10-Gingerol, a Phytochemical Derivative from “Tongling White Ginger

Feb 23, 2017 - Phone: +86-551-62901539. ... Most cell cycle related genes and protein expression significantly decreased, followed by a slight decreas...
0 downloads 0 Views 2MB Size
Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

10-Gingerol, a Phytochemical Derivative from Tongling White Ginger, Inhibits Cervical Cancer: Insights into the Molecular Mechanism and Inhibitory Targets Fang Zhang, KIRAN THAKUR, Fei Hu, Jian-Guo Zhang, and Zhao-Jun Wei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00095 • Publication Date (Web): 23 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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

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

Page 1 of 38

Journal of Agricultural and Food Chemistry

1

Title and authorship

2 3

10-Gingerol, a Phytochemical Derivative from Tongling

4

White Ginger, Inhibits Cervical Cancer: Insights into the

5

Molecular Mechanism and Inhibitory Targets

6

Fang Zhang †, Kiran Thakur †, Fei Hu †, Jian-Guo Zhang †, Zhao-Jun Wei † ‡ *

7



8

230009, People’s Republic of China

9



School of Food Science and Engineering, Hefei University of Technology, Hefei

Agricultural and forestry specialty food processing industry technological innovation

10

strategic alliance of Anhui province, Hefei 230009, People’s Republic of China

11

Fang Zhang [email protected]

12

Kiran Thakur [email protected]

13

Fei Hu hufei@ hfut.edu.cn

14

Jian-Guo Zhang [email protected]

15

*Correspondence: E-mail: [email protected]; Tel: +86-551-62901539; Fax:

16

+86-551-62901539.

17

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

18

Abstract: With an aim to evaluate anti-cancerous activities of 10-gingerol (10-G)

19

against Hela cells, it was purified and identified from Tongling White Ginger by

20

HSCCC, UPLC-TOF-MS/MS and NMR analysis, respectively. 10-G inhibited the

21

proliferation of HeLa cells at IC50 (29.19 µM) and IC80 (50.87 µM) with altered cell

22

morphology, increased cytotoxicity and arrested cell cycle in G0/G1-phase. The most

23

cell cycle related genes and proteins expression significantly decreased, followed by

24

slight decrease in few and without affecting cyclin B1 and cyclin E1 (protein). Both

25

death receptors significantly upregulated and activated apoptosis indicators (caspase

26

family). Furthermore, significant changes in mitochondria dependent pathway

27

markers were observed and led to cell death. 10-G led to PI3K/AKT inhibition and

28

AMPK activation to induce mTOR mediated cell apoptosis in Hela cells. Our results

29

can be an asset to exploit 10-G with other medicinal plant derivatives for future

30

applications.

31 32

Keywords: Tongling White Ginger; 10-Gingerol; Hela cells; Apoptosis;

33

PI3K/AKT/AMPK/mTOR pathways

34

2

ACS Paragon Plus Environment

Page 2 of 38

Page 3 of 38

Journal of Agricultural and Food Chemistry

35

Introduction

36

‘Tongling White Ginger’ in the ideal climatic conditions of Tongling (Anhui province,

37

China) after cultivation for thousands of years enjoys a reputation on the characteristic

38

thin white peel, tender flesh, rich in juice and flavor characters, also traditionally

39

regarded as one of the top gingers in China.1 In general, ginger is widely consumed as

40

a popular spice throughout the world, and had been cultivated for traditional oriental

41

medicinal usages in China, India, Japan and other Asian countries.2, 3 Pharmacological

42

investigations have revealed the chemo-preventive and chemotherapeutic effects of

43

ginger and its major pungent ingredients on variety of cancer cell lines and animal

44

models.4 Previous studies have shown that the extracts of ginger possess variety of

45

biological and pharmacological activities, including antioxidant,5 anti-inflammatory,6

46

anti-cancer,7 glucose lowering,8 musculoskeletal disorder,9 osteoarthritis,10 migraine11

47

and cardiovascular protective activities.12

48

Gingerols have been reported as main functional components from ginger, which

49

share the same vanillyl moiety and possess a similar chemical structure with a

50

different unbranched alkyl carbon chain.13 Based on alkyl side chain lengths,

51

gingerols are assigned as 4, 6, 8, 10, 12-gingerol and so on.14 Previous report

52

suggested that phytochemicals extracted from ginger are better studied for their

53

anti-cancerous attributes.15 Similarly, gingerols have been reported to exhibit

54

anti-oxidant, analgesic, anti-pyretic, anti-inflammatory, anti-neuroinflammatory16 and

55

anti-tumorigenic activities.17 As the major pharmacologically active member of

56

gingerols, 10-G had attracted many attentions and it was reported to exhibit 3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

57

anti-cancerous,18 anti-neuroinflammatory,16 anti-oxidant and anti-inflammatory13

58

attributes. However, the detailed molecular mechanism of its anti-cancerous activities

59

is still in its infancy.

60

The series of protein kinase complexes (cyclin-dependent kinases (CDKs) and their

61

activating partners) regulate cell cycle progression in eukaryotic cells.19 The cyclin

62

A/E - CDK-2 and cyclin D1 - CDK-4/6 complexes play vital role in regulating the

63

G0/G1 checkpoint. Apoptosis, a programmed cell death involves several

64

morphological changes and cellular signaling pathways.20 Among them, intrinsic

65

(mitochondrial related which involves Bcl-2 family members, release of cytochrome c

66

and activation of series of caspases) and extrinsic (death receptor) pathways are

67

important.21, 22 Moreover, PI3K/AKT pathway is important in preventing cells from

68

undergoing apoptosis and leading to the pathogenesis of malignancy. Besides, it is

69

also associated with the regulation of cell cycle progression.17

70

Drugs used in classical chemotherapy are expensive, not target specific and lead to

71

severe systemic side effects.23 Multiple uses of these drugs help the body to develop

72

resistance due to heterogeneity of cell types and clonal selection. Here comes the role

73

of traditional dietary ingredients from plants, besides their safety and easy availability

74

and multiple targets/pathways sites, they are consumed as part of daily diet. Hence,

75

our approach is promising which recommend administration of dietary

76

phytochemicals that possess biological active components for inhibition of cancer

77

cells. However, applications of plant components for cancer treatment/prevention

78

require better understanding of anticancer functions and elucidation of their 4

ACS Paragon Plus Environment

Page 4 of 38

Page 5 of 38

Journal of Agricultural and Food Chemistry

79

mechanisms of action in depths. Despite evidences for the number of biological

80

effects of 10-G as the second major component of gingerols, its anti-cancerous effects

81

related to apoptosis have not been fully explored till date.18

82

This study has reported the separation and identification of 10-G from Tongling White

83

Ginger, and evaluation of its anti-cancerous activities against HeLa cells. The effects

84

of 10-G on the cell proliferation, morphology, cell cycle, and apoptosis were

85

investigated. Furthermore, cell cycle and apoptosis related genes and their proteins

86

were studied by RT-qPCR and western blot, respectively. The anti-cancerous activities

87

of 10-G on the cell proliferation, cytotoxicity, morphology, cell cycle, and apoptosis

88

would extend the utilization of ginger as functional foods in the future.

