Andrographolide Antagonizes TNF-Î±-induced IL-8 via Inhibition of

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Bioactive Constituents, Metabolites, and Functions

Andrographolide Antagonizes TNF-#-induced IL-8 via Inhibition of NADPH Oxidase/ROS/NF-#B and Src/MAPKs/ AP-1 Axis in Human Colorectal Cancer HCT116 Cells Miaomiao Yuan, Wei Meng, Wenzhen Liao, and Sen Lian J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00810 • Publication Date (Web): 19 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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Journal of Agricultural and Food Chemistry

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Andrographolide Antagonizes TNF-α-induced IL-8 via Inhibition of NADPH

2

Oxidase/ROS/NF-κB and Src/MAPKs/AP-1 Axis in Human Colorectal Cancer

3

HCT116 Cells

4 5

Miaomiao Yuan§, ¶, ˧,, Wei Meng†,⁋, ˧, Wenzhen Liaoǁ,*, Sen Lian†,⁋,*

6 7

†

8

Sciences, Southern Medical University, Guangzhou 510515, Guangdong, China;

9

§

Department of Biochemistry and Molecular Biology, School of Basic Medical

Guangdong Provincial Key Laboratory of Cancer Immunotherapy Research,

10

Southern Medical University, Guangzhou 510515, Guangdong, China;

11

ǂ

12

University, Guangzhou 510515, Guangdong, China;

13

ǁ

14

of Tropical Disease Research, School of Public Health, Southern Medical University,

15

Guangzhou 510515, Guangdong, China;

16

¶

17

University, Guangzhou, Guangdong 510515, Guangdong, China;

18

⁋

19

China;

Guangzhou Key Laboratory of Tumor Immunology Research, Southern Medical

Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory

Cancer Research Institute, School of Basic Medical Sciences, Southern Medical

Guangdong Provincial Key Laboratory of Biochip, Guangzhou 510515, Guangdong,

20 21

˧

These authors are joint first authors on this work.

22 23

Co-corresponding authors:

24

Sen Lian

ACS Paragon Plus Environment


25

Department of Biochemistry and Molecular Biology, School of Basic Medical

26

Sciences, Southern Medical University, No.1023 South Shatai Road, Guangzhou

27

510515, China.

28

Tel: (+86) 20- 62789385; Fax: (+86) 20- 62789385;

29

Email: [email protected]

30

Wenzhen Liao

31

Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of

32

Tropical Disease Research, School of Public Health, Southern Medical University,

33

No.1023 South Shatai Road, Guangzhou 510515, China.

34

Tel: (+86) 20-61648309; Fax: (+86) 20-61648324;

35

E-mail: [email protected]; [email protected]


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Abstract

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Andrographis paniculata Nees is used as a functional food in Japan, Korea, India and

38

China. Andrographolide, a naturally occurring phytochemical identified in

39

Andrographis paniculata, has been discovered to present anti-inflammatory and

40

anticancer activities. Highly expressed of interleukin (IL-8) has been detected in

41

colorectal cancer and is implicated in angiogenesis. However, the effect and

42

molecular mechanisms of IL-8 expression by andrographolide remain obscure in

43

human colorectal cancer cells. The present study was aimed to investigate the effects

44

of andrographolide on TNF-α-induced IL-8 expression and its underlying mechanisms.

45

We found that andrographolide concentration-dependently inhibited TNF-α-induced

46

IL-8 mRNA (2.23 ±0.15 fold at 20 μM) and protein expression (4.78 ±0.31 fold at 20

47

μM), and reduced the IL-8 transcriptional activity (2.59 ± 0.25 fold at 20 μM). TNF-α

48

stimulated the membrane translocation of p47phox to activate reactive oxygen species

49

(ROS)-producing NADPH oxidase (NOX). Furthermore, TNF-α induced Src and

50

MAPKs (Erk1/2, p38 MAPK) phosphorylation, and NF-κB and AP-1 binding

51

activities. We found that NF-κB and AP-1 were the critical transcription factors for

