AGS-30, an andrographolide derivative, suppresses tumor angiogenesis and growth in vitro and in vivo

Journal Pre-proofs AGS-30, an andrographolide derivative, suppresses tumor angiogenesis and growth in vitro and in vivo Jingjing Li, Feng Li, Fan Tang, Jinming Zhang, Renkai Li, Dekuan Sheng, Simon Ming-Yuen Lee, Guo-Chun Zhou, George Pak-Heng Leung PII: DOI: Reference:

S0006-2952(19)30393-4 https://doi.org/10.1016/j.bcp.2019.113694 BCP 113694

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

30 September 2019 4 November 2019

Please cite this article as: J. Li, F. Li, F. Tang, J. Zhang, R. Li, D. Sheng, S.M-Y. Lee, G-C. Zhou, G.P-H. Leung, AGS-30, an andrographolide derivative, suppresses tumor angiogenesis and growth in vitro and in vivo, Biochemical Pharmacology (2019), doi: https://doi.org/10.1016/j.bcp.2019.113694

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© 2019 Published by Elsevier Inc.

1

AGS-30, an andrographolide derivative, suppresses tumor angiogenesis and growth in

2

vitro and in vivo

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

Jingjing Lia,1, Feng Lib,1, Fan Tangc,1, Jinming Zhangd, Renkai Lia, Dekuan Shengb, Simon Ming-Yuen Leec,*,Guo-Chun Zhoub,*, George Pak-Heng Leunga,* aDepartment

of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong,

China; bSchool

of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, China; Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao, China; dCollege of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China. cState

1These

authors contributed equally to the paper.

*Corresponding author Dr. Simon Ming-Yuen Lee, N22 Research Building, State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao SAR, China Tel.: +853 88224695 E-mail address:[email protected]

Dr. Guo-Chun Zhou, NO.30, South Puzhu Road, Pukou District, School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, China. Tel.: +86-15850780314 E-mail address:[email protected]

Dr. George Pak-Heng Leung, 2/F, 21 Sassoon Road, Li Ka Shing Faculty of Medicine, Laboratory Block, Faculty of Medicine Buliding, Department of Pharmacology and Pharmacy, University of Hong Kong, Hong Kong SAR, China Tel.: +852 39179024 E-mail address: [email protected]

Key words: andrographolide derivative, anti-angiogenic, colon cancer, VEGF

40 1

41

Abstract

42

Poor bioavailability and limited efficacy are challenges associated with using

43

andrographolide as a therapeutic agent. We recently synthesized AGS-30, a new

44

andrographolide derivative, in our laboratory. In this study we investigated the potential

45

anti-tumor effect of AGS-30 and the underlying mechanisms, particularly those related to

46

angiogenesis. Results from our in vitro experiments showed that AGS-30 exerted

47

anti-angiogenic effects by inhibiting endothelial cell proliferation, migration, invasion, and

48

tube formation. Phosphorylation and activation of angiogenesis-related signaling molecules

49

(e.g., vascular endothelial growth factor [VEGF] receptor 2, mitogen-activated protein kinase

50

kinase 1/2, extracellular signal-regulated kinase 1/2, mechanistic target of rapamycin

51

[mTOR], protein kinase B [Akt], and p38) were markedly reduced by AGS-30. Meanwhile,

52

AGS-30 potently inhibited cell proliferation and phosphorylation of cell survival-related

53

proteins (e.g., Akt, mTOR, and ERK1/2) and decreased the expression of VEGF in HT-29

54

colon cancer cells. AGS-30 blocked microvessel sprouting in a rat aortic ring model and

55

blood vessel formation in zebrafish embryos and a mouse Matrigel plug model. Additionally,

56

AGS-30 suppressed tumor growth and angiogenesis in HT-29 colon cancer cell xenografts in

57

nude mice. These effects were not observed when same concentration of andrographolide, the

58

parent compound of AGS-30, was used. Thus, AGS-30 exerted a strong antitumor effect by

59

inhibiting tumor cell growth and angiogenesis and is a candidate compound for the treatment

60

of cancer.

61 62 2

63

1. Introduction

64

Natural products have great potential for drug development owing to their diversity,

65

complexity, and biocompatibility. The basic chemical structures of natural products can be

66

modified and can serve as a basis for the development of novel agents with improved

67

biological properties and safety profiles [1]. It has been reported that over 50% of approved

68

drugs in clinical use either originate or are derived from natural products [2] and as of the end

69

of 2014, nearly half of the 175 small-molecule compounds approved for the treatment of

70

cancer were natural product-based [3]. Most of the drugs that succeeded in clinical trials (e.g.,

71

sorafenib, imatinib, and gefitinib [4]) were chemically modified from lead compounds.

72 73

Angiogenesis is a basic biological process involving the sprouting and growth of new blood

74

vessels from the pre-existing vascular primordium [5] and plays a key role not only in the

75

regulation of various physiological activities (e.g., embryonic development, wound healing,

76

female reproductive cycle, etc.) but also in tumor growth and metastasis [6, 7]. Tumors that

77

grow beyond a size of 2 mm3 are dependent on oxygen and nutrients supplied by surrounding

78

newly formed blood vessels [8]. Anti-angiogenic therapy involves starving tumor cells by

79

targeting endothelial cells and blocking angiogenesis [9]. Most currently used anti-angiogenic

80

drugs are small-molecule compounds, which have advantages over monoclonal antibodies.

81

For instance, small molecules agents are less expensive and more convenient to administer

82

[10]. However, the development of drug resistance and limited efficacy limit their widespread

83

clinical application [11]. There is therefore a need for novel types of small molecule with

84

anti-angiogenic activity. 3

85

Diterpenoids are a class of bioactive natural compounds that are found in many medicinal

86

herbs and have great potential in the treatment of diseases involving excessive angiogenesis

87

such as cancer. For instance, oridonin, a tetracycline diterpenoid isolated from Rabdosia

88

rubenscens, was shown to induce apoptosis and inhibits the migration and invasion of highly

89

metastatic human breast cancer cells [12]. Oridonin suppressed tumor growth and metastasis

90

by blocking tumor angiogenesis via downregulation of vascular endothelial growth factor

91

(VEGF)-induced Jagged/Notch signaling [13]. We recently showed that oridonin not only

92

synergistically enhances the anti-tumor efficacy of doxorubicin against aggressive breast

93

cancer

94

doxorubicin-induced cardiotoxity in mice [14]. Another example of a natural anti-angiogenic

95

compound is triptolide, a diterpenoid epoxide isolated from Tripterygium wilfordii Hook F.

