After 48h and/or 72 h treatment, the

149

absorbance (A) values in each test group were measured using MTT assay.

150

Absorbance was measured at a wavelength of 570 nm and a reference wavelength of

151

630 nm using a Synergy H4 Hybrid Multi-Mode Microplate Reader (Bio-Tek,

152

Instruments, Inc., Winooski, VT, USA). The relative cell growth (viability) was

153

calculated based on the absorbance of treatment (AT) group vs. the absorbance of

8


Page 9 of 43


154

control (AC) group (vehicle). The percentage of relative cell viability (growth) was

155

calculated using the following formula:

156

Relative cell growth (viability) (%) = AT / AC x 100%.

157

Each experiment was performed in triplicate and repeated at least three times.

158

Migration assay

159

Tumor cell migration was

160

fibronectin-coated polycarbonate filters, using modified transwell chambers according

161

to our previous methods with slight modification.14-17 In brief, cells (5 x 104) were

162

added onto the upper chamber in 220 µL of serum-free medium containing different

163

concentrations of theanine or DTBrC (1 to 16 µM); the lower compartment was filled

164

with 0.67 ml of DMEM media supplemented with 10% of FBS, HGF (40 ng/ml), EGF

165

(40 ng/ml), or EGF+HGF (40 ng/ml) (as a chemoattractant). The cells in control group

166

were treated with vehicle [DMSO at 0.05% (v/v)]. After incubation for 6 h at 37°C, the

167

cells that migrated to the lower surface of the filter were fixed and stained with

168

propidium iodide. The migrated cells were counted and recorded for images under a

169

fluorescent microscope (Nikon，TE2000-U, Japan). Experiments were conducted in

170

triplicate.

171

Detection of cell apoptosis

172

The cell apoptosis was detected following the instructions from the manufacturer of

173

Annexin V-FITC/PI Apoptosis Detection Kit.19 In brief, cells were treated for 48 h with

174

theanine or DTBrC (16 to 250 µM)，Ly294002 (16 µM), Bay (3.2 µM) or in combination

examined by detecting cell

migration through

9



Page 10 of 43 10

175

of DTBrC with Z, Z-VAD-FMK (12.5 µM). The cells in control group were treated with

176

DMSO vehicle [0.05% (v/v)]. The treated cells were stained with Annexin V-FITC and

177

propidium iodide (PI). The apoptotic cell fraction was measured using the Annexin V-

178

FITC/PI Apoptosis Detection Kit. The apoptotic ratio was detected by flow cytometry

179

(Becton Dickinson FACS Vantage SE, San Jose, CA, USA).

180

Western blotting

181

Western immunoblotting experiments were performed as described previously with

182

some modifications. 14, 15, 18 In brief, cells were treated with different concentrations of

183

theanine or DTBrC (16 to 250 µM), Ly294002 (Ly, 16 µM), or Bay (3.2 µM). The cells

184

in control group were treated with vehicle [DMSO at 0.05% (v/v)]. The treated cells

185

were harvested either at 45 minutes for detection of phosphorylation ratio of pMet/Met,

186

pEGFR/EGFR, pVEGFR2/VEGFR2, pAkt/Akt, and pNF-κB (p65)/NF-κB (p65), or at

187

48 h for detection of the protein expressions. Then the cells were treated with HGF

188

(40 ng/ml) and EGF (40 ng/ml) plus HGF (40 ng/ml) for 1 h, respectively. The cells

189

were harvested and washed with PBS. The whole cell lysates and cytosolic fractions

190

were prepared according to the instructions of the manufacturer (Beyotime, Haimen,

191

China). The protein concentration was measured using the Bradford method. Equal

192

amounts of the cell lysates were resolved by electrophoresis in SDS-PAGE and

193

probed with the primary antibodies to the detected proteins mentioned above,

194

respectively.

195

as a loading control. Chemiluminescence was achieved using the ECL Wester

196

Blotting Detection Reagents (GE Healthcare Life Sciences, Piscataway, NJ USA).

Anti-β-actin antibody, Anti-GAPDH, or anti-β-tubulin antibody was used

10


Page 11 of 43


197

Transfection of Akt and NF-κB p65 short hairpin (sh)RNA

198

SMMC7721 cells were transfected with Akt shRNA plasmid and NF-κB p65 shRNA

199

plasmid (Santa Cruz Technology Inc., Shanghai, China), respectively using

200

Lipofectamine as per the manufacturer’s recommendations. Scrambled non targeting

201

shRNA（scAkt）and shRNA (scNF-κB p65) were used as the control, respectively.

202

Thirty-six hours after transfection, cells were subjected to the in vitro migration assay

203

and Western blot analysis mentioned-above.

204

Subcutaneous tumor model

205

Female BALB/c nu/nu mice (age range, 6 weeks) were purchased from Tumor

206

Research Institute, Chinese Academy of Sciences, Beijing, China). The animal

207

protocol was approved by the Animal Care and Use Committee (ACUC) at Yantai

208

University, China. SMMC7721 cells (3 × 106 in 0.1 ml Hank's balanced salt solution)

209

were subcutaneously injected into rear right flank of each BALB/c nu/nu mouse.

210

Twelve days later, the HHC tumor model was established. The tumor-bearing nude

211

mice were randomly divided into three groups (six mice each group): (1) control group

212

(vehicle); (2) theanine group (80 mg/kg/day); (3) DTBrC group (80 mg/kg/day). The

213

mice were administrated with vehicle, theanine and DTBrC by intraperitoneal injection

214

once every day. The therapy was performed for 15 days. Tumor volumes were

215

measured every two days.19, 20 On the 15th day, mice in all groups were killed and

216

tumors were weighed. Results were plotted as relative tumor growth (volume) and

217

weight. The relative tumor weight and growth (volume) are relative to that of the

218

control (vehicle) designated as 100. 19, 20

11



Page 12 of 43 12

219

Statistical analysis

220

The results of cell growth, migration and protein levels as well as tumor volumes and

221

weights are expressed as mean ± standard deviation (S.D.). All experiments were

222

performed three times and the data were analyzed by the SPSS 16.0 software to

223

assess the statistical difference. Statistical analysis was performed by the ANOVA and

224

Bonferroni post-test, or simple linear regression. Statistical significance was assumed

225

at a P value of less than 0.05.