52

TNF-α-induced IL-8 expression. Specific inhibitors and mutagenesis studies indicated

53

that Src, Erk1/2, p38 MAPK are related to TNF-α-induced IL-8. NOX-derived ROS

54

and Src/MAPKs (Erk1/2 and p38 MAPK) functioned as upstream activators of NF-κB

55

and AP-1, respectively. Taken together, andrographolide antagonizes TNF-α-induced

56

IL-8 via inhibition of NADPH oxidase/ROS/NF-κB and Src/MAPKs/AP-1 signaling

57

pathways in HCT116 colorectal cancer cells and then suppresses angiogenesis in the



58

tumor microenvironment.

59

Key words: Andrographolide, TNF-α, IL-8, NADPH oxidase, Angiogenesis

60

INTRODUCTION

61

Colorectal cancer (CRC) is the leading cause of death from gastrointestinal tumor

62

and second cause of tumor-related death worldwide. It was recently suggested

63

that chemokines act as key regulators of CRC, emergence of a vascular supply,

64

and the acquisition of invasive/metastatic properties 1. IL-8, a member of the

65

neutrophil-specific CXC membrane chemokine family, was related to cancer cell

66

migration, invasion and proliferation 2. IL-8 also acts on endothelial cells to

67

promote in vivo angiogenesis 3. Previous studies have revealed that highly

68

metastatic solid tumors constitutively express IL-8 4. An increase in serum and

69

cancer tissue of IL-8 levels was also demonstrated in CRC patients 5. Clinical

70

studies indicated that increased IL-8 expression in primary CRC tumors

71

accelerates risk for metastatic lesions 6. The gene polymorphism of IL-8 and

72

CXCR2 were reported to associate with clinical outcome in patients with

73

metastatic CRC treated with oxaliplatin-based chemotherapy 7. Therefore, agents

74

with the ability to suppress IL-8 expression may contribute to the development of

75

therapeutic strategy for colorectal cancer.

76

Studies show that inflammation contributes to proliferation and survival of

77

malignant cells 8. TNF-α is a potent proinflammatory cytokine and the plasma

78

concentration of TNF-α is elevated in several pathologies, including cancer 9.

79

Wang et al reported that TNF-α induced epithelial–mesenchymal transition (EMT)

80

in CRC

81

of NADPH oxidase and Src

82

NADPH oxidase, Src , MAPKs and NF-κB are critical for the expression of IL-8

10

. In human endothelial cells, TNF-α stimulated ICAM-1 by activation 11

. Several lines of evidence have shown that


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83

12-13

84

mechanisms are yet to be fully elucidated in CRC.

85

. However, the effect of TNF-α on IL-8 expression and the underlying

Andrographis paniculata Nees is used as a health food in Japan, Korea, 14

86

China and other countries in Southeast Asia

87

abundant diterpene lactone in the leaves and stem of Andrographis paniculata

88

and possesses several beneficial properties, including anti-inflammation,

89

antioxidation and anti-tumor properties. Based on this, andrographolide has been

90

reported to inhibit matrix metalloproteinase-9 expression in MCF-7 breast cancer

91

cells 15. Nevertheless, the role of andrographolide in IL-8 expression has not been

92

elucidated. Here, we studied the effect of andrographolide on TNF-α-induced

93

IL-8 expression and explored the underlying signaling molecular mechanisms.

94

This is the first report that andrographolide suppressed IL-8 via blocking NADPH

95

oxidase/ROS/NF-κB and Src/MAPKs/AP-1 axis in CRC, consequently inhibiting

96

endothelial cell proliferation in tumor microenvironment.