96

Triptolide was shown to inhibit angiogenesis and invasion of human anaplastic thyroid

97

carcinoma cells by blocking nuclear factor-κB signaling [15], and induced apoptosis while

98

suppressing the growth and angiogenesis of human pancreatic cancer cells via negative

99

regulation of cyclooxygenase 2 and VEGF expression [16]. These reports demonstrate the

100

through

proapoptotic

and

anti-angiogenic

signaling,

but

also

reduced

potential of diterpenoids as anti-cancer agents.

101 102

Andrographolide (Andro; Scheme 1) is a bioactive labdane diterpenoid. It was previously

103

reported that the Andro content of dried whole Andrographalis paniculata (Burn. f.) plant is

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as high as 4%, suggesting that Andro is the most abundant diterpenoid in this medicinal plant

105

[17]. Andro is known to have anticancer [18, 19], anti-angiogenic [20, 21], anti-inflammatory

106

[22], antidiabetic [23], and neuroprotective [24] activities. Andro and A. paniculate extracts 4

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inhibit tumor angiogenesis by suppressing pro-angiogenic molecules such as VEGF and nitric

108

oxide and enhancing the expression of anti-angiogenic factors such as interleukin 2 and

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TIMP metallopeptidase inhibitor 1 [25]. Moreover, Andro can block tumor angiogenesis by

110

preventing VEGF-A-induced activation of VEGFR2 and downstream mitogen-activated

111

protein kinase (MAPK) signaling [20, 26]. However, despite the therapeutic potential

112

demonstrated by Andro, its development and clinical application are a challenge owing to its

113

poor water solubility, low potency, and limited therapeutic efficacy [27].

114 115

AGS-30 is a recently synthesized Andro derivative [28]. In this study, we investigated the

116

anticancer and anti-angiogenic effects of AGS-30 in vitro and in vivo and the underlying

117

mechanisms of action. Moreover, the present study also indicated that modification of the

118

C14 position in the chemical structure of Andro significantly enhanced its anticancer and

119

anti-angiogenic effects. Our findings provide a basis for the future development of Andro

120

derivatives as novel angiogenesis inhibitors for the treatment of cancer.

121

5

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2. Materials and methods

124

2.1. Ethical statement

125

Ethics approval for animal experiments was obtained from the University of Macau and

126

Chengdu University of Traditional Chinese Medicine. All animal experiments were

127

conducted in compliance with Instructional Animal Care and Use Committee (IACUC).

128 129

2.2. Chemicals and reagents

130

AGS-30 was synthesized from Andro (Scheme 1) [28]. Andro, dimethyl sulfoxide (DMSO),

131

heparin, gelatin, collagen, protease, endothelial cell growth supplement (ECGS), Trypan blue,

132

paraformaldehyde, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), and

133

Drabkin Reagent Kit 525 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

134

Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), Hoechst 33342

135

dye, bicinchoninic acid assay kit (BCA), phenylmethylsulfonyl fluoride (PMSF), protease

136

inhibitor cocktail, phosphate-buffered saline (PBS), penicillin/streptomycin, and 0.25% (w/v)

137

trypsin containing 1 mM EDTA were from Invitrogen (Carlsbad, CA, USA). Growth

138

factor-reduced Matrigel was obtained from BD Biosciences (Franklin Lakes, NJ, USA).

139

VEGF-A was from R&D Systems (Minneapolis, MN, USA). The lactate dehydrogenase

140

(LDH) cytotoxicity assay kit was from Cayman Chemical (Ann Arbor, MI, USA). VEGF

141

receptor (VEGFR) tyrosine kinase inhibitor II (VRI) was from CalBiochem (San Diego, CA,

142

USA). A primary antibody against VEGF-A and a human VEGF enzyme-linked

143

immunosorbent assay (ELISA) kit were obtained from Abcam (Cambridge, MA, USA).

144

Other antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). All 6

145

chemicals were dissolved in appropriate solvent and stored at -20°C until use.

146 147

2.3. Cell culture

148

Human umbilical vein endothelial cells (HUVECs) and HT-29 human colon adenocarcinoma

149

cells were obtained from American Type Culture Collection (Manassas, VA, USA).

150

HUVECs were cultured in F-12K complete medium supplemented with 10 mM L-glutamine,

151

1 μg/mL hydrocortisone hemisuccinate, 50 μg/mL ascorbic acid, 100 µg/mL heparin, 30

152

µg/mL endothelial cell growth supplement, 10% heat-inactivated FBS, and 1%

153

penicillin/streptomycin. Cells from early passages (passages 38) were used in experiments.

154

HT-29 human colon cancer cells were cultured in DMEM supplemented with 10%

155

heat-inactivated FBS and 1% penicillin/streptomycin. Cells were cultured in tissue flasks

156

precoated with 0.1% gelatin at 37°C in a humidified atmosphere of 5% CO2.

157 158

2.4. Cell viability assay

159

HUVECs and HT-29 cells were seeded in 96-well plates at a density of 8 × 103 cells/well and

160

cultured for 24 h until the cells had attached. The cells were then treated with various

161

concentrations (020 µM) of Andro and AGS-30 in low-serum medium (containing 0.5%

162

FBS) for 24 or 48 h. Cell viability was evaluated with the MTT assay according to the

163

manufacturer’s protocol. Briefly, the medium was discarded and the cells were incubated

164

in MTT solution (at a final concentration of 0.5 mg/mL) for 4 h at 37°C. DMSO was then

165

added to lyse the cells and dissolve the violet formazan crystals that formed inside the

166

cells. Absorbance at 570 nm was measured using a SpectraMax M5 Multi-Mode Microplate 7

167

Reader (Molecular Devices, Sunnyvale, CA, USA).