89

Materials and methods

90

Materials

91

Chinese white ginger rhizomes (Zingiber officinale) were procured from Tongling

92

White Ginger Development Co., Ltd. All chemicals used in the study were of

93

analytical grade. All tissue culture reagents, e.g., Dulbecco’s Minimum Essential

94

Medium (DMEM), Roswell Park Memorial Institute (RPMI-1640), fetal bovine

95

serum, trypsin solution without EDTA, penicillin and streptomycin were purchased

96

from Invitrogen (Carlsbad, USA). Pifithrin-µ, Wortmannin, Deguelin, WZ4003 and

97

Temsirolimus were obtained from Abmole Bioscience Inc. (USA).

98

Preparation of crude extraction

99

Fresh ginger rhizomes were sliced, grinded (60 meshes) and vacuum-dried at 50°C, 5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

100

and then addition of α-amylase at 0.5% (IU) concentration, followed by pH to 5.0 ±

101

0.2 which was adjusted with citric acid. Mixed aqueous solution was incubated at

102

50°C for 120 min for collection of enzyme-treated powders. The residue was mixed

103

with 40 mL 90% methanol in an ultrasonic bath at 45°C for 1 h to extract gingerols.

104

After centrifugation, the supernatant was evaporated under reduced pressure at 40°C

105

to remove the methanol, and was freeze-dried for further use.24 Gingers used for

106

experiment were from the same batch.

107

Purification of 10-gingerol

108

10-G was purified from crude extraction according to the procedure of HSCCC

109

separation by Zhan et al.25 with some modification. In present study, the HSCCC

110

system was carried out using binary solvent system which consisted of

111

n-hexane-chloroform-methanol-water (2:5.5:6.5:1, v/v/v/v) system for the isolation of

112

10-G. 100.0 mg of sample in 5 ml solvent mixtures of lower phase and upper phase

113

(1:1, v/v) was added. After the mixed solvents were thoroughly equilibrated at room

114

temperature, the upper stationary phase at a flow rate of 10 mL/min was pumped into

115

multilayer coiled column until fully filled with the fluid, and the apparatus was rotated

116

at 800 rpm. Keeping the volume of the stationary phase under the reach of

117

equilibration, the lower mobile phase was eluted in a descending mode at a flow rate

118

of 1.3-2.0 mL/min followed by immediate injection of the samples. The effluent was

119

continuously monitored with a UV detector at 280 nm to obtain chromatograms and

120

the fraction of peak fraction was collected.

6

ACS Paragon Plus Environment

Page 6 of 38

Page 7 of 38

Journal of Agricultural and Food Chemistry

121

HPLC analysis of ginger extraction

122

HPLC analysis was performed on a Shimadzu LC-8A system, equipped with 515

123

HPLC pump, 20 µL injection loop and SPD-m10AVP UV detector with system

124

controller SCL-10AVP. HPLC analysis was performed according to the description by

125

Pawar et al.26 The sample was identified with a reverse phase Alltech-C18 column

126

with 5 µm particle size. The mixture solution of acetonitrile: water (75:25) were used

127

as the mobile phase at a flow rate of 0.8 mL/min for chromatographic separation of

128

10-G. The sample of ginger extraction was dissolved in methanol for detection with

129

the wavelength under 280 nm, and the injection volume was 20 µL per sample which

130

was executed for 20 min of each procedure.

131

UPLC-TOF-MS/MS analysis of ginger extraction

132

The ginger extraction dissolved in methanol (HPLC grade) and was analyzed by

133

Waters ACQUITY UPLC system (Waters, Milford, MA, USA) and LCT Premier XE

134

time-of-flight (TOF) mass spectrometer (Waters, Milford, MA, USA) equipped with

135

vacuum degasser, binary pump, auto-sampler, column compartment, and diode array

136

detector. ACQUITY UPLC BEH-C18 column (5 cm, 2.1 mm, 1.7 µm) was used for

137

chromatographic analysis by ACQUITY UPLC BEH-C18 column (5 cm, 2.1 mm, 1.7

138

µm) at a flow rate of 0.3 mL/min at 30°C. A binary gradient elution system composed

139

of eluent A (deionized water) and eluent B (acetonitrile) was applied as followed27:

140

maintaining 70% of A and 30% of B at the beginning 3 min, and then the content of B

141

was increased from 30% to 70% at 3.2 min and maintained to 3.5 min. The injection

7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

142

volume was set at 10 µL, and each run was followed by equilibration time of 5 min.

143

And the elution system was supplied according to same system given for 10-G

144

detection. The MS incorporated with electrospray ionization (ESI) interfaces. The

145

positive and negative ion polarity mode was set for the ESI source with the voltage on

146

the ESI interface maintained at approximately 5.5 kV. The conditions were set as

147

followed: de-solvation gas flow at 600 L/h at 350°C, cone gas flow at 60 L/h and

148

source temperature at 110°C, collision energy 10.0/35 V, nebulizer pressure 50 psi and

149

scan range m/z 100-1000.

150

Nuclear Magnetic Resonance

151

1

152

MHz). Chemical shifts in parts per million (in ppm) of the 1H-NMR spectra were

153

referenced to tetramethylsilane (δ = 0 ppm) in CDCl3 as an internal standard. And the

154

13

155

Cell culture

156

Hela cell and HEK293 cell lines were obtained from Shanghai wei atlas biological

157

technology co., LTD. The Hela cells was grown in RPMI-1640 medium supplemented

158

with 10% fetal bovine serum (FBS) and 100 µg/mL streptomycin and 100 U/ml

159

penicillin G at 37°C in a humidified atmosphere of 5% CO2.28 The Human embryonic

160

kidney HEK293 cells was cultured in Dulbecco minimum essential medium (DMEM)

161

containing FBS (10%), streptomycin and penicillin G at 37°C in a CO2-incubator. The

162

culture medium was revived twice in a week and detached by trypsin, and then plated

H-NMR spectra and 13C-NMR spectra were recorded with Bruker AVIII-600 (600

C-NMR spectra were calibrated with CDCl3 (δ = 77.00 ppm).

8

ACS Paragon Plus Environment

Page 8 of 38

Page 9 of 38

Journal of Agricultural and Food Chemistry

163

in 6- or 96-well plates before experiments. To evaluate the effect of inhibitors

164

targeting signal transduction pathway on 10-G induced apoptosis in Hela cells, the

165

cells were pre-treated with respective experimental conditions as per protocol for each

166

inhibitor.

167

Determination of cancer cell inhibition

168

The cell proliferation was assayed according to the manufacturer’s instructions of the

169

Cell Counting kit-8 assay kit (DOJINDO Corp.). Cells were dispensed at the initial

170

density of 2 × 105 cells per well of a 96-well microplate with the volume of 100 µL

171

and pre-incubated at 37°C. After 10 hours, medium was replaced with RPMI-1640 or

172

DMEM 10% FBS containing different concentrations of 10-G (5-120 µM) or

173

5-fluorouracil (5-FU, 50 µM) solution for Hela or HEK293 cells, respectively. And

174

then the cells were further continuously cultivated for 48 h. Number of viable cells

175

were monitored using WST tetrazolium salt (CCK-8) followed by method Johnson et

176

al.29 Inhibition ability was expressed as the percentage of absorbance in the treated

177

cells compared to negative control:

178

Cell inhibition ability (%) = (ODnegative control - ODtreatment)/(ODstandard - ODblank)×100%.

179

All the experiments were performed in triplicate. The inhibition rate of 10-G on the

180

cell lines can be evaluated as follow: y =

181

A1 - A 2 1+e

( x − x 0 ) / dx

+ A2

182

Where y is the inhibition rate of 10-Gingerol at concentration (x), A1 and A2 are the

183

maximum and initial inhibition value, respectively. X0 is the concentration required to

184

reach half of the maximum inhibition intensity, and dx is the apparent first-order 9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

185

aggregation constant.