97

Materials and Methods

98

Chemicals

99

PD98059 (PD), SB203580 (SB) and BAY11-7082 (BAY) were from Calbiochem (San

100

Diego, CA); SR11302 (SR) was from Tocris (Bristol, UK); Andrographolide and the

101

other chemicals were from Sigma-Aldrich (St. Louis, MO);

102

Cell Culture

103

The HCT116 CRC cell line and EAhy.926 endothelial cell line were purchased from

104

the American Type Culture Collection (Manassas, VA). HCT116 cells were cultured

105

in Mc-Coy’s 5A medium (Thermo Fisher Scientific, Waltham, MA). EAhy.926 cells

106

were cultured in DMEM medium (Hyclone, Logan, UT). All of the medium was

. Andrographolide is the most



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107

supplemented

108

penicillin–streptomycin (Hyclone, Logan, UT), and the cell lines were maintained at

109

37 °C in a humidified atmosphere consisting of 5% CO2 and 95% air.

110

Cell Viability Assay

111

The viability of HCT116 cells was examined by MTT assay. Afterward, the cell

112

viability assay was performed according to our previous study 16.

113

Isolation of Cell Fractions

114

Homogenization buffer A (200 μL; 20 mM Tris–HCl, pH 8.0, 10 mM EGTA, 2 mM

115

EDTA, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 μg/ml aprotinin,

116

10 μg/mL leupeptin) was added to each dish, and the cells were scraped into a 1.5 mL

117

tube. Cells were centrifuged at 5000× g for 15 min at 4 °C. The pellet was collected as

118

the nuclear fraction. The supernatant was centrifuged at 15000× g at 4 °C for 60 min

119

to yield the pellet (membrane fraction) and the supernatant (cytosolic fraction).

120

Detection of ROS

121

ROS was performed by using the probe H2DCFDA as described previously 17. Images

122

were acquired using the Laser Scanning Microscope 5 PASCAL program (Carl Zeiss)

123

on a confocal microscope. DCF fluorescence was excited at 488 nm with an argon

124

laser, and the evoked emission was filtered with a 515 nm long pass filter.

125

Cell Transfections

126

For transfection of siRNA, cells were seeded at a density of 1.5×105 cells/well in

127

24-well plates and grown until 50% confluent. Cells were transfected with 100 nm

128

siRNA against Src, p47phox (Santa Cruz, Dallas, TX), Erk1/2, P38 and NF-κB P65

with

10%

FBS

(Gibco,

Gaithersburg,


MD)

and

0.6%

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129

(Cell Signaling, Danvers, MA) by using Lipofectamine 2000 (Invitrogen, Carlsbad,

130

CA) according to the manufacturer’s protocol. The phosphorothioate double-stranded

131

ODNs with the sequences against the AP-1 binding site (5'-CAC TCA GAA GTC

132

ACT TC-3' and 3'-GAA GTG ACT TCT GAG CTG-5') were prepared (Genotech, St.

133

Louis, MO) and annealed (AP-1 decoy ODNs).

134

Luciferase Activity analysis

135

An IL-8 promoter-luciferase reporter construct (PGL2-IL-8) was used to determine

136

the transcriptional regulation of IL-8

137

plasmid (Clontech, Palo Alto, CA) were used for the analysis. Luciferase activity

138

analysis was performed according to our previous study 19.

139

IL-8 Quantitative Analysis

140

An enzyme-linked immunosorbent assay (ELISA) kit (R&D system, Minneapolis,

141

VA) was used to determine the release of IL-8.

142

Reverse Transcription-Polymerase Chain Reaction and qPCR

143

Briefly, total RNA was isolated from the cells using TRIzol reagent (Invitrogen,

144

Carlsbad, CA). Synthesis of complementary DNA (cDNA) was performed using

145

M-MLV Reverse Transcriptase kit (Promega, Madison, WI). The specific primer

146

sequences were GAPDH sense, 5′-TTG TTG CCA TCA ATG ACCCC-3′; GAPDH

147

antisense, 5′-TGA CAA AGT GGT CGT TGA GG-3′ (836 bp); IL-8 sense, 5′-ACT

148

TCC AAG CTG GCC GTG GCT-3′ and IL-8 antisense, 5′-TCA CTG GCA TCT

149

TCA CTG ATT-3′ (345 bp). The cycling conditions comprised 94 °C for 30 s, 57 °C

150

for 30 s, and 72 °C for 45 s. The products were resolved on 1.5% agarose gel and

18

. The NF-κB and AP-1 luciferase reporter



151

photographed under ultraviolet light using Image Quant TL analysis software

152

(Amersham Bioscience, Uppsala). qPCR was carried out using Taqman SYBR Green

153

Master Mix (Applied Biosystems, Foster City, CA). The threshold fluorescence level