168 169

2.5. Cell proliferation assay

170

HUVECs were seeded in a 48-well plate at a density of 3 × 104 cells/well and cultured for 24

171

h until they had attached; they were then cultured overnight in low-serum medium to induce

172

quiescence. The cells were treated with various concentrations of Andro and AGS-30 in

173

low-serum medium containing VEGF (20 ng/mL) for 48 h and then stained using Trypan

174

blue and observed under a microscope. Blue cells were considered non-viable.

175 176

2.6. Lactate dehydrogenase (LDH) assay

177

Cellular injury was assessed by measuring the activity of LDH released into the culture

178

medium upon cellular damage using a commercial kit according to the manufacturer’s

179

instructions. Absorbance at 490 nm was measured on a microplate reader.

180 181

2.7. Transwell migration and invasion assays

182

Cell migration and invasion assays were performed using HUVECs as previously described

183

[29]. Transwell inserts (8-μm pores) were obtained from BD Biosciences (Franklin Lakes, NJ,

184

USA). For the cell migration assay, the upper and lower sides of the Transwell membrane

185

were pre-coated with 0.1% collagen. In the invasion assay, the upper and lower sides of the

186

membrane were pre-coated with 100 μL Matrigel (20% in blank medium). HUVECs were

187

seeded in the Transwells at a density of 5 × 104 cells/well and cultured in low-serum medium

188

containing VEGF (20 ng/mL) and different concentrations of Andro and AGS-30. After 8

189

incubation for 24 h at 37°C, cells on the upper surface of the Transwell membrane were

190

removed using cotton swabs. The membranes were fixed with 4% paraformaldehyde for 15

191

min and then stained with Hoechst 33342 (10 μg/mL) for 15 min and mounted on microscope

192

slides, and images were captured on a fluorescence inverted microscope (Axiovert 200; Carl

193

Zeiss, Oberkochen, Germany) with a charge-coupled device camera (AxioCam HRC; Carl

194

Zeiss). Cell migration and invasion were quantified by counting the number of cells per insert

195

using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

196 197

2.8. Tube formation assay

198

To evaluate angiogenic potential, 15-well microslides (ibidi, Fitchburg, WI, USA) were

199

coated with Matrigel (50% in blank medium) and incubated for 30 min at 37°C to induce

200

polymerization. HUVECs (2 × 105 cells/mL) were resuspended in low-serum medium with

201

different concentrations of Andro and AGS-30 in the presence of 20 ng/mL VEGF, and then

202

seeded on the Matrigel-coated slides. After incubation for 46 h, tube-like structures formed

203

that were connected at both ends, which were defined as endothelial cords. Images were

204

acquired before and after drug treatment on an inverted microscope. Tube formation was

205

quantified by counting the number of branch points in three randomly selected fields of view.

206 207

2.9. Western blot analysis

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Total protein was extracted from cells using lysis buffer containing 1% PMSF and 1%

209

protease inhibitor. Lysates were centrifuged at 12,500 × g for 20 min at 4°C and the

210

supernatant was collected. Protein concentration was determined with the bicinchoninic acid 9

211

assay. Equal amounts of protein were resolved by sodium dodecyl sulfate–polyacrylamide gel

212

electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio-Rad

213

Laboratories, Hercules, CA, USA) that was blocked with 5% non-fat milk in Tris-buffered

214

saline containing 0.1% Tween-20 (TBST) for 1 h. The membrane was then incubated

215

overnight at 4°C with primary antibodies against VEGF-A, VEGFR2, phosphorylated

216

(p-)VEGFR2 (Tyr1175), mechanistic target of rapamycin (mTOR), p-mTOR (Ser2448),

217

protein kinase B (Akt), p-Akt (Ser473), extracellular signal-regulated kinase (ERK)1/2,

218

p-ERK1/2 (Thr202/Tyr204), MAPK kinase (MEK)1/2, p-MEK1/2 (Ser217/221), p38, p-p38

219

(Thr180/Tyr182), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All the primary

220

antibodies were diluted with TBST buffer at a ratio of 1:1000. After washing with PBS, the

221

membrane was incubated with horseradish peroxidase-conjugated secondary antibodies

222

(1:2000 dilution) for 1 h at room temperature. After multiple washes with PBS, proteins were

223

visualized by enhanced chemiluminescence. Images of protein bands were captured and

224

densitometric measurements of signal intensity were performed using the Molecular Imager

225

ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA).

226 227

2.10.

ELISA

228

HT-29 colon cancer cells were treated with AGS-30 (0–5 μM) for 48 h, and the levels of

229

secreted VEGF-A in the culture medium were measured and quantified using a commercial

230

ELISA kit according to manufacturer’s instructions.

231 232

2.11.

Zebrafish maintenance and embryo handling 10

233

The Tg(fli1:EGFP) transgenic zebrafish line expressing enhanced green fluorescent protein

234

(EGFP) in endothelial cells was provided by the Zebrafish Information Network (Eugene, OR,

235

USA). Wild-type zebrafish were purchased from a local pet store. The zebrafish were

236

maintained in a controlled environment at 28.5°C on a 14:10-h light/dark cycle and were fed

237

twice a day with brine shrimp and occasionally with general tropical fish food. Embryos were

238

generated through natural mating and reared in embryo medium at 28.5°C. Dead, unfertilized,

239

or abnormally shaped embryos were removed at 4 h post-fertilization (hpf).

240 241

2.12.

Morphological observation of zebrafish embryos

242

Morphological observation of zebrafish embryos was performed as previously described [30].

243

At 24 hpf, Tg(fli-1:EGFP) embryos (12 per group) were distributed in 12-well plates. The

244

groups were treated with different concentrations of AGS-30 (3–30 µM) or Andro (300 µM)

245

dissolved in embryo medium for 24 h. Embryos treated with DMSO (0.1%) served as the

246

control, and those treated with 250 ng/mL VRI were the positive control. The embryos were

247

observed for morphological changes and images were captured with a spinning disk confocal

248

microscope system (Olympus, Tokyo, Japan). Intact and defective intersegmental vessels

249

(ISVs) in each zebrafish embryo were counted as previously described [27].