226

RESULTS

227

DTBrC showed greater inhibitory activity, compared to theanine on HHC cell

228

migration and growth

229

In this study, we synthesized a novel small molecule fluorescent compound, (R) - 2-

230

(6, 8 - dibromo -2- oxo- 2H-benzopyran - 3-carboxamido) -5- (ethylamino)- 5- oxo-

231

pentanoic ethyl ester (short for DTBrC), to improve the ability of the parental

232

compound theanine to inhibit the migration and growth of cancer cells. The scheme of

233

DTBrC synthesis and chemical structure are shown in Fig. 1A. DTBrC shows the very

234

strong in vitro and in vivo fluorescence characteristics as shown in Fig. 1B. In

235

previous studies, including our own, theanine displayed some anticancer activities.9-12

236

Because the high water solubility of theanine and the structure of 6, 8-substituted

237

dibromo-coumarin-3-carboxylic acid (DBrC) could limit the antitumor activity in vitro

238

and in vivo, we synthesized the theanine derivative, DTBrC by esterification of the

239

carboxyl group of theanine with ethanol and acylation of the amino group of theanine

240

with DBrC. We hypothesized and confirmed that the semi-synthesized DTBrC would

12


Page 13 of 43


241

greatly enhance the anticancer activity in vitro, ex vivo, and in vivo.

242 243

We first compared the in vitro activity of theanine, DBrC, and DTBrC against cancer

244

cell migration. Theanine and DTBrC (1 to 16 µM) suppressed the migration of HHC

245

SMMC7721 and HepG2 cell lines in a dose-dependent manner (Fig. 1C, 1D),

246

although DBrC did not show any significant inhibition of the migration of both HHC

247

cell lines at the same concentrations (data not shown). The ratios of cancer cell

248

migration were reduced by 26.3% and 48.6% in SMMC7721 cells, and by 22.2% and

249

54.8% in HepG2 cells, respectively, in response to the treatment of theanine and

250

DTBrC at 16 µM, although 24 h treatment with theanine and DTBrC at the same

251

concentration did not significantly repress the growth of both SMMC7721 and HepG2

252

cell lines (data not shown). DTBrC displayed much stronger inhibition than theanine

253

on the migration of SMMC7721 and HepG2 cells.

254 255

We next tested the in vitro effects of theanine, DBrC, and DTBrC on HHC cell growth.

256

The result indicated that 48 h and 72 h treatment with DTBrC significantly suppressed

257

the growth of SMMC7721 and HepG2 cell lines in dose- and time-dependent

258

manners. While the HHC cells were treated with DTBrC (16 to 250 µM) for 48 and 72

259

h, the IC50 values (72 h) were 54 µM and 105 µM for SMMC7721 and HepG2 cells,

260

respectively (Fig 2A, 2B). In contrast, both theanine and DBrC had no IC50 values

261

even at 250 µM. DTBrC showed more than 5-fold higher inhibition of the SMMC7721

262

and HepG2 cell growth compared to theanine and DBrC (Fig. 2A, 2B). In addition,

13



Page 14 of 43 14

263

theanine and DTBrC did not display any growth inhibition of normal human peripheral

264

blood lymphocytes (PBL) but DBrC significantly repressed the PBL growth at the

265

concentration of 250 µM (data not shown).

266 267

Then, we evaluated the ex vivo effects of theanine, DBrC, and DTBrC on the growth

268

of SMMC7721 and HepG2 cells. The ex vivo assay indicated that DTBrC exhibited its

269

significant inhibition of SMMC7721 and HepG2 cell growth for 8 h and the inhibitory

270

peak of the HHC cell growth at 5 h after its oral administration in rats. In contrast,

271

theanine and DBrC showed the suppression of SMMC7721 and HepG2 cell growth

272

for only 1 h and 3 h, respectively, and the inhibitory peak of the HHC cell growth at 1 h

273

( for theanine) and 3 h (for DBrC) after their oral administration in rats. DTBrC

274

displayed much stronger inhibitory effects than theanine and DBrC against the growth

275

of the HHC cell lines ex vivo (Fig. 2C, 2D). Importantly, theanine and DTBrC

276

significantly enhanced the repression of SMMC7721 and HepG2 cell growth in

277

combination with anticancer drug pirarubicin (Fig. 2E, 2F) although DBrC did not

278

show any enhancement (data not shown). DTBrC displayed much stronger

279

enhancement with pirarubicin than theanine on the growth inhibition of the HHC cell

280

lines (Fig. 2E, 2F).

281 282

The in vitro and ex vivo results above have shown that theanine and DTBrC have the

283

similar effects on the growth and migration in both HHC cell lines of SMMC7721 and

284

HepG2. In addition, the preliminary experiments displayed the SMMC7721 tumor

14


Page 15 of 43


285

growth speed in nude mice much faster than HepG2. Based on these results, we

286

focused on uncovering the mechanisms of action of theanine and DTBrC against

287

SMMC7721 cells. We analyzed their effects on SMMC7721 cell apoptosis using

288

FACS by Annexin V-FITC/PI double-staining assay. The results indicated that

289

theanine and DTBrC (16 to 250 µM) displayed evident induction of apoptosis in

290

SMMC7721 cells after the cells were treated for 48 h. In response to the treatment of

291

theanine and DTBrC at 250 µM, the ratios of cell apoptosis increased by 7.75% and

292

30.48% in SMMC7721 cells, respectively (Fig. 3A, 3B). DTBrC showed much

293

stronger effects than theanine on induction of apoptosis in SMMC7721 cells.