154

was set manually for each plate using Sequence Detection System software, version

155

1.7 (Applied Biosystems, Foster City, CA). The comparative Ct method was used to

156

calculate the relative abundance of mRNA and compared with that of GAPDH

157

expression 20.

158

Western Blot Analysis

159

Cell lysates were prepared and Western blot analysis was performed as described

160

previously 21. Clathrin HC, c-jun and c-fos antibodies were from Santa Cruz (Dallas,

161

TX). The other antibodies were from Cell Signaling (Danvers, MA).

162

Endothelial Cell Proliferation Assay

163

Detection of endothelial proliferation was performed by using a MTT assay as

164

described in ref 13.

165

Statistics Analysis

166

All measurements were performed using one-way analysis of variance (ANOVA)

167

followed by Tukey’s honestly significant difference tests between individual groups.

168

Data were expressed as mean ± SEM. A value of P＜0.05 was considered to be

169

significant. The statistical software package Prism 5.0 (GraphPad Software, La Jolla,

170

CA) was used for analyzes.

171

Results and Discussion

172

Andrographolide Inhibits TNF-α-stimulated IL-8 Expression in HCT116 Cells


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Phytochemicals, derived from plants, have become a key source of

174

anti-inflammation and anti-tumor therapies, with a lot of current therapies being

175

composed of or derived from natural products. Andrographolide is the most abundant

176

diterpene lactone in the leaves and stem of A. paniculata and possesses several

177

biological activities, especially in tumor therapy. The anticancer activity of

178

andrographolide has been an attractive research topic which is because that: i)

179

Andrographolide appears to be active against a broad spectrum of tumors, including

180

liver cancer, and breast cancer

181

damage via inhibition of ROS in vitro and in vivo

182

various genes that are related to tumor development

183

enhances chemotherapy. In previous studies, andrographolide enhanced TRAIL- and

184

5-fluorouracil-induced apoptosis in hepatocellular carcinoma cells

185

the inhibitory effect of andrographolide on IL-8 expression, andrographolide was

186

pretreated to HCT116 cells before exposure to TNF-α. TNF-α-stimulated IL-8 were

187

suppressed by andrographolide in a concentration-dependent manner (Figure 1A, B, C,

188

D). The concentrations of andrographolide used in this study did not affect cell

189

viability (Figure 1E). These results indicated that andrographolide inhibited

190

TNF-α-induced IL-8 expression in colorectal cancer HCT116 cells.

191

Andrographolide Inhibits TNF-α-stimulated IL-8 by Suppressing NADPH

192

Oxidase Activation and ROS Generation

193 194

22-23

ii) Andrographolide protects against oxidative 11, 24

. iii) Andrographolide inhibits 25-26

. iv) Andrographolide

27-28

. To determine

Oxidative stress acts an important part in the pathogenesis of cancer Activation of NOX is a main source of ROS in some cases


30

29

.

. Membrane


Page 10 of 28

195

translocation of p47phox plays a crucial role in the activation of NOX

196

the effect of andrographolide on TNF-α-stimulated ROS generation, we pretreated

197

cells with 20 μM andrographolide for 12 h and incubated them with 10 μM

198

H2DCFDA for 10 min before challenging the cells with TNF-α for another 20 min. As

199

shown in Figure 2A, B, andrographolide treatment inhibited TNF-α-stimulated ROS

200

generation. NAC and DPI were used as positive controls. NAC or DPI treatment

201

abrogated TNF-α-induced IL-8 gene expression (Figure 2C). Then, the data in Figure