186

Effect of 10-gingerol on cytotoxicity assessment

187

Hela cells and HEK293 cells were cultured at the density of 2 × 105 cells per well in

188

96-well plates for 10 h, and were replaced into the medium including 10-G at

189

different concentration or 5-FU solution (65 µM). After 48 h, the culture medium was

190

collected and LDH cytotoxicity was determined by a microplate reader at 490 nm

191

using the cytotoxicity LDH assay kit (Dojindo).30 All experiments were repeated three

192

times.

193

Effect of 10-gingerol on cancer cell cycle

194

Cell cycle distribution was detected by the classical propidium iodide (PI) staining

195

method and flow cytometric analysis. 1×106 cells were fixed in cold 70%ethanol,

196

RNase treated, and stained with propidium iodide (BD) followed the manufacturer’s

197

instructions Cell Cycle Analysis kit (BD Biosciences). Cells were analyzed for DNA

198

content by Flow Cytometry (BD FACSCalibur, USA).31 All the experiments were

199

performed in triplicates and expressed as mean ± SD. Proportion of the cells in G0/G1,

200

S, and G2/M phases were analyzed by the Flowjo software.

201

Cell apoptotic analysis by flow cytometry

202

Cellular apoptosis was determined following the manufacturer’s instruction of

203

Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences) and finally analyzed

204

by FACScalibur Flow Cytometer (Becton Dickinson).19 Cells were incubated in the

205

medium containing 10-G (30 µM) or 5-FU (50 µM) for 12, 24 and 48 h, respectively. 10

ACS Paragon Plus Environment

Page 10 of 38

Page 11 of 38

Journal of Agricultural and Food Chemistry

206

Then, the cells were harvested by trypsinization (0.25% trypsin without EDTA)

207

(Invitrogen), and washed twice with cold PBS. The solution containing 5 µL of

208

annexin V-FITC and 10 µL of propidium iodide was added to the cells, re-suspended

209

in 400 µL binding buffer, and incubated in the dark at 4°C for 10 min. Finally, the

210

cells were detected with flow cytometer within 1 h. All assays were performed at least

211

three times.

212

Assay for RT-qPCR

213

Total RNA extracted using Trizol Reagent (Invitrogen, Life Technologies, USA)

214

followed by first-strand cDNA synthesis using the Prime Script 1st Strand cDNA

215

Synthesis kit and the Oligo dT-adaptor primer in a series of standard 10 µL reverse

216

transcription reactions. Changes in the steady-state expression concentration of

217

mRNA in cyclin A, cyclin B1, cyclin D1, cyclin E1, CDK-1, CDK-2, CDK-4, CDK-6,

218

p15, p16, p21, p27, GSK-3β, and β-catenin were evaluated by reverse-transcription

219

PCR (RT-qPCR), which was carried out using EvaGreen Master Mix (Biotium,

220

Hayward, CA, USA).32 The primers are presented in Table 1. RT-qPCR was

221

performed using the ABI Step One Plus system (Applied Biosystems) followed by

222

melting curve analysis with the following cycling program: initial activation at 95°C

223

for 3 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C

224

for 20 s. GAPDH served as a control for sample loading and integrity.

225

Assay for Western blotting

226

The method of Western blot was according to Lin et al.32 with some modification.

227

Following the treatment with 10-G (15 µM, 30 µM and 50 µM) for 48 h, Hela cells 11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

228

were washed 3 times in cold PBS and lysed with an ice-cold

229

radioimmunoprecipitation (RIPA) buffer containing a protein phosphatase inhibitor

230

and a complete protease inhibitor mixture for 30 min over ice. To obtain the cytosol

231

fraction, the cell lysates were centrifuged at 15,000 rpm/min for 15 min at 4°C. The

232

cytosolic proteins were boiled in a loading buffer, and then the denatured proteins

233

were separated by sodium dodecyl sulfate–polyacrylamide (SDSP) gel electrophoresis

234

and transferred to a 0.45 µm polyvinylidene difluoride (PVDF) membrane. After 2 h

235

at room temperature of incubation in a blocking buffer (150 mM NaCl, 20 mM

236

Tris-HCl, 0.1% Tween 20, and 5% skim milk, the membranes were incubated with the

237

specific primary antibodies overnight at 4°C. Subsequently, the blot was washed 3

238

times with TBST (150 mM NaCl, 20 mM Tris-HCl, and 0.1% Tween 20), followed by

239

incubated with the appropriate secondary antibody for 3 h.33 Immunoreactivity was

240

detected using the Amersham ECL Prime Western Blot detection reagent (GE

241

Healthcare, Fairfield, CT, USA) according to the manufacturer’s instructions.

242

Statistical analysis

243

Statistical analysis was carried out by using SPSS 18.0 software. All the data were

244

expressed as mean ± standard deviation (SD) (n ≥ 3). One-way analysis of variance

245

(ANOVA) was per-formed using the Origin Lab (Origin Pro 8.0) software at

246

significance level p < 0.05.

247

Results and discussion

248

10-Gingerol separated from Tongling White Ginger 12

ACS Paragon Plus Environment

Page 12 of 38

Page 13 of 38

Journal of Agricultural and Food Chemistry

249

The α-amylase enzyme (0.5%) was used to pre-treat the ginger before extracting the

250

crude extraction of ginger; and then, 10-G was separated using HSCCC

251

chromatogram with mobile phase of n-hexane-chloroform-methanol-water system.

252

The separated 10-G was determined by HPLC (Figure 1A), and the main peak located

253

at 24.092 min, which was calculated as 90.06% of the full components. The separated

254

sample was further determined with UPLC-TOF-MS/MS analysis, and the quasi

255

molecular ions of sample was (MW 350) m/z = 349 [M-H]+ (Figure 1B).

256

Identification and recognition of the structure of purified ginger extraction was

257

performed with 1H and 13C NMR. The NMR data of the product were obtained as

258

followed. 1H NMR (400 MHz) δ: 0.87 (3H, t, J=6.4 Hz, H-14), 1.35-1.66 (14H, m,

259

H-7 ~H-14), 1.49 (2H, m, H-6), 2.42-2.65 (2H, m, H-4), 2.75 (2H, dd, J=10 Hz and

260

7.8 Hz, H-2), 2.88 (2H, dd, J=20, 7.8 Hz, H-1), 3.91 (3H, s, OCH3), 4.06 (1H, m,

261

H-5), 6.69 (1H, dd, J=9.6, 2 Hz, H-6’), 6.72 (1H, s, H-2’), 6.85 (1H, d, 8, H-5’); 13C

262

NMR (100 MHz) δ: 14.27 (C-14, q), 22.93 (C-13, t), 25.57 (C-7, t), 29.0, 29.1 (C-11,

263

t; C-10, t), 29.3 (C-9, t), 29.71 (C-8, t), 32.46 (C-1, t), 36.54 (C-6, t), 45.57 (C-2, t),

264

49.47 (C-4, t), 56.00 (OCH3, q), 67.68 (C-5, d), 112.52(C-2’, d), 114.30 (C-5’, d),

265

132.76 (C-1’, s), 144.08 (C-4’, s), 146.45 (C-3’), 211.61 (C-3, s). Based on the above

266

data, the obtained product could be identified as 10-G.