294

Furthermore, a caspase inhibitor Z-VAD-FMK (Z, 12.5 µM) completely reduced the

295

apoptosis in DTBrC-treated SMMC-7721 cells (Fig. 3A, 3B). In addition, the inhibitors

296

of NF-κB (Bay) and PI3K/Akt (Ly) remarkably induced apoptosis in SMMC7721 cells.

297

The induction of apoptosis in SMMC7721 cells by theanine and DTBrC could greatly

298

contribute to the growth inhibition in the HHC cells (Fig. 2A), which may be associated

299

with repression of PI3K/Akt-NF-KB signaling in SMMC7721 cells.

300

Theanine and DTBrC displayed down-regulation of the protein levels of Bcl-2

301

and cyclin D1 and up-regulation of the protein levels of Bax, cytosolic

302

cytochrome c, caspase-3, and PARP-1 in SMMC7721 cells

303

To understand the molecular mechanisms by which theanine and DTBrC inhibit cell

304

growth and induce apoptosis in HHC cells, we studied the effects of theanine and

305

DTBrC on the expressions of proteins related to cell growth and apoptotic pathways

306

in SMMC7721 cells. Theanine and DTBrC markedly reduced the Bcl-2 protein levels

15



Page 16 of 43 16

307

in SMMC7721 cells at the concentrations of 16 to 250 µM (Fig. 4A). Moreover,

308

theanine and DTBrC remarkably up-regulated the expression levels of Bax (Fig. 4A),

309

cytosolic cytochrome c (Fig. 4B), caspase-3 (Fig. 4C), and the cleavage of

310

poly(ADP-ribose) polymerase-1 (PARP-1) (Fig. 4D) in SMMC7721 cells. In addition,

311

theanine and DTBrC greatly reduced the protein expression level of cyclin D1 (Fig. 4E)

312

in SMMC7721 cells. DTBrC displayed higher activity than theanine on the regulation

313

of the protein expressions of Bcl-2/Bax ratio, cytochrome c, caspase-3, PARP-1, and

314

cyclin D1 in SMMC7721 cells. Furthermore, the inhibitors of NF-κB (Bay) and

315

PI3K/Akt (Ly) showed the significant repression of the protein expressions of Bcl-2,

316

and cyclin D1 and increase in the protein levels of Bax, cytosolic cytochrome c,

317

caspase-3, and the cleavage of PARP-1 in SMMC7721 cells (Fig.4). It suggests that

318

the changes of these apoptosis-associated proteins may be involved in the

319

PI3K/Akt-NF-KB signaling in SMMC7721 cells.

320

Theanine and DTBrC exhibited down-regulation of the phosphorylation and/or

321

protein expressions of Met, VEGFR2, Akt, and NF-κB in SMMC7721 cells

322

In order to clarify the receptors regulated in theanine and DTBrC-treated SMMC7721

323

cells, we studied the effects of theanine and DTBrC on the related receptor

324

expressions in SMMC7721 cells. Theanine and DTBrC at the concentrations of 16 to

325

250 µM remarkably reduced the expressions of hepatocyte growth factor receptor,

326

Met (Fig. 5A) and vascular endothelial growth factor receptor, VEGFR2 (Fig. 5B), and

327

the phosphorylation and expressions of Akt (Fig.5C) in SMMC7721 cells. Moreover,

328

theanine and DTBrC greatly reduced NF-κB protein expression (Fig. 5D) in

16


Page 17 of 43


329

SMMC7721 cells. DTBrC displayed higher activity than theanine on the regulation of

330

the protein expressions of Met, VEGFR2, Akt, and NF-κB in SMMC7721 cells.

331

Theanine and DTBrC showed suppression of HGF- and EGF+HGF-enhanced

332

migration and Akt/NF-κB signaling in SMMC7721 cells

333

In order to determine whether suppression of Met/EGFR/VEGFR-Akt/NF-κB signaling

334

pathways is essential for theanine and DTBrC's anticancer effect, we examined the

335

effects of theanine and DTBrC on the migration and the protein expressions related to

336

the signaling pathways in SMMC7721 cells that were treated with HGF and

337

EGF+HGF. We demonstrated that theanine and DTBrC at 250 µM significantly

338

inhibited HGF-and EGF+HGF-enhanced migration in SMMC7721 cells after 6 h

339

treatment (Fig. 6A). Ly and Bay which are the inhibitors of PI3K/Akt and NF-κB,

340

respectively also repressed the HGF- and EGF+HGF-enhanced migration in

341

SMMC7721 cells. Theanine and DTBrC significantly enhanced the suppression of

342

SMMC7721 cell migration in combination with Ly and Bay (Fig.6A). Moreover,

343

theanine

344

EGF+HGF-enhanced the phosphorylation of Met (Fig. 6B), EGFR (Fig. 6C), VEGFR2

345

(Fig. 6D), pAkt (Fig. 6E), and pNF-κB (Fig. 6F) in SMMC7721 cells. In addition,

346

theanine and DTBrC at 250 µM significantly repressed HGF-and EGF+HGF-induced

347

reduction of p53 protein level and enhanced the p53 protein expression in

348

SMMC7721 cells (Fig. 6G). Furthermore, Akt knockdown decreased the migration by

349

63.4% (Fig. 7A) and the CD44 expression by 75% (Fig. 7B) in SMMC7721 cells.

350

NF-κB knockdown reduced the migration by 45.5% (Fig. 7C) and NF-κB expression

and

DTBrC

at

250

µM

remarkably

reduced

the

HGF-

and

17



Page 18 of 43 18

351

by 70% (Fig. 7D) in SMMC7721 cells. Theanine (T) and DTBrC at 250 µM remarkably

352

reduced the cell migration (Fig. 7A, 7C) and protein expressions of Akt, CD44, and

353

NF-κB (Fig. 7B, 7D) in SMMC7721 cells. Theanine and DTBrC at 250 µM reduced the

354

CD44 expression by 14% and 59%, respectively in the cancer cells (Fig. 7B).