202

2D showed that TNF-α stimulated p47phox membrane translocation. Furthermore,

203

p47phox siRNA abolished IL-8 mRNA (Figure 2E). Andrographolide inhibits

204

TNF-α-induced p47phox membrane translocation (Figure 2F). These results

205

demonstrated that andrographolide may through suppress NADPH oxidase activation

206

and ROS production to inhibit TNF-α-induced IL-8 expression. Previously,

207

andrographolide was reported to induce HO-1 and GCLM expression in human

208

endothelial cells

209

oxidase and ROS by andrographolide should be investigated in future studies.

210

Andrographolide Inhibits TNF-α-stimulated IL-8 by Suppressing Src, Erk1/2

211

and p38 MAPK Activation

11

31

. To delineate

. Thus, the mechanisms involved in the inhibition of NADPH

212

The Src tyrosine kinase has well established roles in the progression of human

213

cancers 32. In particular, inhibition of Scr activation leads to decreased cell migration

214

and invasion 33. MAPKs signaling pathways are predominant oncogenic routes

215

regulate IL-8 expression 35. Pharmacological inhibitors of Src, including PP1 and PP2

216

were used to determine the molecular mechanisms by which TNF-α induces IL-8

217

expression. As shown in Figure 3A, treatment of PP1 or PP2 abrogated


34

and

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218

TNF-α-induced IL-8 mRNA expression. Transfection of Src siRNA inhibited

219

TNF-α-induced IL-8 promoter activity (Figure 3B). Furthermore, TNF-α-induced

220

IL-8 expression was partially blocked by PD and SB (Figure 3C). Additionally,

221

transfection of Erk1/2 siRNA and P38 siRNA inhibited TNF-α-induced IL-8 promoter

222

activity (Figure 3D). Then andrographolide inhibited Src, Erk1/2 and P38 activation

223

(Figure 3E, F). These findings demonstrated that andrographolide may inhibit IL-8 by

224

blocking Src, Erk1/2 and p38 MAPK activation. Aberrant activation of epidermal

225

growth factor receptor has been correlated with tumor progression

226

and activation of PKCs are highly implicated in tumor metastasis

227

beneficial to investigate additional signaling modulators in TNF-α-induced IL-8

228

expression in HCT116 cells.

229

Andrographolide Inhibits TNF-α-stimulated IL-8 By Suppressing ROS-mediated

230

NF-кB Activation

36

. Upregulation

37

. It would be

231

The inhibitory effects of BAY (a NF-κB inhibitor) on IL-8 expression were

232

detected to determine the role of NF-κB in TNF-α-induced IL-8 expression (Figure

233

4A). Results presented in Figure 4B showed that transfection of NF-кB P65 siRNA

234

suppressed IL-8 expression. NAC or DPI treatment blocked the activation of NF-κB

235

induced by TNF-α (Figure 4C). Additionally, treatment of andrographolide attenuated

236

the activation of NF-κB (Figure 4D, E). Our results indicated that ROS-mediated

237

NF-кB activation was involved in the inhibition of IL-8 by andrographolide in

238

HCT116 cells.

239

Andrographolide Inhibits TNF-α-stimulated IL-8 By Suppressing Src/MAPKs

240

(Erk1/2, p38)-mediated AP-1 Activation

241

The inhibitory effects of SR11302 (an AP-1 inhibitor) on TNF-α-induced IL-8



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expression were examined to explore the role of AP-1 in TNF-α-induced IL-8

243

expression. Treatment of cells with SR11302 inhibited IL-8 mRNA expression

244

(Figure 5A). Similarly, TNF-α-induced IL-8 promoter activity was suppressed by the

245

decoy oligonucleotide (Figure 5B). Src tyrosine kinase plays well established roles in

246

the progression of different human cancers. HCT116 cells were treated with PP1 or

247

PP2 (Src inhibitors), prior to TNF-α exposure. The activation of Erk1/2 and P38

248

(Figure 5C), stimulated by TNF-α was markedly suppressed in the cells treated with