267

Effect of 10-gingerol on the inhibition of cancer cell proliferation

268

CCK-8 assay was used to evaluate the effects of 10-G for dose-dependent manner on

269

the proliferation of HeLa cells (Figure 2A,B). The cells were highly sensitive to 10-G

13

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

270

treatment at IC50 = 29.19 µM and IC80 = 50.87 µM in a concentration-dependent

271

manner (Figure 3B). The treatments of 5-FU (50 µM) and starvation were considered

272

as control and the inhibition of cells proliferation after 48 h of treatment reached to

273

73.52% (5-FU) and 32.38% (starvation), respectively. 40 µM of 10-G showed 70.08%

274

inhibition rate on HeLa cells, which is almost identical to the effect of 5-FU, while,

275

above 40 µM, the inhibitory effects of 10-G on the proliferation of HeLa cells were

276

higher than 5-FU (50 µM) (Figure 3A). Being used as normal cell line, HEK293 cells

277

were used as control to determine the effects of 10-G treatment, whereas it almost

278

showed no inhibition on the proliferation (Figure 3B). The above results clearly

279

demonstrated that 10-G displayed specific anti-cancerous properties, but no inhibition

280

on normal cells. The anti-cancerous properties of ginger make it possible to serve as a

281

broad-spectrum cytotoxic agent against cancer cell lines, such as prostate LNCaP

282

cells,34 breast MDA-MB-231 cells,35 as well as lung A549, ovarian SK-OV-3, and

283

melanoma SK-MEL-2 cells.36 As compared to other ginger extracts, 10-G showed the

284

remarkable anti-cancerous effects. In our previous study, the IC50 of 10-G (29.19 µM),

285

6-G (96.32 µM), 8-G (43.17 µM), crude extract of ginger leaves (165.91 µg/mL) and

286

roots (176.58 µg/mL) demonstrated 10-G as the best inhibitor of Hela cell

287

proliferation (data not shown). Above results suggest that Tongling White Ginger is

288

suitable to be used as an important ingredient for functional food.

289

Previous reports suggested that higher alkyl chain leads to increase in lipophilic

290

character (log P) of homologous series of gingerols which further greatly impact the

291

inhibition of tumor cell proliferation, by affecting the entry of these substances in the 14

ACS Paragon Plus Environment

Page 14 of 38

Page 15 of 38

Journal of Agricultural and Food Chemistry

292

cells through plasma membrane. Subsequently, the higher log P value corresponds to

293

the higher permeation in biological membranes.37, 38 Kim and coworkers concluded

294

that IC50 for 10-G (13 µM to 40 µΜ) was more effective than 8-G in inhibiting the

295

proliferation of human tumor cells lines.36 Similarly, in another study, Wei and

296

coworkers also observed the inhibition of HL-60 proliferation by IC50 for 10-G (56.5

297

µM) and 8-G (87.9 µM)”.39 Thus, from our results we can conclude that the

298

anti-cancerous properties of ginger can be attributed to 10-G.

299

Effect of 10-gingerol on the toxicity to cancer cells

300

The release of LDH is usually regarded as an index to assess the integrity of cell

301

membranes and also to evaluate the efficacy of cytotoxicity on cancer cells by plant

302

derived extractions. As shown in Figure 3C, after exposure to 10-G for 48 h, the LDH

303

vitalities were quantitatively assessed for the cytotoxicity on treated cells increased

304

gradually in dose dependent manner with the comparison between 5-FU and untreated

305

cells. When the concentration increased to 100 µM, 10-G displayed the similar

306

cytotoxicity on Hela cells by 5-FU (80 µM) (Figure 3C), and as compared to 5-FU,

307

10-G had shown remarkable anti-cancer properties.

308

Effect of 10-gingerol on cancer cell morphology

309

Hela cells subjected to 10-G (30 µM) for 24, 48 and 60 h exhibited significant

310

abnormal forms as compared to untreated and 5-FU-treated cells as negative and

311

positive controls (Figure 3). After treatment of 10-G for 48 h, Hela cells displayed

312

distinct changes as compared to the negative cells. After 60 h, the damage to HeLa 15

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

313

cells by 10-G (30 µM) was found to be more severe than those treated by 5-FU

314

(Figure 3) (increased number of dead cells, apoptotic cells or cell spherical

315

suspensions). Cells were reduced in number, appeared round and distorted, lost their

316

contact with adjacent cells, and more floating cells, pyknosis, and apoptotic bodies

317

were observed. Furthermore, appearance of vacuoles in the cytoplasm of treated cells

318

was also noticed (Figure 3).

319

Effects of 10-gingerol on cancer cell cycle

320

The effects of 30 µM 10-G on HeLa cell cycle progression was examined by flow

321

cytometric measurement of cellular DNA content (Figure 4A), and the percentage of

322

cells in G0/G1 stage was summarized in Figure 4B. With the increase in time, the

323

significant changes appeared in treated cells when compared with the untreated at

324

G0/G1 stage.

325

The HeLa cells stimulated with 10-G (30 µM) revealed a slight increase at the stage

326

of G0/G1: 1.8% after 6 h, and quick rise to 9.8% at 12 h. After cultivated with 10-G

327

for 18 h, 67.48% HeLa cells were arrested at G0/G1 stage, and apoptotic cells were

328

about two times of the cells present in control group (Figure 4B). After treatment with

329

10-G for 24 h, the apoptotic cells increased to 33.62%, which could be associated

330

with the block from G0/G1-phase progress into S-phase induced by 10-G. The above

331

results suggested that 10-G can induce HeLa cells cycle arrest in G0/G1-phase. The

332

in-depth studies to know more about the mechanisms of blocking would be of great

333

importance.

334

As 10-G arrested Hela cells in G0/G1 phase, we further studied the molecular 16

ACS Paragon Plus Environment

Page 16 of 38

Page 17 of 38

Journal of Agricultural and Food Chemistry

335

regulation mechanism by using different concentrations of 10-G, and also by

336

evaluating expression of several important cell cycle-related genes (cyclin A, cyclin

337

B1, cyclin D1, cyclin E1, CDK-1, CDK-2, CDK-4, CDK-6, p15, p16, p21, p27,

338

GSK-3β, and β-catenin) using RT-qPCR analysis. As shown in Figure 4C, cyclin A,

339

cyclin D1, cyclin E1 CDK-2, CDK-4, CDK-6, p15 and p21 mRNA expression was

340

significantly decreased in a dose-dependent manner, while CDK-1, p16, p27, GSK-3β,

341

and β-catenin mRNA expression presented a slight decrease, and nevertheless the

342

cyclin B1 was not evidently changed.

343

Subsequently, the effects of 10-G on the protein levels of the above target genes were

344

studied by western blot analysis. Our results showed the decrease in cyclin A and

345

cyclin D1 protein levels in treated cells (Figure 4D,E), which are the essential markers

346

of G1/S phase, whereas, the cyclin E1 protein had shown on alteration (Figure 4D,E),

347

Similarly, the expression of cyclin B1 as well as the G2/M phase marker did not show

348

any significant change (Figure 4D,E).

349

These results lead to an understanding that 10-G play an important role in controlling

350

the expression of cycle-related targets both at the transcriptional and

351

posttranscriptional level in cancer cells.