250 251

2.13.

Rat aortic ring angiogenesis assay

252

A 48-well plate was precoated with 100 µL Matrigel per well and polymerized at 37°C for 30

253

min. Aortic rings were isolated from 6- to 8-week-old male Sprague-Dawley rats and cut into

254

1.5-mm pieces that were rinsed at least three times in cold PBS and then transferred to a well 11

255

and overlaid with 100 µl of Matrigel for sealing. Serum-free culture medium containing

256

VEGF (100 ng/ml), with or without Andro or AGS-30 (5 μM), was added to the wells. The

257

medium was changed every 2 days. After 10 days, microvessel growth was photographed on

258

an inverted microscope and the number of branching sites was quantified with ImageJ

259

software.

260 261

2.14.

Mouse Matrigel plug angiogenesis assay

262

Growth factor-reduced Matrigel (0.5 mL) containing heparin (100 U) and VEGF (250 ng)

263

with or without Andro and AGS-30 was subcutaneously injected into the ventral area of

264

8-week-old male C57BL/6 mice (n = 5). After 14 days, mice were sacrificed and the Matrigel

265

plugs were removed. To quantify the formation of functional blood vessels, the hemoglobin

266

concentration in the plug was measured using Drabkin Reagent Kit 525.

267 268

2.15.

Inhibition of tumor growth in vivo

269

The subcutaneous dorsum of male nude mice (4-weeks-old; 16 g) was inoculated with HT-29

270

human colon cancer cells (1 × 106 cells/mouse). Tumors were allowed to grow to

271

approximately 200 mm3, and the xenografted mice were then randomly assigned to one of

272

three groups (n = 5 each) that were intraperitoneally injected with saline, Andro (2 mg/kg), or

273

AGS-30 (2 mg/kg) once every 2 days for 20 days. Body weight and tumor volume ([width]2

274

× [length]/2) were recorded every 2 days. After treatment, mice were sacrificed by overdose

275

of barbiturates administered intraperitoneally and then photographed, primary tumors and

276

major organs were excised and weighted. Tumor tissue was fixed with 4% (v/v) 12

277

formaldehyde and cut into 6-μm sections. Tumors and major organs sections were stained

278

with hematoxylin and eosin (H&E) to examine tissue architecture. The sections were also

279

labeled with primary antibodies against VEGF, p-VEGFR2, and CD31 to evaluate

280

angiogenesis at the tumor site.

281 282

2.16.

Statistical analysis

283

Data are expressed as mean ± SD of at least three independent experiments and were

284

analyzed with Prism v.5.0 software (GraphPad, La Jolla, CA, USA). The difference between

285

groups was evaluated by one-way analysis of variance and P < 0.05 was considered

286

significant.

287 288 289

13

291

3. Results

292

3.1. Effects of AGS-30 on the viability of HUVECs and HT-29 cancer cells

293

Cell viability was evaluated with the MTT assay in HUVECs and HT-29 colon cancer cells

294

treated with various concentrations (0–20 μM) of Andro or AGS-30 for 24 or 48 h. The

295

half-maximal inhibitory concentration (IC50) of AGS-30 in HT-29 cancer cells after 24 and

296

48 h of treatment was 8.72 ± 3.35 and 4.13 ± 2.25 μM, respectively (Fig. 1A). IC50 values for

297

Andro were much higher at these time points (> 40 μM). Meanwhile, the viability of

298

HUVECs was decreased by treatment for 24 and 48 h with AGS-30 at concentrations greater

299

than 5 μM, but was unaffected by Andro in the concentration range of 0–20 μM (Fig. 1B). To

300

further evaluate the toxicity of Andro and AGS-30, we examined LDH levels in HUVECs

301

and observed no cellular damage upon treatment with 1.25–5 μM AGS-30 and 5–20 μM

302

Andro, respectively (Fig. 1C). These concentrations were therefore considered as safe for

303

HUVECs and were used in subsequent experiments evaluating angiogenesis.

304 305

3.2. Effects of AGS-30 on VEGF-induced proliferation

306

Endothelial cell proliferation plays a critical role in angiogenesis. We therefore examined the

307

effects of Andro and AGS-30 on endothelial cell proliferation by counting cell numbers.

308

HUVEC proliferation was increased by 38% in the presence of VEGF (20 ng/mL) (Fig. 1D).

309

This effect was reversed by AGS-30 and Andro in a concentration-dependent manner; the

310

lowest effective concentrations of AGS-30 and Andro were 2.5 and 20 μM, respectively.

311

These data demonstrated that AGS-30 more potently inhibited HUVEC proliferation than

312

Andro. 14

314

3.3. Effects of AGS-30 on VEGF-induced endothelial cell migration, invasion, and tube

315

formation

316

The effects of AGS-30 and Andro on endothelial cell migration and invasion were evaluated

317

with the Transwell assay. The migratory and invasive capacities of HUVECs were increased

318

by 145% and 210%, respectively, in the presence of VEGF (20 ng/mL) (Fig. 2A, B). This

319

effect was reversed by AGS-30 in a concentration-dependent manner, with the lowest

320

effective concentration at 2.5 μM (Fig. 2D, E). However, VEGF-induced endothelial cell

321

migration and invasion was unaffected by Andro (5 μM).

322 323

We next investigated the effect of AGS-30 and Andro on the construction of cord-like

324

networks by HUVECs. After treatment for 6 h, endothelial cells in the control and 20 ng/mL

325

VEGF-treated groups formed capillary-like structures and cord-like networks (Fig. 2C).

326

However, VEGF-induced tube formation was abolished by AGS-30 in a dose-dependent

327

manner, whereas Andro (5 μM) had no effect (Fig. 2F). These data suggested that AGS-30

328

had more potent anti-angiogenic activity than Andro in vitro.