249

PP1 or PP2. Furthermore, PD and SB treatment inhibited the TNF-α-induced

250

activation of c-fos (Figure 5D). Therefore, Erk1/2 and p38 MAPK are the upstream

251

signallings

252

TNF-α-induced activation of c-fos and c-jun (Figure 5E) and AP-1 promoter activity

253

significantly (Figure 5F). Our results suggested that Src/MAPK (Erk1/2 and p38

254

MAPK)-mediated AP-1 was related to the inhibition of IL-8 by andrographolide in

255

HCT116 cells. Previous study reported that transcription factor NF-IL-6 binds to the

256

IL-8 promoter

257

phosphorylation, implicating IL-8 induction in HCT116 cells 39.

258

Effect of Andrographolide on Angiogenesis in vitro

of

AP-1

38

by TNF-α.

Andrographolide

pretreatment

suppressed

. Nguyen et al indicated that lithocholic acid blocked STAT3

259

To determine the role of andrographolide on angiogenesis in vitro, we explored

260

the role of the conditioned medium (CM) from HCT116 cells exposed to TNF-α with

261

or without andrographolide on the proliferation of EAhy.926 endothelial cells. CM

262

from TNF-α-activated HCT116 cells promoted the in vitro growth of EAhy.926

263

endothelial cells, whereas the CM from andrographolide treatment group or an IL-8


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264

neutralizing antibody treatment group abolished the in vitro growth of EAhy.926.

265

Furthermore, exogenous IL-8 treatment restored the inhibition on endothelial cell

266

proliferation by CM from TNF-α-activated HCT116 cells pretreated with

267

andrographolide (Figure 6A). These results illustrated that andrographolide

268

suppresses the angiogenesis caused by TNF-α-stimulated HCT116 cells by

269

specifically inhibiting IL-8. It is of interest to investigate the effect of andrographolide

270

on angiogenesis in vivo.

271

In summary, as shown in Figure 6B, C, this study demonstrates that

272

andrographolide effectively suppressed IL-8 expression and angiogenesis in the tumor

273

microenvironment by inhibition of NADPH oxidase, ROS, Erk1/2, P38 MAPK,

274

NF-кB, and AP-1 activation. Our findings represent a novel therapeutic approach to

275

repress angiogenesis and may provide useful evidence for developing new anticancer

276

therapeutics for CRC.

277

ACKNOWLEDGEMENTS

278

This research was supported by National Natural Science Foundation of China

279

(No.81702413, 81701836), and Scientific Research Starting Foundation of Southern

280

Medical

281

Program-Research Foundation for Advanced Talents (C1034220, C1034214).

282

Notes

283

The authors declare no competing ﬁnancial interest.

University (No.

C1034409),

and

2017

High

Level

University

284 285

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FIGURE LEGENDS

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Figure 1. Andrographolide inhibits TNF-α-induced IL-8 expression in HCT116 cells.

408

HCT116 cells were pretreated with andrographolide (5, 10, and 20 μM) for 1 h

409

followed by incubation with 10 ng/mL TNF-α for 4 or 12 h. IL-8 mRNA level (A) and

410

(B), protein level (C), and promoter activity (D) were measured by RT-PCR, qPCR,

411

ELISA, and luciferase activity assay, respectively. (E) The cells were incubated with

412

0-100 µM andrographolide for 24 h, and then the viability was tested by the MTT

413

method. *P < 0.05 versus control; **P < 0.05 versus only TNF-α. The above data

414

represent means ±SEM from triplicate measurements.