352

Effects of 10-gingerol on cancer cell apoptosis

353

It is well-known that apoptosis is implicated for multistage carcinogenesis and

354

phytochemical complexity of plant foods conferring health-promoting benefits

355

including chemo preventive and anticancer effects. To investigate the effects of

356

cytotoxicity of apoptosis by 10-G, the ratios of the sum of apoptotic cells after 17

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

357

exposure were assessed by flow cytometry using annexin V-FITC and PI labeling

358

after treatments for 12, 24 and 48 h (Figure 5). The apoptosis cells distributed in early

359

stage and late stage were summarized in Table 2. Compared to the negative control,

360

the apoptotic cells induced by 10-G increased dramatically (Figure 5 and Table 2).

361

The apoptotic cells showed no distinctly different attributes after treatment for 12 h

362

with 5-FU and 10-G. When the exposure time was extended, the apoptotic cells

363

numbers (treated by 10-G) were higher to those treated by 5-FU. Particularly, after 48

364

h, the apoptotic cells both in early and late stage were higher than corresponding

365

numbers by 5-FU (Figure 5 and Table 2). The above results indicated that 10-G can

366

induce apoptosis of cancer HeLa cells. Previously confirmed anti-cancerous activities

367

of 10-G against HCT116 human colon cancer cells revealed that inhibition of the

368

proliferation of cells was accompanied by the morphological changes indicative of

369

apoptosis.18

370

Apoptosis is biologically regulated process which comprises two major pathways: the

371

extrinsic and intrinsic pathway.40 Death receptor 3 and 5 (DR3 and DR5), also known

372

as tumor necrosis factor receptors are present on cell surface and mediate apoptotic

373

signaling and differentiation.41 Our study revealed that, 10-G could significantly up

374

regulate both the apoptotic proteins (DR3 and DR5) and resulted in the activation of

375

apoptosis indicators (cleaved caspases-3, -8, and -9) (Figure 6). The increased

376

expression of the caspase cascade leads to 10-G induced apoptosis process.

377

Furthermore, the expression of cytochrome c, Bid, Bad, Bax and Bcl-2 was evaluated

378

to study the mitochondria dependent pathway and significant increase in the 18

ACS Paragon Plus Environment

Page 18 of 38

Page 19 of 38

Journal of Agricultural and Food Chemistry

379

expression of these proteins was observed (Figure 6) except Bcl-2 which decreased

380

significantly followed by slight decrease in Bid and latter resulted in increased

381

cleaved Bid which ultimately activates Bax. Our results conclude that 10-G activated

382

the intrinsic apoptotic signaling pathways in the Hela cells and led to cell death due to

383

mitochondrial dysfunction.

384

PI3K/AKT pathway as a vital component in the regulation of tumorigenesis and its

385

progression as well as known as upstream signaling molecules of the mTOR pathway

386

get activated on their specific sites through phosphorylation.32 The anti-cancerous

387

activities of 10-G could be associated with a control of signal transduction of

388

PI3K/AKT pathway and this led us to investigate the change of the survival pathway

389

associated proteins. Previous studies suggested that 6-gingerol had no effect on the

390

expression of PI3K, p85α, however, 6-gingerol increased phosphorylation of AKT,

391

which is regulated by PI3K17. Activated AKT promotes the cell survival by

392

anti-apoptotic mechanism and also inactivates the proapoptotic proteins.17 Our present

393

study showed treated cells significantly inhibited the phosphorylation of PI3K (Figure

394

6). The decreases in phosphorylation level of AKT and P70S6K down regulated PI3K

395

while triggered the mTOR (Figure 6). On the other hand, PI3K also changed PKCε,

396

thus regulated the decrease of NF-kβ expression (Figure 6). As known previously, that

397

10-G affected Ca2+ and led to cell death in human colorectal cancer cells,4 and our

398

results showed that it led to induction of AMPK (1.8-fold increase) which finally

399

inhibited mTOR phosphorylation (0.6-fold decrease) signaling.

400

The above results confirmed the anti-cancerous potential of 10-G, and suggested that 19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

401

Tongling White Ginger can be utilized as functional food for anticancer effects. We

402

also emphasize the exploitation and utilization of fresh ginger or its components to be

403

explored for other health disorders or ailments and also to preserve the native bio

404

heritage and medicinal properties of native Chinese plants by using suitable animal

405

models followed by clinical trials. This study would be an asset to establish synergy

406

of 10-G and other medicinal plant products for future applications.

407

Effect of inhibitors targeting signal transduction pathway on 10-G induced

408

apoptosis

409

To gain insights into the relationship between 10-G and apoptosis-related pathways,

410

we used the p53 inhibitor (Pifithrin-µ), PI3K inhibitor (Wortmannin), AKT inhibitor

411

(Deguelin), AMPK inhibitor (WZ4003) and mTOR inhibitor (Temsirolimus) to

412

pretreat the Hela cells before 10-G treatment. As shown in Figure 7, these results

413

indicated that treatment with these five inhibitors to a certain extent, could influence

414

the expression of their corresponding proteins. Particularly, Pifithrin-µ and WZ4003

415

dramatically decreased 10-G induced activation of the p53 and p-AMPK protein,

416

respectively. Moreover, cells pre-treatment with Wortmannin could reduce the

417

expression level of p-PI3K, followed by slight augmentation in the inhibitory effects

418

of 10-G on Hela cells. In addition, exposure with Deguelin and Temsirolimus could

419

weakened the inhibitory attributes of 10-G on the expression of p-AKT and p-mTOR.

420

Thus, we speculated, it might be due to their special structure-activity relationship

421

between 10-G and inhibitor, which caused the corresponding competitive binding and

422

masked their synergetic effect. On the other hand, compared with 10-G treatment 20

ACS Paragon Plus Environment

Page 20 of 38

Page 21 of 38

Journal of Agricultural and Food Chemistry

423

alone, 10-G significantly enhanced the inhibition effect of Deguelin and Temsirolimus

424

on correspondent protein expression. Our results further confirmed 10-G induced

425

apoptosis by PI3K/AKT/AMPK/mTOR signaling pathways in Hela cells.

426

Overall results showed that 10-G triggered mTOR mediated cell apoptosis by

427

inhibiting PI3K/AKT and activating AMPK. After analyzing the cell cycle and

428

apoptosis mechanisms of 10-G, present investigation presented an outline of

429

mechanisms involved in anti-cancerous properties of 10-G (Figure 8).

430

Abbreviations Used 10-G

10-Gingerol

HPLC

High Performance Liquid Chromatography

HSCCC

High-speed Countercurrent Chromatography

UPLC-TOF-MS/MS

Ultra-Performance Liquid Chromatography Time-of-Flight Mass Spectrometer

NMR

Nuclear Magnetic Resonance

DMEM

Dulbecco’s Minimum Essential Medium

FBS

Fetal bovine serum

5-FU

Fluorouracil

CCK-8

Cell Counting kit-8

LDH

Lactate dehydrogenase

PI

Propidium iodide

431

Acknowledgements

432

This study was supported by the Major Projects of Science and Technology in Anhui

433

Province (15czz03115), the grants from the National Natural Science Foundation of 21

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

434

China (31371947 and 31272111), the Key projects of Natural Science Research of

435

Anhui Province (KJ2016A575) and the Special Fund for Agro-scientific Research in

436

the Public Interest of China (201403064).

437

There is no conflict of interest to declare.