355

Theanine and DTBrC showed inhibition of tumor growth in mice

356

Theanine and DTBrC significantly inhibited the tumor growth in SMMC7721

357

tumor-bearing mice. The relative inhibitory rates of tumor volumes (Fig. 8A, 8B) and

358

weights (Fig. 8C) in SMMC772 increased by 33.2% and 29.2% (for theanine

359

treatment), and 62.7% and 61.2% (for DTBrC treatment), respectively after treatment

360

with theanine and DTBrC for 15 days. DTBrC showed more than 2-fold higher in

361

reduction of SMMC7721 tumor weights compared to theanine (Fig. 8C). Moreover,

362

theanine and DTBrC did not have any toxicity to the mice according to the changes of

363

body weights and the pathological dissection of the mice.

364

DISCUSSION

365

In this study, we have synthesized a novel theanine derivative DTBrC, and evaluated

366

the anticancer activities of DTBrC by comparing it with theanine against HHC cells in

367

vitro, ex vivo, and in vivo. DTBrC has the very strong fluorescence characteristics in

368

vitro and in vivo (Fig. 1B). Theanine and DTBrC can significantly inhibit the in vitro

369

migration (Fig. 1C, 1D) and in vitro (Fig. 2A, 2B) and ex vivo (Fig. 2C, 2D) growth in

370

SMMC7721 and HepG2 cells. Theanine and DTBrC can significantly enhance the

371

effectiveness of anticancer drug pirarubicin on the growth suppression of SMMC7721

372

and HepG2 cells (Fig. 2E, 2F). DTBrC has displayed much stronger activity than

18


Page 19 of 43


373

theanine on repression of the in vitro migration and in vitro and ex vivo growth in

374

SMMC7721 and HepG2 cells. In addition, Theanine and DTBrC can induce apoptosis

375

in SMMC7721 cells, which DTBrC has also exhibited much stronger activity than

376

theanine on the induction (Fig. 3A, 3B).

377 378

Why dose DTBrC possess higher anticancer activities than theanine? We consider

379

that both esterification of the carboxyl group of theanine with ethanol and acylation of

380

the amino group of theanine with 6, 8-dibromo-coumarin-3-carboxylic acid (DBrC)

381

may enhance the inhibitory effects of theanine on in vitro cell migration and growth as

382

well as in vivo tumor growth. The esterification of the carboxyl group of theanine with

383

ethanol would increase the liposoluble solubility of theanine and thus the suppression.

384

In the chemical structure of DTBrC, the acylation of the amino group of theanine with

385

DBrC would prolong the time of the effective compound in vivo and thus enhance the

386

compound's efficacy. The ex vivo experiment has confirmed that DTBrC showed

387

much stronger activities than either theanine or DBrC on growth inhibition of HHC cell

388

lines, which DTBrC displayed the significant suppression of SMMC7721 and HepG2

389

cell growth for 8 h and the inhibitory peak of the HHC cell growth at 5 h after its oral

390

administration in rats, while theanine and DBrC showed the repression of SMMC7721

391

and HepG2 cell growth for only 1 h and 3 h, respectively, and the inhibitory peak of

392

the HHC cell growth at 1 h (for theanine) and 3 h (for DBrC) after their oral

393

administration in rats (Fig.2C, 2D). In addition, the double bromo groups would also

394

enhance the inhibition of the HHC cell growth because the preliminary experiments

19



Page 20 of 43 20

395

confirmed that DBrC had much higher activity than coumarin-3-carboxylic acid

396

without the double bromo groups against the cancer cell growth.

397 398

In order to understand the molecular mechanisms of theanine and DTBrC action, we

399

have further analyzed the expressions of proteins related to the migration and growth

400

as well as apoptosis in SMMC7721 cells. The ratio of antiapoptotic Bcl-2 and

401

proapoptotic Bax has great impacts on regulation of apoptosis. The decrease in Bcl-2

402

protein and increase in Bax protein determine the susceptibility to apoptosis. The

403

release of cytochrome c from the mitochondria can propagate the apoptotic pathway

404

21, 22

. The increased protein expressions of cytochrome c, caspase-3, and PARP-1 in 21, 22

405

the cytosol are the important hallmarks of the apoptosis.

. In the present study,

406

our results showed that theanine and DTBrC significantly up-regulated the level of

407

proapoptotic Bax protein and down-regulated the level of antiapoptotic Bcl-2 protein,

408

leading to reduction of Bcl-2/Bax ratio in SMMC7721 cells (Fig. 4A). Moreover,

409

theanine and DTBrC remarkably enhanced the release of cytocrome c from the

410

mitochondria (Fig. 4B) and greatly increased the protein expressions of caspase-3

411

(Fig. 4C) and PARP-1 (Fig. 4D) in SMMC7721 cells. Furthermore, theanine and

412

DTBrC significantly down-regulated the expression of cyclin D1 protein in SMMC7721

413

cells (Fig. 4E). In addition, the inhibitors of PI3K/Akt (Ly) and NF-κB (Bay) also

414

displayed significant regulation on these protein expressions in SMMC7721 cells (Fig.

415

4A, 4B, 4C, 4D, 4E). Our present results indicated that theanine and DTBrC

416

significantly repressed the growth of SMMC7721 cells and markedly induced the

20


Page 21 of 43


417

apoptotic cell death by decreasing Bcl-2/Bax ratio, activating the mitochondrial and

418

caspase-3 pathways, and reducing the protein expression of cyclin D1 in SMMC7721

419

cells. DTBrC showed significant higher activities than theanine on regulation of these

420

protein expressions in SMMC7721 cells

421 422

The activated signaling pathways of Met/EGFR/VEGFR – Akt/ NF-κB display very

423

important effects on the migration and growth of cancer cells including HHC cells.3, 5, 7,

424

26-28

Inhibition of these receptor protein expressions in HHC cells (Fig. 5A, 5B, Fig.6B,

425

6C, 6D) by theanine and DTBrC could inhibit these receptors-mediated downstream

426

signaling pathways, resulting in the inhibition of the migration and growth of HHC cells.