329 330

3.4. Effect of AGS-30 on VEGFR2-mediated angiogenesis signaling

331

To investigate the anti-angiogenic mechanisms of AGS-30, we examined the effect of

332

AGS-30 on the expression levels of key proteins involved in VEGFR2-mediated

333

angiogenesis signaling by western blotting. The phosphorylation of VEGFR2, mTOR, Akt,

334

MEK1/2, ERK1/2, and p38 in HUVECs was increased by VEGF (50 ng/mL) (Fig. 3A).

335

However, this was abrogated by AGS-30 in a concentration-dependent manner (1.25–5 μM 15

336

(Fig. 3C–F). In addition, the VEGF-induced phosphorylation of VEGFR2 and p38 was

337

inhibited by AGS-30 at concentrations as low as 1.25 μM (Fig. 3B, G). However, overall

338

expression of these proteins was unaltered by AGS-30.

339 340

3.5. Effects of AGS-30 on cancer cell proliferation and survival pathways

341

To investigate the mechanism underlying the effect of AGS-30 on cancer cell proliferation

342

and survival, we examined the expression levels of proteins in cancer cell proliferation and

343

survival pathways. The total amounts of these proteins were unaffected by AGS-30. However,

344

mTOR

345

concentration-dependent manner 48 h after treatment with AGS-30 (1.25–5 μM) (Fig. 4A).

346

Meanwhile, p-ERK1/2 and p-Akt were downregulated by AGS-30 (5 μM), although the

347

levels were unaltered by AGS-30 concentrations of 1.25 and 2.5 μM (Fig. 4B, D).

phosphorylation

in

HT-29

colon

cancer

cells

was

reduced

in

a

348 349

3.6. Effect of AGS-30 on VEGF expression in HT-29 cells

350

VEGF plays a critical role not only in angiogenesis but also in tumor growth. To clarify the

351

effect of AGS-30 on VEGF expression in HT-29 cells, we assessed intra- and extracellular

352

VEGF levels in HT-29 cells by western blotting and ELISA, respectively. VEGF levels in

353

HT-29 colon cancer cell lysates and culture medium were decreased by AGS-30 treatment in

354

a concentration-dependent manner (Fig. 4E, F).

355 356

3.7. Effect of AGS-30 on blood vessel formation in zebrafish model

357

To investigate the anti-angiogenic activity of AGS-30 in vivo, we examined the effect of 16

358

AGS-30 on blood vessel formation in a transgenic zebrafish model. Zebrafish embryos at 24

359

h hpf were treated with vehicle (0.1% DMSO), the positive control VRI (250 ng/mL), Andro

360

(300 μM), or AGS-30 (3–30 μM) for 24 h. Intact ISVs were observed in the control and

361

Andro groups (Fig. 5A, C), whereas defective ISVs were present in the VRI and

362

AGS-30-treated groups (Fig. 5B). ISV formation in the AGS-30 group was markedly

363

inhibited even at a concentration as low as 3 μM (Fig. 5D–F). Quantitative analysis revealed

364

that the number of intact ISVs decreased with increasing AGS-30 concentration (Fig. 5M).

365

We analyzed the anti-angiogenic phenotype of embryos (i.e., with incomplete ISVs) in each

366

group and found that in the VRI-treated group, the growth of intact ISVs was reduced, with

367

nearly 90% showing an anti-angiogenic phenotype. On the other hand, the fraction of ISVs

368

exhibiting the phenotype in the AGS-30-treated group was increased from 42% to 72% when

369

the concentration was increased from 3 to 30 μM (Fig. 5N). Blood vessel formation in the

370

zebrafish model was unaffected by Andro, even a very high concentration (300 μM). These

371

results confirmed that AGS-30 had more potent anti-angiogenic activity than Andro in vivo.

372 373

3.8. Effects of AGS-30 on VEGF-induced angiogenesis ex vivo and in vivo

374

To investigate whether AGS-30 blocks angiogenesis ex vivo, we evaluated the effect of

375

AGS-30 on microvessels sprouting in a rat aortic ring model. Microvessels sprouting was

376

induced by treatment with VEGF (100 ng/mL) for 10 days, resulting in the formation of a

377

complete network of microvessels surrounding aortic rings. This was abolished in the

378

presence of 5 μM AGS-30 (Fig. 6B) but was unaffected by 5 μM Andro treatment.

379 17

380

The anti-angiogenic activity of AGS-30 in vivo was assessed using a mouse Matrigel plug

381

model. Matrigel plugs containing different compounds were implanted in mice; those

382

containing VEGF (250 ng) were dark red and filled with blood vessels after 14 days (Fig. 6C),

383

suggesting that abundant microvessels were formed in the plugs. In contrast, Matrigel plugs

384

containing VEGF and 5 μM AGS-30 were light red and nearly transparent, indicating that

385

fewer blood vessels had formed. To quantify functional blood vessel formation, we measured

386

hemoglobin levels in the Matrigel plugs and found that they were reduced by AGS-30 and

387

Andro by 74% and 36%, respectively, compared to the VEGF-treated group (Fig. 6D).

388 389

3.9. Effects of AGS-30 on tumor growth in a nude mouse model

390

Based on the in vitro and in vivo data suggesting that AGS-30 has anti-angiogenic activity,

391

we further investigated the anti-tumor efficacy of AGS-30 and Andro in nude mice bearing

392

HT-29 colon cell-derived tumors in which tumor growth is closely associated with

393

angiogenesis. Average tumor volume in the control group exceeded 1,300 mm3 after 20 days;

394

treatment with 2 mg/kg of AGS-30 and Andro reduced tumor weight by 52% and 21%,

395

respectively (Fig. 7B). The inhibitory effect of AGS-30 on tumor volume was greater than

396

that of Andro (Fig. 7C). However, neither AGS-30 nor Andro affected the body weight of

397

mice (Fig. 7D).

398 399

We performed a histological analysis in order to verify the mechanisms underlying the

400

anti-angiogenic and anti-tumor effects of AGS-30 in the mouse xenograft model. H&E

401

staining showed no necrosis in tumors of both control and Andro-treated mice, whereas large 18

402

areas of necrosis were observed in tumors of mice treated with AGS-30 (Fig. 8A). A

403

quantitative analysis showed that the necrotic area was increased nearly 2 fold in the

404

AGS-30-treated group compared to the control and Andro-treated groups (Fig. 8B). Blood

405

vessels in tumors were labeled with an antibody against CD31, an endothelial cell marker.