438

References

439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471

(1) Feng, T.; Su, J.; Ding, Z. H.; Zheng, Y. T.; Li, Y.; Leng, Y.; Liu, J. K. Chemical constituents and their bioactivities of "Tongling White Ginger" (Zingiber officinale). J Agric Food Chem 2011, 59, 11690-5. (2) Shukla, Y.; Singh, M. Cancer preventive properties of ginger: a brief review. Food Chem Toxicol 2007, 45, 683-90. (3) Lee, E.; Surh, Y. J. Induction of apoptosis in HL-60 cells by pungent vanilloids, [6]-gingerol and [6]-paradol. Cancer Lett 1998, 134, 163-8. (4) Chen, C. Y.; Li, Y. W.; Kuo, S. Y. Effect of [10]-gingerol on [ca2+]i and cell death in human colorectal cancer cells. Molecules 2009, 14, 959-69. (5) Sekiwa, Y.; Kubota, K.; Kobayashi, A. Isolation of novel glucosides related to gingerdiol from ginger and their antioxidative activities. J Agric Food Chem 2000, 48, 373-7. (6) Baliga, M. S.; Haniadka, R.; Pereira, M. M.; D'Souza, J. J.; Pallaty, P. L.; Bhat, H. P.; Popuri, S. Update on the chemopreventive effects of ginger and its phytochemicals. Crit Rev Food Sci Nutr 2011, 51, 499-523. (7) Gundala, S. R.; Mukkavilli, R.; Yang, C.; Yadav, P.; Tandon, V.; Vangala, S.; Prakash, S.; Aneja, R. Enterohepatic recirculation of bioactive ginger phytochemicals is associated with enhanced tumor growth-inhibitory activity of ginger extract. Carcinogenesis 2014, 35, 1320-9. (8) Shishodia, S.; Sethi, G.; Aggarwal, B. B. Curcumin: getting back to the roots. Ann N Y Acad Sci 2005, 1056, 206-17. (9) Surh, Y. J. Anti-tumor promoting potential of selected spice ingredients with antioxidative and anti-inflammatory activities: a short review. Food Chem Toxicol 2002, 40, 1091-7. (10) Aggarwal, B. B.; Shishodia, S. Molecular targets of dietary agents for prevention and therapy of cancer. Biochem Pharmacol 2006, 71, 1397-421. (11) Semwal, R. B.; Semwal, D. K.; Combrinck, S.; Viljoen, A. M. Gingerols and shogaols: Important nutraceutical principles from ginger. Phytochemistry 2015, 117, 554-568. (12) Stoilova, I.; Krastanov, A.; Stoyanova, A.; Denev, P.; Gargova, S. Antioxidant activity of a ginger extract (Zingiber officinale). Food Chem 2007, 102, 764-770. (13) Dugasani, S.; Pichika, M. R.; Nadarajah, V. D.; Balijepalli, M. K.; Tandra, S.; Korlakunta, J. N. Comparative antioxidant and anti-inflammatory effects of [6]-gingerol, [8]-gingerol, [10]-gingerol and [6]-shogaol. J Ethnopharmacol 2010, 127, 515-20. (14) Sang, S.; Hong, J.; Wu, H.; Liu, J.; Yang, C. S.; Pan, M. H.; Badmaev, V.; Ho, C. T. Increased growth inhibitory effects on human cancer cells and anti-inflammatory potency of shogaols from Zingiber officinale relative to gingerols. J Agric Food Chem 2009, 57, 10645-50. (15) Eren, D.; Betul, Y. M. Revealing the effect of 6-gingerol, 6-shogaol and curcumin on mPGES-1, 22

ACS Paragon Plus Environment

Page 22 of 38

Page 23 of 38

Journal of Agricultural and Food Chemistry

472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515

GSK-3beta and beta-catenin pathway in A549 cell line. Chem Biol Interact 2016, 258, 257-65. (16) Ho, S. C.; Chang, K. S.; Lin, C. C. Anti-neuroinflammatory capacity of fresh ginger is attributed mainly to 10-gingerol. Food Chem 2013, 141, 3183-91. (17) Park, Y. J.; Wen, J.; Bang, S.; Park, S. W.; Song, S. Y. 6-Gingerol induces cell cycle arrest and cell death of mutant p53-expressing pancreatic cancer cells. Yonsei Med J 2006, 47, 688-97. (18) Ryu, M. J.; Chung, H. S. [10]-Gingerol induces mitochondrial apoptosis through activation of MAPK pathway in HCT116 human colon cancer cells. In Vitro Cell Dev Biol Anim 2015, 51, 92-101. (19) Feng, Y.; Xu, X.; Zhang, Y.; Ding, J.; Wang, Y.; Zhang, X.; Wu, Z.; Kang, L.; Liang, Y.; Zhou, L.; Song, S.; Zhao, K.; Ye, Q. HPIP is upregulated in colorectal cancer and regulates colorectal cancer cell proliferation, apoptosis and invasion. Sci Rep 2015, 5, 9429. (20) Thornberry, N. A.; Rano, T. A.; Peterson, E. P.; Rasper, D. M.; Timkey, T.; Garcia-Calvo, M.; Houtzager, V. M.; Nordstrom, P. A.; Roy, S.; Vaillancourt, J. P.; Chapman, K. T.; Nicholson, D. W. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem 1997, 272, 17907-11. (21) Balaban, R. S.; Nemoto, S.; Finkel, T. Mitochondria, oxidants, and aging. Cell 2005, 120, 483-95. (22) Reuter, S.; Eifes, S.; Dicato, M.; Aggarwal, B. B.; Diederich, M. Modulation of anti-apoptotic and survival pathways by curcumin as a strategy to induce apoptosis in cancer cells. Biochem Pharmacol 2008, 76, 1340-51. (23) Johnson, J. J.; Mukhtar, H. Curcumin for chemoprevention of colon cancer. Cancer Lett 2007, 255, 170-81. (24) Nagendra chari, K. L.; Manasa, D.; Srinivas, P.; Sowbhagya, H. B. Enzyme-assisted extraction of bioactive compounds from ginger (Zingiber officinale Roscoe). Food Chem 2013, 139, 509-514. (25) Zhan, K.; Xu, K.; Yin, H. Preparative separation and purification of gingerols from ginger (Zingiber officinale Roscoe) by high-speed counter-current chromatography. Food Chem 2011, 126, 1959-1963. (26) Pawar, N.; Pai, S.; Nimbalkar, M.; Dixit, G. RP-HPLC analysis of phenolic antioxidant compound 6-gingerol from different ginger cultivars. Food Chem 2011, 126, 1330-1336. (27) Lv, L.; Chen, H.; Soroka, D.; Chen, X.; Leung, T.; Sang, S. 6-gingerdiols as the major metabolites of 6-gingerol in cancer cells and in mice and their cytotoxic effects on human cancer cells. J Agric Food Chem 2012, 60, 11372-7. (28) Jung, J. H.; Lee, J. O.; Kim, J. H.; Lee, S. K.; You, G. Y.; Park, S. H.; Park, J. M.; Kim, E. K.; Suh, P. G.; An, J. K.; Kim, H. S. Quercetin suppresses HeLa cell viability via AMPK-induced HSP70 and EGFR down-regulation. J Cell Physiol 2010, 223, 408-14. (29) Johnson, N.; Li, Y. C.; Walton, Z. E.; Cheng, K. A.; Li, D.; Rodig, S. J.; Moreau, L. A.; Unitt, C.; Bronson, R. T.; Thomas, H. D.; Newell, D. R.; D'Andrea, A. D.; Curtin, N. J.; Wong, K. K.; Shapiro, G. I. Compromised CDK1 activity sensitizes BRCA-proficient cancers to PARP inhibition. Nat Med 2011, 17, 875-82. (30) Wakatsuki, S.; Furuno, A.; Ohshima, M.; Araki, T. Oxidative stress-dependent phosphorylation activates ZNRF1 to induce neuronal/axonal degeneration. J Cell Biol 2015, 211, 881-96. (31) Petrocca, F.; Visone, R.; Onelli, M. R.; Shah, M. H.; Nicoloso, M. S.; de Martino, I.; Iliopoulos, D.; Pilozzi, E.; Liu, C. G.; Negrini, M.; Cavazzini, L.; Volinia, S.; Alder, H.; Ruco, L. P.; Baldassarre, G.; Croce, C. M.; Vecchione, A. E2F1-regulated microRNAs impair TGFbeta-dependent cell-cycle arrest and apoptosis in gastric cancer. Cancer Cell 2008, 13, 272-86. (32) Lin, H.-C.; Lin, M.-H.; Liao, J.-H.; Wu, T.-H.; Lee, T.-H.; Mi, F.-L.; Wu, C.-H.; Chen, K.-C.; Cheng, C.-H.; Lin, C.-W. Antroquinonol, a Ubiquinone Derivative from the Mushroom Antrodia camphorata, 23