427

Our present results have shown that theanine and DTBrC significantly reduced the

428

phosphorylation and expression of Akt (Fig. 5C) and the NF-κB (p65) expression (Fig.

429

5D) in HHC cells. In addition, the inhibitors of PI3K/Akt (Ly) and NF-κB (Bay)

430

significantly suppressed the phosphorylation and protein expression of Akt and the

431

NF-κB (p65) expression in HHC cells (Fig. 5C, 5D). Furthermore, Akt knockdown

432

greatly decreased the migration (Fig. 7A) and CD44 expression (Fig. 7B) in

433

SMMC7721 cells. NF-κB knockdown markedly reduced the migration (Fig. 7C) in

434

SMMC7721 cells. The enhanced CD44 (HCAM) expression is associated with the

435

migration, invasion, anti-apoptosis, chemosensitivity, and metastasis during the

436

cancer progression.23-25 Theanine and DTBrC at 250 µM remarkably reduced the

437

protein expressions of CD44, Akt, and NF-κB (Fig. 7B, 7D), leading to the reduction of

438

cell migration (Fig. 7A, 7C) in SMMC7721 cells.

21



Page 22 of 43 22

439

Akt as a cytosolic signal transduction protein kinase has the important influences on

440

cell survival pathways. 26 The activated Akt controls various cellular functions such as

441

gene transcription, protein synthesis, and apoptosis through the phosphorylation of

442

downstream substrates such as NF-κB26. NF-κB can regulate Bcl-2 transcription27.28.

443

The activation of signaling pathway of pAkt and NF-κB/Bcl-2 results in repression of

444

chemotherapy-induced apoptosis, leading to the therapy resistance related to various

445

cancers including HHC. 26,27,28 Our present results demonstrated that theanine and

446

DTBrC significantly suppressed HGF- and EGF+HGF-enhanced migration and

447

pAkt/NF-κB signaling in HHC cells by reducing the HGF- and EGF+HGF-enhanced

448

protein phosphorylation of Met, EGFR, VEGFR2, Akt, and NF-κB in HHC cells. We

449

also found that HGF and HGF+EGF reduced the expression of p53 protein, the tumor

450

suppressor in SMMC7721 cells, which could lead to the resistance to apoptosis and

451

reduction of repressing proliferation and/or migration in the HHC cells, and theanine

452

and DTBrC significantly repressed the reduction of p53 protein and even increased

453

the p53 protein levels in SMMC7721 cells. Our results showed that theanine and

454

DTBrC significantly inhibited the migration and growth, and induced apoptosis in HHC

455

cells, which correlated with a suppression of the PI3K/Akt and NF-κB pathway by

456

reducing the phosphorylation and expression of Met, VEGFR2, EGFR, Akt and the

457

NF-κB. Suppression of Met, EGFR, and VEGFR2 receptors-activated Akt-NF-κB

458

signaling pathways by theanine and DTBrC might be one mechanism for the

459

inhibition of the migration and growth of HHC cells as well as HHC tumor growth in

460

tumor-bearing mice.

22


Page 23 of 43


461

In summary, our new synthetic small fluorescent molecule compound DTBrC and its

462

parental compound theanine have the activities against HHC in vitro, ex vivo, and in

463

vivo. Theanine and DTBrC significantly inhibit the migration and growth of HHC cell

464

lines. At the molecular level, theanine and DTBrC suppress the Met, EGFR, and

465

VEGFR2 receptors-mediated signaling pathways of Akt-NF-κB in HHC cells.

466

Theanine and DTBrC do not exhibit any toxicity to the mice but strongly suppress the

467

tumor growth in the SMMC7721 tumor-bearing mice. In addition, theanine and DTBrC

468

significantly enhanced the growth inhibition of HHC cell lines when combined with

469

conventional anticancer drug pirarubicin. All these findings suggest that theanine and

470

DTBrC may have the potential of adjuvant therapeutic applications in the treatment of

471

HHC.

472

ABBREVIATIONS

473

P, pirarubicin; PI, propidium iodide; Cyto C, cytochrome c;

474

8-dibromo- 2-oxo- 2H-benzopyran-3-carboxamido)-5-(ethylamino)-5-oxo-pentanoic

475

ethyl ester; DBrC, 6,8-substituted dibromo-coumarin-3-carboxylic acid; NF-κB,

476

nuclear factor κB; VEGFR, vascular endothelial growth factor receptor; HGF,

477

hepatocyte growth factor; EGF, epidermal growth factor; EGFR, epidermal growth

478

factor receptor; PARP, poly(ADP-ribose) polymerase; Ly, Ly294002 ， Bay, Bay

479

11-7082; MTT, 3- [4, 5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; HHC,

480

Human hepatocellular carcinoma.

481

ACKNOWLEDGEMENTS

482

This work is supported in part by grant (863) from the Ministry of Science and

DTBrC, (R)- 2-(6,

23



Page 24 of 43 24

483

Technology of the People’s Republic of China to G.Z.(2012AA020206), from the

484

Ministry of Human Resources and Social Security of the People’s Republic of China

485

to G..Z., Projects of Yantai University to G.Z., Project from the National Natural

486

Science Foundation of China to G.Z.(No.30973553), and grants from the Department

487

of Science and Technology of Shandong Province to G.Z. (ZR2015HM004;

488

2009GG10002087).