406

The immunohistochemical analysis revealed that VEGF expression was slightly reduced by

407

Andro, while a marked decreased was observed in microvessel density and VEGFR2

408

phosphorylation (Fig. 8A). A quantitative analysis showed that microvessel density and

409

VEGF and p-VEGFR2 levels were decreased by 82%, 77%, and 91%, respectively, in the

410

presence of AGS-30 (Fig. 8C–E).

411 412

Finally, we investigated the toxicity of AGS-30 and Andro in vivo by examining

413

morphological changes in the major organs. No significant damage was observed in the heart,

414

liver, spleen, lung, and kidney of mice treated with AGS-30 or Andro (Fig.9).

19

416

4. Discussion

417

VEGFR2 is an important drug target for angiogenesis inhibitors; to date, a large number of

418

drugs targeting VEGFR2 have been approved by the U.S. Food and Drug Administration

419

(FDA) including monoclonal antibodies, small molecule compounds, and microRNAs [31].

420

Sorafenib, a small molecule drug that acts on multiple kinases, was shown to inhibit tumor

421

angiogenesis by suppressing VEGFR-mediated signaling [32]. In the present study, we

422

showed that AGS-30, a 14β-(2'-chlorophenoxy) derivative of andrographolide, potently

423

inhibited tumor angiogenesis by blocking VEGFR2-induced endothelial cell proliferation,

424

migration, invasion, and tube formation. Previous studies have demonstrated that both

425

Akt/mTOR- and MEK/ERK-dependent signaling pathways regulate cell survival and

426

proliferation in endothelial and cancer cells [33-36], and that p38 MAPK regulates

427

angiogenesis by modulating endothelial cell permeability and migration [37, 38]. The present

428

study showed that AGS-30 decreased VEGF-induced phosphorylation of Akt, mTOR,

429

MEK1/2, ERK1/2, and p38 MAPK in endothelial cells. Sorafenib, a small molecule drug,

430

inhibited tumor growth and angiogenesis by blocking RAF/MEK/ERK-dependent signaling

431

in hepatocellular carcinoma [39]. Interestingly. we found that AGS-30 also inhibited the

432

activation of Akt, mTOR, and ERK1/2 in HT-29 cells. In addition to the blockade of

433

VEGFR2-mediated angiogenesis in endothelial cells, such inhibition of Akt/mTOR and

434

ERK1/2-dependent signaling in colon cancer cells may directly contribute to the anticancer

435

effect of AGS-30. The primary target of AGS-30 on HT-29 colon cancer cells is not known

436

and further study is required. It is well established that receptor tyrosine kinases (RTKs) are a

437

class of cell membrane receptors that play important roles in the regulation of cell survival, 20

438

proliferation and migration by activating the downstream PI3K/Akt/mTOR and MAPK/ERK

439

signaling pathways. We speculate that AGS-30 not only inhibits VEGFR2 but also other

440

RTKs, such as epidermal growth factor receptors (EGFR), which are found in cancer cells.

441

To support this notion, previous studies have demonstrated the potential blocking activities of

442

Andro on EGFR in cancer cells [40]. In addition, the inhibition of cancer cell proliferation via

443

the direct blocking of Akt/mTOR and ERK pathways is not impossible.

444 445

VEGF plays a critical role in tumor angiogenesis, growth, and metastasis. Preclinical and

446

clinical studies have shown that solid tumors including colon cancer express high levels of

447

VEGF in the tumor microenvironment that stimulate endothelial cells for angiogenesis [41].

448

VEGF is the most important angiogenic factor in the progression of human colon cancer and

449

its overexpression has been linked to tumor metastasis and poor prognosis [42]. Bevacizumab,

450

an anti-angiogenic agent targeting VEGF, was first approved by the FDA in 2004 and is used

451

as first-line therapy for metastatic colorectal cancer [43]. In the present study, we

452

demonstrated that VEGF is overexpressed in HT-29 colon cancer cells. Interestingly, VEGF

453

was potently suppressed by AGS-30 in these cells as well as in the tumor tissue of nude mice.

454

Thus, AGS-30 showed promise as an inhibitor of tumor angiogenesis not only through

455

blockade of VEGFR2-mediated signaling but also by reducing VEGF expression in tumor

456

cells.

457 458

The in vitro and in vivo anti-angiogenic activities of Andro have been previously

459

demonstrated.

Andro

was

shown

to

inhibit 21

tumor

angiogenesis

by

blocking

460

VEGF/VEGFR2-mediated MAPK signaling [20]; it also blocked VEGF-induced endothelial

461

cell migration and tube formation at 2.5 μM and cell proliferation at 7.5 μM [20]. However, 5

462

μM Andro had no effect on endothelial cells in our study. The reason is unclear but it is likely

463

due to the different sources of Andro. In the earlier study, Andro was isolated from

464

Andrographis paniculata by the authors [20]. However, we used Andro (purity ≧ 98%)

465

purchased commercially. The purity of Andro used in Shen et.al. was not known and we

466

cannot completely exclude the possibility that it might contain other bioactive ingredients that

467

could enhance the anti-angiogenic activity. However, our findings are in good agreement

468

with another report that Andro has anti-angiogenic activity in HUVECs at concentrations

469

higher than 25 μM and that Andro had no effects on blood vessel formation in zebrafish even

470

at a concentration as high as 300 μM [44].

471 472

Rat aortic rings and Matrigel plugs are two classic models for evaluating angiogenesis ex vivo

473

and in vivo, respectively [45]. To date, there are no reports on the anti-angiogenic effects of

474

Andro or its derivatives in the rat aortic ring model. Our results showed that 5 μM Andro had

475

no effects in this model whereas AGS-30 suppressed VEGF-induced microvessel sprouting.