415

Figure 2. Andrographolide inhibits TNF-α-induced IL-8 expression by suppressing

416

NADPH oxidase activation and ROS generation. (A) Synchronized quiescent cells,

417

after being treated with 20 μM andrographolide for 4 h or 5 mM NAC or 5 μM DPI

418

for 1 h, were incubated with 5 ng/mL TNF-α for another 15 min. The cells were then

419

incubated in the dark for 10 min with 10 μM H2DCFDA. The H2DCF fluorescence

420

was imaged with a confocal laser scanning fluorescence microscope. (B)

421

Quantification of the ROS level as detected by H2DCFDA fluorescence intensities. (C)

422

Cells pretreated with NAC or DPI for 1 h were incubated with TNF-α for 4 h. After

423

incubation, IL-8 mRNA levels in the cell lysates were determined by qPCR. (D) Cells

424

were incubated with TNF-α for the indicated times and the membrane and cytosol

425

fractions were prepared and subjected to western blotting using an anti-p47phox

426

antibody. (E) Cells were transfected with si-con or si-p47phox and then incubated with

427

TNF-α for 4 h, after which qPCR was performed to detect IL-8 expression. (F) Cells



428

pretreated with 20 μM andrographolide for 1 h were incubated with TNF-α for 15 min

429

and the the membrane and cytosol fractions were prepared and subjected to western

430

blotting using an anti-p47phox antibody. *P < 0.05 versus control; **P < 0.05 versus

431

only TNF-α. The above data represent means ±SEM from triplicate measurements.

432

Figure 3. Andrographolide inhibits TNF-α-induced IL-8 expression by suppressing

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Src, Erk1/2 and p38 MAPK activation. (A) Cells pretreated with 0-10 μM PP1 and

434

PP2 for 1h were incubated with TNF-α for 4 h. Following incubation, IL-8 mRNA

435

levels in the cell lysates were determined by qPCR. (B) Cells transfected with

436

scrambled (si-con) or Src siRNA were transiently transfected with a PGL2-IL-8

437

reporter construct and incubated with TNF-α for 4 h. The luciferase activity was

438

measured using a luminometer. (C) Cells pretreated with PD (20 μM) or SB (20 μM)

439

for 1 h were incubated with TNF-α for 4 h. After incubation, IL-8 mRNA levels in the

440

cell lysates were determined by qPCR. (D) Cells transfected with scrambled (si-con)

441

or Erk1/2 siRNA or P38 siRNA were transiently transfected with a PGL2-IL-8

442

reporter construct and incubated with TNF-α for 4 h. The luciferase activity was

443

measured using a luminometer. (E) Cells pretreated with andrographolide (5, 10, and

444

20 μM) for 1 h were incubated with TNF-α for 15 min and the expression of

445

phospho-Src Tyr416 and Src was analyzed by western blotting. (F) Cells pretreated

446

with andrographolide (5, 10, and 20 μM) for 1 h were incubated with TNF-α for 15

447

min and the expression of phospho-Erk1/2, Erk1/2, phospho-p38, and p38 was

448

analyzed by western blotting. *P < 0.05 versus control; **P < 0.05 versus only TNF-α.

449

The above data represent means ±SEM from triplicate measurements.


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450

Figure 4. Andrographolide inhibits TNF-α-induced IL-8 expression through the

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suppression of ROS-mediated NF-кB activation. (A) HCT116 cells were treated with

452

0–20 μM BAY11-7082 prior to incubation with TNF-α for 4 h. After incubation, IL-8

453

mRNA levels in the cell lysates were determined by qPCR. (B) Cells transfected with

454

scrambled (si-con) or NF-кB P65 siRNA were transiently transfected with a

455

PGL2-IL-8 reporter construct and incubated with TNF-α for 4 h. The luciferase

456

activity was measured using a luminometer. (C) Cells pretreated with NAC or DPI for

457

1 h were incubated with TNF-α for 30 min. The whole cell proteins were extracted

458

and analyzed by western blot using antibodies against phospho-p65 (ser 536). (D)

459

Cells were treated with andrographolide prior to exposure to TNF-α and the

460

expression of phospho-p65 (Ser 536), phospho-IкB-α (Ser 32), and IкB-α was

461

analyzed by western blotting. (E) Cells were transiently transfected with the pNF-кB

462

luciferase reporter construct, after being pretreated with andrographolide, and then

463

incubated with TNF-α for 4 h. After incubation, the cells were lysed and luciferase

464

activity was determined. *P < 0.05 versus control; **P < 0.05 versus only TNF-α.