489

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490

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[2] Ferlay, J.; Shin, H.R.; Bray, F.; Forman, D.; Mathers, C. Parkin, D.M.: Estimates

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epigallocatechin-3-gallate in breast cancer patients under radiotherapy. Current Mol.

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on the proliferation of human lung cancer cells and its molecular mechanisms of

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Y. ，Wang, Q., Zhao, X., Wan, X. Inhibition of lung tumor growth by targeting

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EGFR/VEGFR-Akt/NF-κB pathways with novel theanine derivatives. Ongotarget 2014,

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conserved homolog, Bax, that accelerates programmed cell death. Cell 1993, 74,

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inhematologic malignancies. Semin. Hematol. 1997, 34, S9-S19.

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T.C.; Poon, R.T.; Pang, R.W. The Enhanced metastatic potential of hepatocellular

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carcinoma (HCC) cells with sorafenib resistance. PLoS One 2013, 8(11):e78675.

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enhances apoptosis, chemosensitivity, and reduces tumorigenesis and invasion.

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Cancer Chemother Pharmacol. 2008,62(6):949-57.

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[26] West, K.A.; Castillo, S.S.; Dennis, P.A. Activation of the PI3K/Akt pathway and

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chemotherapeutic resistance. Drug Resist. Updat 2002, 5:234–248.

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[27] Marsden, V.S.; O'Connor, L.; O'Reilly, L.A.; Silke, J.; Metcalf, D.; Ekert, P.G.;

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Huang, D.C.; Cecconi, F.; Kuida, K.; Tomaselli, K.J.; Roy, S.; Nicholson, D.W.; Vaux,

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D.L.; Bouillet, P.; Adams, J.M.; Strasser, A. Apoptosis initiated by Bcl-2-regulated

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apoptosis: potentiation by inhibition of NF-kappaB. Science 1996, 274, 784-787.

581

FIGURE LEGENDS

582

Figure 1 Scheme of synthesis of the theanine derivative DTBrC and inhibitory

583

effects of theanine and DTBrC on the migration of human hepatocellular

584

carcinoma (HHC) cell lines.

585

(A) Scheme of synthesis of the theanine derivative DTBrC. DTBrC was prepared as

586

outlined in the scheme. L-Theanine (1) treated with SOCl2 in ethanol to generate its

587

ethyl

588

8-dibromo-coumarin-3-carboxylic acid (3/DBrC) under classical peptide coupling

589

condition to afford the target derivative DTBrC (4). The compound DTBrC was

590

purified by column chromatography and the structure was characterized by 1H NMR,

591

13

ester

(2).

The

amino

group

of

2

was

acylated

with

6,

C NMR and MS. (B) The in vitro (DTBrC at 8 mg/ml in tube) and in vivo fluorescent

28


Page 29 of 43


592

signals in mouse (3 h after injection of DTBrC at 80 mg/kg) were recorded under 530

593

nanometer excitation and 600 nanometer emission by a Kodak Image Station 4000

594

Multi-Modal Imaging System. (C) Inhibitory effects of theanine (T) and DTBrC on

595

migration of SMMC7721 cells. (D) Inhibitory effects of theanine (T) and DTBrC on

596

migration of HepG2 cells. The effects of theanine and DTBrC on the migration of

597

SMMC7721 and HepG2 cells were examined by the migration assay as described in

598

the section of “Materials and methods”. (C1，， D1) The photos (200X) show the

599

propidium iodide-stained SMMC7721 (C1) and HepG2 (D1) cells that migrated

600

through fibronectin-coated transwell chamber. (C2，，D2) The quantitative analysis of

601

inhibition of SMMC7721 (C2) and HepG2 (D2) cell migration by theanine and DTBrC.

602

The cells were treated for 6 h with the indicated concentrations of theanine (T) and

603

DTBrC (1–16 µM). The control received vehicle. The data are presented as the mean

604

± S.D. (Bar) (n=6). The figure is the representative of 3 similar experiments performed.

605

Statistical analysis was performed using simple linear regression. Values with

606

different letters (a–f) differ from each other significantly (P < 0.05).

607

Figure 2 Inhibitory effects of theanine, DBrC and DTBrC on the growth of HHC

608

cell lines.

609

(A-B) In vitro effects of theanine (T), DBrC and DTBrC on the growth of HHC

610

SMMC7721 (A) and HepG2 (B) cells. (C-D) Ex vivo effects of the sera from theanine-,

611

DBrC-, or DTBrC-treated rats at 0, 0.5, 1, 3, 5 and 8 h after oral administration of

612

theanine, DBrC or DTBrC on the growth of SMMC7721 (C) and HepG2 (D) cells. (E-F)

613

Theanine (64 µM), and DTBrC (16 µM) enhanced the growth inhibition of SMMC7721

29



Page 30 of 43 30

614

(E) and HepG2 (F) cells in combination with anticancer drug pirarubicin (P, 12.5 to 25

615

nM). The cells were treated for both 48 h and 72 h with the indicated concentrations

616

of theanine, DBrC or DTBrC in (A) and (B), or only 72 h in (C, D, E, F). The cells in

617

control group were treated with vehicle. The rate of relative cell growth was

618

determined by the MTT assay. The data are presented as the mean ± S.D. (Bar) (n=6).

619

The figure is the representative of 3 similar experiments performed. Values with

620

different letters (a–g) differ from each other significantly (P < 0.05).

621

Figure 3 Induction of apoptosis in SMMC7721 cells treated by theanine (T) and

622

DTBrC.

623

(A) FACS analysis of apoptosis in SMMC7721 cells after the cells were treated for 48

624

h with the indicated concentrations of theanine/T and DTBrC (16–250 µM), DTBrC+Z

625

(DTBrC/250 µM + Z/12.5 µM; Z /Z-VAD-FMK, a caspase inhibitor),Ly294002 (16 µM)

626

and Bay (3.2 µM) as the positive control. The cells in control group were treated with

627

DMSO vehicle. (B) The summary of total percent apoptosis (early apoptosis/Q4 plus

628

late apoptosis/Q2) in the cells treated with theanine and DTBrC, DTBrC+Z, as well as

629

the inhibitors (Ly and Bay) of PI3K/Akt and NF-κB. *, P < 0.05; ***, P < 0.001.