476

In previous work, Andro at a concentration of 5 μM decreased blood vessel formation by

477

20% in the Matrigel plug model; moreover, Andro (3 mg/kg) blocked tumor growth and

478

reduced tumor weight by approximately 30% in nude mice [19]. These data are consistent

479

with our results, which demonstrated that 5 μM Andro inhibited VEGF-induced angiogenesis

480

by 15% in the Matrigel plug model and that 2 mg/kg Andro decreased tumor weight by 20%.

481

Importantly, we found that AGS-30 had a more potent anti-angiogenic effect in the Matrigel 22

482

plug assay and anti-tumor effects in nude mice bearing human colon tumors than its parent

483

compound Andro. AGS-30 suppressed tumor growth and decreased tumor weight by 55%

484

compared to the control group, whereas body weight was unaffected by treatment with

485

AGS-30 for 20 days. Additionally, AGS-30 did not cause any organ damage in mice,

486

highlighting the safety of this compound. The results of the immunohistochemical analysis

487

showed that AGS-30 inhibited blood vessel formation and VEGF and p-VEGFR2 expression

488

in the tumor microenvironment. The large necrotic areas observed in the tumor tissue of

489

AGS-30-treated mice may have resulted from the decreased supply of oxygen and nutrients

490

to tumor cells caused by the inhibition of angiogenesis.

491 492

In our previous study, AGP-40, a 14-phenoxy andrographolide acetal derivative of Andro,

493

was shown to exert anti-angiogenic activities in vitro and in vivo; the lowest effective

494

concentration of AGP-40 in both HUVECs and zebrafish was 10 μM [46], which is 3- to

495

4-fold

496

3,19-acetonylidene-14α-(2-methoxymethoxy) andrographolide, was shown to inhibit

497

VEGF-induced endothelial cell proliferation, migration, invasion, and tube formation at a

498

concentration as low as 2.5 μM [27], which is similar to AGS-30. However, the average

499

effective concentration of 3,19-acetonylidene-14α-(2-methoxymethoxy) andrographolide for

500

inhibiting angiogenesis in zebrafish was 103.9 ± 5.9 μM, which is much higher than that of

501

AGS-30 (7.5 ± 2.3 μM). These data indicated that AGS-30 was the most potent

502

anti-angiogenic and anticancer agent among all Andro derivatives tested to date.

higher

than

what

was

observed

503 23

here.

Another

derivative

of

Andro,

504

In conclusion, a new derivative of Andro, AGS-30, was synthesized by modifying the 14

505

position of Andro. In vitro and in vivo studies showed that this derivative was a more potent

506

anti-angiogenic and antitumor agent than its parent compound. AGS-30 strongly suppressed

507

tumor cell growth and proliferation by inhibiting Akt/mTOR and ERK-dependent pathways.

508

In addition, AGS-30 reduced VEGF expression in tumor cells and inhibited VEGF-induced

509

endothelial cell proliferation, migration, invasion, and tube formation by blocking

510

VEGFR2-mediated signaling. These findings highlighted the therapeutic potential of AGS-30

511

in the treatment of cancer.

512 513

5. Competing interests

514

The authors declare that they have no conflicts of interest.

24

516

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VEGFA/VEGFR2-MAPKs

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cascade,

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a dual-action inhibitor that targets RAF/MEK/ERK pathway in tumor cells and tyrosine

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640

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641

30

643

Scheme 1 O HO

O

HO a

HO

H

HO

644

Andrographolide (Andro)

Cl

O

O

O b

O

H

O

H O

O Intermediate

645

31

AGS-30

O O

647

Figure 1

648

32

650

Figure 2

651 652

33

654

Figure 3

655

34

657

Figure 4

658

35

660

Figure 5

661 662 663 664 36

665

Figure 6

666

37

668

Figure 7

669

38

671

Figure 8

672

39

674

Figure 9

675

40

677

Scheme 1. Synthesis of AGS-30. Reagents and conditions: (a) previously described; (b)

678

anhydrous tetrahydrofuran, intermediate (1.0 eq.), 2-chlorophenol (1.5 eq.), diisopropyl

679

azodicarboxylate (1.5 eq.), triphenylphosphine (1.5 eq.), 0°C to room temperature.

680 681 682

Figure 1. Effect of AGS-30 on the viability of HT-29 colon cancer cells and HUVECs. (A,

683

B) HT-29 cells (A) and HUVECs (B) were treated with various concentrations of Andro and

684

AGS-30 (0–20 μM) for 24 and 48 h, and cell viability was evaluated with the MTT assay. (C)

685

Cellular damage in HUVECs detected with the LDH assay. (D) HUVECs were starved in

686

low-serum medium (0.5%) for 24 h and then left untreated (control) or treated with VEGF

687

(20 ng/mL) in the presence of various concentrations of Andro (5–20 μM) and AGS-30

688

(1.25–5 μM) for 48 h. Viable cell number was quantified by Trypan Blue staining. Data are

689

presented as a percentage of the control group (mean ± SD of three independent experiments).

690

#P

< 0.05 vs. control; *P < 0.05 vs. VEGF treatment.

691 692 693

Figure 2. Effect of AGS-30 on VEGF-induced endothelial cell migration, invasion, and

694

tube formation. (A) Transwell migration assay. HUVECs were left untreated (control) or

695

treated with VEGF (20 ng/mL) in the presence of Andro (5 μM) or various concentrations of

696

AGS-30 (1.25–5 μM) for 24 h. The migration of HUVECs was detected by nuclear staining

697

of cells on the lower side of the polycarbonate membrane coated with collagen in the

698

Transwell system using 10 μg/mL Hoechst 33342. (B) Transwell invasion assay. HUVECs 41

699

were left untreated (control) or treated with VEGF (20 ng/mL) in the presence of Andro (5

700

μM) or various concentrations of AGS-30 (1.25–5 μM) for 24 h. The invasion of HUVECs

701

was detected by nuclear staining as described above. (C) HUVECs seeded on microslides

702

coated with Matrigel were left untreated (control) or treated with VEGF (20 ng/mL) in the

703

presence of Andro (5 μM) and various concentrations of AGS-30 (1.25–5 μM) for 6 h. (D–F)

704

Quantitative analysis of HUVEC migration (D), invasion (E), and tube formation (F). Scale

705

bar: 200 μm. Data are presented as percentage of the control group (mean ± SD of three

706

independent experiments). #P < 0.05 vs. control; *P < 0.05 vs. VEGF treatment.