465

The above data represent means ±SEM from triplicate measurements.

466

Figure 5. Andrographolide inhibits TNF-α-induced IL-8 expression through the

467

suppression of Src/MAPKs (Erk1/2, p38)-mediated AP-1 activation. (A) HCT116

468

cells were treated with 0–5 μM SR for 1 h prior exposure to TNF-α for 4 h. After

469

incubation, IL-8 mRNA levels in the cell lysates were determined by qPCR. (B) AP-1

470

decoy oligonucleotides were co-transfected with pGL2-IL-8 into cells. After

471

incubation with TNF-α for 4 h, the luciferase activities were determined using a



472

luminometer. (C) Cells pretreated with 10 μM PP1 or PP2 for 1 h were incubated with

473

TNF-α for 15 min, and afterward the whole cell proteins were extracted and analyzed

474

by western blot using antibodies against phospho-Erk1/2, Erk1/2, phosphor-P38 and

475

P38. (D) Cells were treated with 0-20 μM PD or 0-20 μM SB for 1 h prior to

476

treatment with TNF-α for 15 min. The whole cell proteins were extracted and

477

analyzed by western blot using antibodies against phospho-c-fos, and c-fos. (E) Cells

478

were treated with andrographolide prior to exposure to TNF-α and the expression of

479

phospho-c-fos, c-fos, phospho-c-jun, and c-jun was analyzed by western blotting. (F)

480

Cells were transiently transfected with the pAP-1 luciferase reporter construct, after

481

being pretreated with andrographolide, and then incubated with TNF-α for 4 h. After

482

incubation, the cells were lysed and luciferase activity was determined. *P < 0.05

483

versus control; **P < 0.05 versus only TNF-α. The above data represent means ±

484

SEM from triplicate measurements.

485

Figure 6. Schematic representation of the mechanisms underlying the inhibition of

486

TNF-α-induced IL-8 expression by andrographolide in HCT116 cells and the

487

mechanisms underlying the inhibition of tumor-derived IL-8-induced angiogenesis by

488

andrographolide in the tumor microenvironment. (A) HCT116 cells were incubated in

489

Mc-Coy’s 5A medium with 1% FBS and stimulated with vehicle (PBS) or 5 ng/mL

490

TNF-α in the presence or absence of andrographolide. After 24 h, CM was harvested

491

and immediately frozen until use. EAhy.926 cells were incubated with CM for 24 h

492

and the number of cells was determined using MTT. To neutralize IL-8, CM was

493

pre-incubated with nonspecific IgG or anti-IL-8 antibody (1 mg/mL) for 1 h before


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494

use. To confirm the role of IL-8 in endothelial cell proliferation, exogenous IL-8 (5

495

ng/mL) was added to the CM from andrographolide-treated group.

496

control; **P < 0.05 versus only TNF-α; ***P < 0.05 versus TNF-α and

497

andrographolide-co-incubated group. The above data represent means ± SEM from

498

triplicate measurements. (B) Andrographolide inhibits TNF-α-induced IL-8 via

499

inhibition of NADPH oxidase/ROS/NF-κB and Src/MAPKs/AP-1signaling pathways

500

in HCT116 colorectal cancer cells. (C) Secretion of IL-8 from cancer cells enhances

501

the proliferation of endothelial cells to promote angiogenesis in the tumor

502

microenvironment. Andrographolide inhibits the expression of tumor-derived IL-8,

503

thereby inhibiting angiogenesis in the tumor microenvironment.

504


*P < 0.05 versus


505 506 507

Figure 1

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Figure 2



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513 514

Figure 3


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516 517 518

Figure 4

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520 521 522

Figure 5

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Figure 6



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Table of Contents Graphic

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Andrographolide Antagonizes TNF-Î±-induced IL-8 via Inhibition of

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