630

Figure 4. Inhibitory effects of theanine and DTBrC on protein expressions of

631

Bcl-2/Bax (A), cytosolic cytochrome c (Cyto c) (B), caspase-3/pro-caspase-3 (C),

632

PARP-1 (D) and cyclin D1 (E) in HHC SMMC7721 cells.

633


634

DTBrC (T16/DTBrC16 – T250/DTBrC250: 16–250 µM), Ly (16 µM) and Bay (3.2 µM).

635

The protein expressions were analyzed by Western Blotting. The optical density (OD)

30


Page 31 of 43


636

of the band is normalized with respective β-actin and is expressed as relative optical

637

density (OD). The OD value of the band shown as mean ± S.D. is relative to that of

638

the control (DMSO vehicle) designated as 100%. Ly and Bay are the inhibitors of

639

PI3K/Akt and NF-κB, respectively. Values with different letters (a–f) differ from each

640

other significantly (P < 0.05).

641

Figure 5. Inhibitory effects of theanine and DTBrC on protein expressions

642

and/or phosphorylation of Met (A), VEGFR2 (B), pAkt/Akt (C), and NF-κB (D) in

643

HHC SMMC7721 cells.

644


645

DTBrC (T16/DTBrC16 – T250/DTBrC250: 16–250 µM), Ly (16 µM) and Bay (3.2 µM).

646

The protein expressions were analyzed by Western Blotting. Values with different

647

letters (a–f) differ from each other significantly (P < 0.05).

648

Figure 6. Inhibition of EGF+HGF-enhanced migration and Met/EGFR/VEGFR-

649

Akt/NF-κB signaling in SMMC7721 cells by theanine and DTBrC.

650

(A) The in vitro migration assay using fibronectin-coated transwell chamber confirmed

651

that theanine (T) and DTBrC at 16 µM significantly repressed EGF-, HGF- and

652

EGF+HGF- (40 ng/ml) enhanced migration in SMMC7721 cells after 6 h treatment; Ly

653

and Bay are the inhibitors of PI3K/Akt and NF-κB, respectively. (B-G) Theanine and

654

DTBrC significantly reduced EGF+HGF-enhanced phosphorylation of Met, EGFR,

655

VEGFR2, Akt, and NF-κB as well as increased the EGF+HGF-reduced p53 protein

656

expression in SMMC7721 cells. After SMMC7721 cells were pretreated for 30

657

minutes with theanine (T) and DTBrC at 250 µM, the cells were treated with HGF (40

31



Page 32 of 43 32

658

ng/ml) or EGF (40 ng/ml) +HGF (40 ng/ml) or without EGF+HGF treatment for 1 h.

659

The protein expressions of pMet/Met (B), pEGFR/EGFR (C), pVEGFR2/ VEGFR2 (D),

660

pAkt/Akt (E), pNF-κB/NF-κB (F), and p53 (G) in SMMC7721 cells were analyzed by

661

Western blotting as described in the section of “Materials AND Methods”. Values with

662

different letters (a–g) differ from each other significantly (P < 0.05).

663

Fig.7 Akt and NF-κB knockdown as well as theanine (T) and DTBrC reduced

664

SMMC7721 cell migration (A-C) and protein expressions (B-D) of Akt, CD44, and

665

NF-κB. Equal number of control (scAkt or scNF-κB) and shAkt or shNF-κB knock

666

down cells were assayed for migration by the in vitro migration assay

667

mentioned-above. The photos (200X) show the propidium iodide-stained SMMC7721

668

cells (A, C) that migrated through fibronectin-coated transwell chamber. The protein

669

expressions were detected by Western blot analysis. Values with different letters (a–c)

670

differ from each other significantly (P < 0.05).

671

Fig.8 in vivo suppression of HHC SMMC7721 xenograft tumor growth in

672

tumor-bearing nude mice by theanine and DTBrC.

673

(A-C) SMMC7721 cells (3 × 106) were implanted in the rear right flank of each nude

674

mouse. After 12 days, the tumor model was established. The tumor-bearing nude

675

mice were randomly divided into three groups: (1) control group (vehicle); (2)

676

theanine group (80 mg/kg/day); (3) DTBrC group (80 mg/kg/day). The mice were

677

treated with vehicle, theanine and DTBrC by intraperitoneal injection once every day.

678

The therapy was conducted for 15 days. The relative tumor volume and weight are

679

relative to that of the control (vehicle) designated as 100. (A) Relative tumor volumes

32


Page 33 of 43


680

in each treatment group. (B) Excised tumors derived from the mice in each treatment

681

group. (C) Relative tumor weights in each treatment group. Tumor volumes and

682

weights were compared among groups using the ANOVA and Bonferroni post-test.

683

Values are shown as mean ± S.D. for each group (n=6). Values with different letters

684

(a–c) differ from each other significantly (P < 0.05). All statistical tests were two-sided.