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542

Inhibits Colon Cancer Stem Cell-like Properties: Insights into the Molecular Mechanism and Inhibitory Targets. J Agric Food Chem 2016. (33) Lee, D.; Go, G.-W.; Imm, J.-Y. Tricin, a methylated cereal flavone, suppresses fat accumulation by downregulating AKT and mTOR in 3T3-L1 preadipocytes. Journal of Functional Foods 2016, 26, 548-556. (34) Shukla, Y.; Prasad, S.; Tripathi, C.; Singh, M.; George, J.; Kalra, N. In vitro and in vivo modulation of testosterone mediated alterations in apoptosis related proteins by [6]-gingerol. Mol Nutr Food Res 2007, 51, 1492-502. (35) Lee, H. S.; Seo, E. Y.; Kang, N. E.; Kim, W. K. [6]-Gingerol inhibits metastasis of MDA-MB-231 human breast cancer cells. J Nutr Biochem 2008, 19, 313-9. (36) Kim, J. S.; Lee, S. I.; Park, H. W.; Yang, J. H.; Shin, T. Y.; Kim, Y. C.; Baek, N. I.; Kim, S. H.; Choi, S. U.; Kwon, B. M.; Leem, K. H.; Jung, M. Y.; Kim, D. K. Cytotoxic components from the dried rhizomes of Zingiber officinale Roscoe. Arch Pharm Res 2008, 31, 415-8. (37) Hansch, C.; Fujita, T. p-σ-π Analysis. A Method for the Correlation of Biological Activity and Chemical Structure. J Am Chem Soc 1964, 86, 1616-1626. (38) Roh, T.; Kim, S. W.; Moon, S. H.; Nam, M. J. Genistein induces apoptosis by down-regulating thioredoxin-1 in human hepatocellular carcinoma SNU-449 cells. Food Chem Toxicol 2016, 97, 127-134. (39) Wei, Q. Y.; Ma, J. P.; Cai, Y. J.; Yang, L.; Liu, Z. L. Cytotoxic and apoptotic activities of diarylheptanoids and gingerol-related compounds from the rhizome of Chinese ginger. J Ethnopharmacol 2005, 102, 177-84. (40) Murugan, K.; Vanithakumari, G.; Sampathraj, R. Biochemical changes in epididymis following treatment with combined extracts of amaranthus spinosus roots and dolichos biflorus seeds. Anc Sci Life 1993, 13, 154-9. (41) Nishikii, H.; Kim, B. S.; Yokoyama, Y.; Chen, Y.; Baker, J.; Pierini, A.; Alvarez, M.; Mavers, M.; Maas-Bauer, K.; Pan, Y.; Chiba, S.; Negrin, R. S. DR3 signaling modulates the function of Foxp3+ regulatory T cells and the severity of acute graft-versus-host disease. Blood 2016, 128, 2846-2858.

543

24

ACS Paragon Plus Environment

Page 24 of 38

Page 25 of 38

Journal of Agricultural and Food Chemistry

544

Figure captions

545

Figure 1 HPLC chromatograms and Mass spectrum of extracts from Tongling White

546

Ginger. (A) HPLC chromatograms; (B) Mass spectrum. Column:

547

Alltech-C18 column with 5 µm particle size; mobile phase: acetonitrile:

548

water (58:42); flow rate: 0.8 mL/min; injected volume: 20 µL samples

549

solution; detection wavelength: 280nm.

550

Figure 2 The inhibition and cytotoxicity assessment of 10-gingerol on Hela cells. (A)

551

inhibition of 10-gingerol on the growth of HeLa cells; (B) inhibition curve

552

fitting, HeLa and HEK293 cells were used to analysis; (C) the cytotoxicity of

553

10-gingerol and 5-FU on HeLa cells. All the data were collected after

554

treatment for 48 h, and expressed as mean ± SD of three replicates.

555

Figure 3 The morphological changes of HeLa cell on exposure to 10-gingerol. These

556

were observed by an Inverted Optic Microscope (original magnification ×40).

557

Cells were reduced in number, appeared round and distorted, lost their

558

contact with adjacent cells, and more floating cells (green arrow), pyknosis

559

(white arrow), and apoptotic bodies (blue arrow) were observed. Furthermore,

560

appearance of vacuoles in the cytoplasm of treated cells (yellow arrow) was

561

also noticed. The concentration of 10-gingerol and 5-FU used for treatment

562

of cells were 30 µM and 50 µM, respectively.

563

Figure 4 (A) 10-Gingerol induces cell cycle arrest in HeLa cell lines. The

564

concentration of 10-gingerol used for treatment of cells was 30 µM. (B) The

565

rate of cell cycle arrest in apoptosis and G0/G1-phase. (C) The mRNA

566

expression of cell cycle related genes with increasing concentrations of 25

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

567

10-gingerol (15 µM, 30 µM and 50 µM) for 48 h as compared to untreated

568

cells. Relative gene-expression levels are expressed with GAPDH as an

569

internal reference. (D) Effect of 10-gingerol on phosphorylation of cell cycle

570

related proteins in Hela cells treated for 48 h analyzed by western blot using

571

tubulin as an internal control. (E) The expression level of the targeted

572

proteins with increasing concentrations of 10-gingerol (15 µM, 30 µM and

573

50 µM) for 48 h as compared to untreated cells. Each value is presented as a

574

mean ± standard deviation (n = 3). Values of a-d represent significantly

575

different under different treatments within same gene, P < 0.05.

576

Figure 5 The effects of 10-gingerol on HeLa cell apoptosis. The concentration of

577

10-gingerol and 5-FU used for treatment of cells were 30 µM and 50 µM,

578

respectively.

579

Figure 6 (A) Effect of 10-gingerol on phosphorylation of apoptosis related proteins in

580

Hela cells treated for 48 h analyzed by western blot using tubulin as an

581

internal control. (B) The expression level of the targeted proteins with

582

increasing concentrations of 10-gingerol (15 µM, 30 µM and 50 µM) for 48 h

583

as compared to untreated cells. Each value is presented as a mean ± standard

584

deviation (n = 3). Values of a-d represent significantly different under

585

different treatments within same gene, P < 0.05.