33



Page 34 of 43 34

The TOC graphic

34


Page 35 of 43

Fig.1


Scheme of Preparation of (R)-ethyl 2-(6,8-dibromo-2-oxo-2H-chromene3-carboxamido)-5-(ethylamino)-5-oxopentanoate (DTBrC) and the effects of theanine and DTBrC on the migration of HHC cell lines

A. Scheme of Preparation of DTBrC

B. In vitro & in vivo fluorescence signal of DTBrC

R1=R2=Br, 1 = theanine (T), 2 = Ethyl L-theanine, 3 = 6,8-dibromo-coumarin-3-carboxylic acid (DBrC),

4=(R)-ethyl 2-(6, 8-dibromo-2-oxo-2H-chromene-3-carboxamido)5-(ethylamino)-5-oxopentanoate (DTBrC)

C. Inhibition of migration of SMMC7721 cells by theanine and DTBrC C1

C2 Control

T16/16 µM

DTBrC16/16 µM

D．Suppression of migration of HepG2 cells by theanine and DTBrC D1

D2 Control

T16/16 µM

DTBrC16/16 µM


Agricultural Chemistry Fig. 2 Effects of theanine (T), Journal DBrC of and DTBrCand on Food the growth of HHC cell lines

C．Ex vivo effects on the growth of SMMC7721 cells.

d e e

40

f

20

DTBrC250

DTBrC125

0

DTBrC64

DTBrC250

DTBrC125

DTBrC64

DTBrC16

DBrC250

DBrC125

DBrC64

DBrC16

T250

T125

T64

g

72h

60

DTBrC16

f f

DBrC250

e e

c

80

DBrC125

d

DBrC64

c

100

DBrC16

bc

b

T250

aa bb

T125

aa

48h

aa aa aa bb bb aa aa bb bbb bb

T64

bc

HepG2 cells

T16

ab

aa aa

72h

120

0

aa

T16

120 100 80 60 40 20 0

48h

B. In vitro effects on the growth of HepG2 cells.

Relative cell growth (%)

SMMC7721 cells

0

Relative cell growth (% )

A. In vitro effects on the growth of SMMC7721 cells.

Page 36 of 43

D. Ex vivo effects on the growth of HepG2 cells.


100

a aa

80

SMMC7721 cells a a a a b b

60 40

Theanine

20

DBrC DTBrC

a

a a

a b

c

c d e

0 0h

0.5h

1h

3h

5h

8h

Time after oral administration of drugs in rats(h)

E. Enhanced growth inhibition of SMMC7721 cells.


HepG2 cells 120

120 100

a

a

a a

80

a

b c

60 40

c d

Theanine DBrC DTBrC

20

a

a

b

b

a

a

a

a

e

0 0h

0.5h

1h

3h

5h

8h

Time after oral administration of drugs in rats (h)

F. Enhanced growth inhibition of HepG2 cells.


Page 37 of 43


Fig.3. Effects of theanine (T) and DTBrC on apoptosis in HHC SMMC7721 cells

A.

B.


Fig.4 Effects of thanine (T) and DTBrC on protein expressions of Bcl-2/Bax, cytosolic cytochrome c Journal of Agricultural and Food Chemistry Page 38 of 43 (Cyto C), caspase-3, PARP-1, and cyclin D1 in SMMC7721 cells A．Bcl-2/Bax

C. Caspase-3

B. Cyto C

D. PARP-1

E. Cyclin D1


Page 39 of 43


Fig.5 Effects of theanine (T) and DTBrC on protein phosphorylation and/or expressions of Met, VEGFR2, Akt and NF-κB in SMMC7721 cells A. Met

C. Akt

B. VEGFR2

D. NF-κB


Fig. 6 Inhibition of HGF+EGF-enhanced migration and Met/EGFR/VEGFR-Akt/NF-κB signaling in SMMC7721 Journal of Agricultural and Food Chemistry Page 40 of 43

cells by theanine (T)）and DTBrC.

d c

a f g

Bay+DTBrC

g

Bay+T

f

Ly+DTBrC

f

Ly

DTBrC

T250

f

HGF+EGF+DTBrC

EGF+DTBrC

e

e

Ly+T

e f

Bay

e

EGF+T

f

HGF+T250

HGF+EGF

EGF

e

HGF+DTBrC

a

HGF+EGF+T

b

HGF

180 160 140 120 100 80 60 40 20 0

Control

Relative migration (%)

A-G. Inhibition of HGF+EGF-enhanced migration (A) and phosphorylation of Met (B), EGFR (C), VEGFR2 (D), Akt (E), and NF-κB (F) as well as decrease in p53 protein expression (G). A.

Treatment

B. pMet

E. pAkt

C. pEGFR

F. pNF-κB


D. pVEGFR2

G. p53

Page 41 of 43


Fig. 7 Akt and NF-κB knockdown reduced migration and expressions of Akt, CD44 and NF-κB in SMMC7721 cells. A-B． Akt knockdown reduced migration (A) and expressions (B) of Akt and CD44. A.

B.

(C-D) NF-κB knockdown reduced migration (C) and expression (D ) of NF-κB. C. D.



Fig. 8 In vivo suppression of SMMC7721 xenograft tumor growth in tumor-bearing mice by theanine and DTBrC.

A.

Relative tumor volume

120

a

100 b

80 60

c

40 20 0 Control

Theanine

DTBrC

Treatment

B.

C.

Realtive tumor weight

120

a

100 b

80 60

c

40 20 0 Control

Theanine

DTBrC

Treatment


Page 42 of 43

Page 43 of 43


Theanine (T) and its derivative DTBrC inhibited growth and migration as well as Akt/NF-κB signaling in human hepatocellular carcinoma cells

R=CH2CH3, R1=R2=Br, 1 = theanine (T), 2 = Ethyl L-theanine, 3 = 6,8-dibromo-coumarin-3-carboxylic acid (DBrC), 4=(R)-ethyl 2-(6, 8-dibromo-2-oxo-2H-chromene-3carboxamido)-5-(ethylamino)-5-oxopentanoate (DTBrC) 100

a aa

80

SMMC7721 cells a a a a b b

60 40

Theanine

20

DBrC DTBrC

a

a a

a

c

c d e

0 0h Pirarubicin(nm) -

-

-

12.5

25

12.5

25

12.5

25

0.5h

1h

3h

120

b

5h

8h

Time after oral administration of drugs in rats(h)


Realtive tumor weight


120

a

100 b

80 60

c

40 20 0 Control

Theanine

Treatment

DTBrC

Repression of Human Hepatocellular Carcinoma Growth by

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