586

Figure 7 (A) Effect of 10-gingerol on phosphorylation of apoptosis related proteins in

587

Hela cells treated with or without inhibitors (Pifithrin-µ for p53, Wortmannin

588

for PI3K, Deguelin for AKT, WZ4003 for AMPK and Temsirolimus for 26

ACS Paragon Plus Environment

Page 26 of 38

Page 27 of 38

Journal of Agricultural and Food Chemistry

589

mTOR) by western blot using tubulin as an internal control. (B) The

590

expression level of the targeted proteins treated by 10-gingerol (with or

591

without inhibitor) at 30 µM as compared to untreated cells. Each value is

592

presented as a mean ± standard deviation (n = 3). Values of a-d represent

593

significantly different under different treatments within same gene, P < 0.05

594 595

Figure 8 Proposed mechanism for the effects of 10-gingerol on cell cycle and apoptosis with a series of pathways involved.

596

27

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

597 598

Table 1 Primers for real-time PCR Gene Cyclin A Cyclin B1 Cyclin D1 Cyclin E1 CDK-1 p21 p27 GAPDH CDK-2 CDK-4 CDK-6 P15 P16 GSK-3β β-catenin

Primer Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

Sequence (5’-3’) AGACTGAGTGGTTGGATGGCA TGTCCACAGTCAGCAATGGTG AAAGGCGTAACTCGAATGGA CCGACCTTTTATTGAAGAGCA ATGGAACACCAGCTCCTGTGCTGC TCAGATGTCCACGTCCCGCACGT GGATTATTGCACCATCCAGAGGCT CTTGTGTCGCCATATACCGGTCAA TCCGCAACAGGGAAGAAC GAGCCTTTTTAGATGGCTGCT CCACAGCGATATCCAGACATTC GAAGTCAAAGTTCCACCGTTCTC AGCGACCTGCTGCAGAAGAT TTACGTCTGGCGTCGAAGGC TGCCCTCAACGACCACTTTG TACTCCTTGGAGGCCATGTG CTTTGGAGTCCCTGTCCGTA CGAAAGATCCGGAAGAGTTG TGCACAGTGTCACGAACAGA ACCTCGGAGAAGCTGAAACA CATCGTTCACCGAGATCTGA CCAACACTCCACATGTCCAC GCGGCAGCTCCTGGAAG GGGTCGGCACAGTTGG CTTCCTGGACACGCTGGT ATCTATGCGGGCATGGTTACT TCCATTCCTTTGGGATCTGCC ATCAGCTCTGGTGCCCTGTAGTAC GCTGATTTGATGGAGTTGGACATGG GCCAAACGCTGGACATTAGTGG

599

28

ACS Paragon Plus Environment

Page 28 of 38

Page 29 of 38

Journal of Agricultural and Food Chemistry

600 601

Table 2 The Statistical analysis of effects of 10-gingerol on HeLa cell apoptosis

602 12 h of treatment Rate of cells

24 h of treatment

Early Late stage Normal Normal stage apoptosis cells cells (%)apoptosis cells (%) (%) cells (%)

48 h of treatment

Early Late stage Normal stage apoptosis cells apoptosis cells (%) (%) cells (%)

Early Late stage stage apoptosis apoptosis cells (%) cells (%)

Control

93.76

1.10

2.04

87.11

2.36

6.58

82.36

2.68

10.40

5-FU

86.07

1.84

8.06

72.27

4.38

10.05

53.91

7.26

16.51

10-gingerol 72.98

5.19

8.78

63.19

4.33

22.31

33.82

16.09

26.35

603

29

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 1 HPLC chromatograms and Mass spectrum of extracts from Tongling White Ginger. (A) HPLC chromatograms; (B) Mass spectrum. Column: Alltech-C18 column with 5 µm particle size; mobile phase: acetonitrile: water (58:42); flow rate: 0.8 mL/min; injected volume: 20 µL samples solution; detection wavelength: 280nm. 177x199mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 30 of 38

Page 31 of 38

Journal of Agricultural and Food Chemistry

Figure 2 The inhibition and cytotoxicity assessment of 10-gingerol on Hela cells. (A) inhibition of 10-gingerol on the growth of HeLa cells; (B) inhibition curve fitting, HeLa and HEK293 cells were used to analysis; (C) the cytotoxicity of 10-gingerol and 5-FU on HeLa cells. All the data were collected after treatment for 48 h, and expressed as mean ± SD of three replicates. 160x47mm (300 x 300 DPI)

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 3 The morphological changes of HeLa cell on exposure to 10-gingerol. These were observed by an Inverted Optic Microscope (original magnification ×40). Cells were reduced in number, appeared round and distorted, lost their contact with adjacent cells, and more floating cells (green arrow), pyknosis (white arrow), and apoptotic bodies (blue arrow) were observed. Furthermore, appearance of vacuoles in the cytoplasm of treated cells (yellow arrow) was also noticed. The concentration of 10-gingerol and 5-FU used for treatment of cells were 30 µM and 50 µM, respectively. 277x190mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 32 of 38

Page 33 of 38

Journal of Agricultural and Food Chemistry

Figure 4 (A) 10-Gingerol induces cell cycle arrest in HeLa cell lines. The concentration of 10-gingerol used for treatment of cells was 30 µM. (B) The rate of cell cycle arrest in apoptosis and G0/G1-phase. (C) The mRNA expression of cell cycle related genes with increasing concentrations of 10-gingerol (15 µM, 30 µM and 50 µM) for 48 h as compared to untreated cells. Relative gene-expression levels are expressed with GAPDH as an internal reference. (D) Effect of 10-gingerol on phosphorylation of cell cycle related proteins in Hela cells treated for 48 h analyzed by western blot using tubulin as an internal control. (E) The expression level of the targeted proteins with increasing concentrations of 10-gingerol (15 µM, 30 µM and 50 µM) for 48 h as compared to untreated cells. Each value is presented as a mean ± standard deviation (n = 3). Values of a-d represent significantly different under different treatments within same gene, P < 0.05. 160x187mm (300 x 300 DPI)

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 5 The effects of 10-gingerol on HeLa cell apoptosis. The concentration of 10-gingerol and 5-FU used for treatment of cells were 30 µM and 50 µM, respectively. 160x133mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 34 of 38

Page 35 of 38

Journal of Agricultural and Food Chemistry

Figure 6 (A) Effect of 10-gingerol on phosphorylation of apoptosis related proteins in Hela cells treated for 48 h analyzed by western blot using tubulin as an internal control. (B) The expression level of the targeted proteins with increasing concentrations of 10-gingerol (15 µM, 30 µM and 50 µM) for 48 h as compared to untreated cells. Each value is presented as a mean ± standard deviation (n = 3). Values of a-d represent significantly different under different treatments within same gene, P < 0.05. 160x198mm (300 x 300 DPI)

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 7 (A) Effect of 10-gingerol on phosphorylation of apoptosis related proteins in Hela cells treated with or without inhibitors (Pifithrin-µ for p53, Wortmannin for PI3K, Deguelin for AKT, WZ4003 for AMPK and Temsirolimus for mTOR) by western blot using tubulin as an internal control. (B) The expression level of the targeted proteins treated by 10-gingerol (with or without inhibitor) at 30 µM as compared to untreated cells. Each value is presented as a mean ± standard deviation (n = 3). Values of a-d represent significantly different under different treatments within same gene, P < 0.05. 160x80mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 36 of 38

Page 37 of 38

Journal of Agricultural and Food Chemistry

Figure 8 Proposed mechanism for the effects of 10-gingerol on cell cycle and apoptosis with a series of pathways involved. 160x137mm (300 x 300 DPI)

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Graphic for table of contents 45x24mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 38 of 38