707 708 709

Figure 3. Effect of AGS-30 on VEGFR2-mediated angiogenesis. HUVECs were cultured

710

in low-serum medium (0.5%) for 3 h and then treated with DMSO (0.1%, vehicle control) or

711

different concentrations of AGS-30 (1.25–5 μM) for 8 h, then stimulated with VEGF (50

712

ng/mL) for 15 min. (A) Expression levels of the major proteins involved in

713

VEGFR2-mediated angiogenesis in HUVECs as determined by Western blotting. (B–G)

714

Quantitative analysis of protein expression levels. Data are presented as a percentage of the

715

control group (mean ± SD of three independent experiments). #P < 0.05 vs. control; *P < 0.05

716

vs. VEGF treatment.

717 718 719 720 42

721

Figure 4. Effect of AGS-30 on the expression of VEGF and other proteins related to

722

colon cancer cell survival and proliferation. (A) HT-29 human colon cancer cells were

723

treated with various concentrations of AGS-30 (1.25–5 μM) for 48 h, and the expression

724

levels of p-Akt, Akt, p-mTOR, mTOR, p-ERK1/2, ERK1/2, and intracellular VEGF were

725

detected by Western blotting. (B–E) Quantitative analysis of protein levels. (F) Extracellular

726

VEGF levels of HT-29 colon cancer cells were measured by ELISA. Data are presented as a

727

percentage of the control group (mean ± SD of three independent experiments). *P < 0.05 vs.

728

control group.

729 730

Figure 5. Effect of AGS-30 on blood vessel formation in a transgenic zebrafish model.

731

(A–F) At 24 hpf, Tg(fli-1: EGFP) zebrafish embryos were treated with 0.1% DMSO (vehicle

732

control), 250 ng/mL VRI (positive control), 300 μM Andro, or various concentrations of

733

AGS-30 (3–30 μM) for 24 h. The embryos were photographed under an inverted fluorescence

734

microscope. (G–L) Magnified views of panels A to F. Yellow arrows indicate intersegmental

735

vessels (ISVs), dorsal aorta (DA), and dorsal longitudinal anastomotic vessel (DLAV); white

736

asterisk indicates ISV loss. (M) Quantitative analysis of blood vessel loss in zebrafish,

737

performed by counting the number of defective and intact ISVs in each embryo at 48 hpf. *P

738

< 0.05 vs. defective ISVs in control group; #P < 0.05 vs. intact ISVs in control group. (N)

739

Quantitative analysis of the anti-angiogenic phenotype of zebrafish. Embryos with any

740

incomplete ISVs were defined as exhibiting an anti-angiogenic phenotype. Scale bar: 500 μm.

741

Data are presented as the mean ± SD of ≥ 12 independent experiments *P < 0.05 vs. control.

742 43

743

Figure 6. Effect of AGS-30 on angiogenesis ex vivo and in vivo. (A) Representative

744

photographs of microvessel sprouting in rat aortic rings. The rings were embedded in

745

Matrigel and treated with 5 µM Andro and AGS-30 in the presence of VEGF (100 ng/mL)

746

for 10 days. Microvessel growth was recorded with an inverted microscope and the number

747

of sprouting microvessels was quantified with ImageJ software. Aortic rings received DMSO

748

(0.1%) and VEGF (100 ng/mL) served as negative control and positive control, respectively.

749

Scale bar: 100 µm. (B) Quantitative analysis of sprouting microvessels in each group. Data

750

are presented as a percentage of the control group (mean ± SD of three independent

751

experiments). $P < 0.05 vs. control; *P < 0.05 vs. VEGF treatment; #P < 0.05 vs. Andro. (C)

752

Representative photographs of blood vessel formation in Matrigel plugs. The 8-week-old

753

C57BL/6N female mice (n = 5) were subcutaneously injected with 0.5 mL of growth

754

factor-reduced Matrigel containing VEGF (250 ng) and heparin (150 Unit) with or without 5

755

µM Andro and AGS-30. After 14 days, the mice were sacrificed and the plugs were removed

756

and photographed. Scale bar: 2 mm. (D) Quantitative analysis of hemoglobin levels in

757

Matrigel plugs. To quantify the formation of functional blood vessels, the amount of

758

hemoglobin was measured using Drabkin reagent. Data are presented as a percentage of the

759

control group (mean ± SD of five independent experiments). $P < 0.05 vs. control; *P < 0.05

760

vs. VEGF treatment; #P < 0.05 vs. Andro.

761 762 763 764 44

765

Figure 7. Effect of AGS-30 on tumor growth in colon tumor cell-derived xenograft mice.

766

The subcutaneous dorsum of nude mice (4-weeks-old; 16 g) was inoculated with HT-29

767

colon cancer cells. Tumors were allowed to grow to approximately 200 mm3, and the mice

768

were intraperitoneally injected once every 2 days for 20 days with saline, 2 mg/kg Andro, or

769

AGS-30. (A) Images of excised tumors obtained at the end of the experiment. (B) Tumor

770

weight measured at the end of the experiment. (C) Time course of tumor growth progression.

771

(D) Time course of body weight. Data are presented as mean ± SD of five independent

772

experiments. *P < 0.05 vs. saline; #P < 0.05 vs. Andro.

773 774 775

Figure 8. Immunohistochemical analysis of tumor tissues. (A) Tumor tissue (n = 5) was

776

fixed with 4% (v/v) formaldehyde and cut into 6-μm sections that were stained with H&E to

777

evaluate tissue architecture. The sections were labeled with anti-CD31 antibody to visualize

778

blood vessels and with anti-VEGF and anti-p-VEGFR2 antibodies to evaluate tumor

779

angiogenesis. (B–E) Quantitative analysis of necrotic area, blood vessel formation, and

780

VEGF and p-VEGFR2 expression in each group. Data were quantified with ImageJ software.

781

*P

< 0.05 vs. saline; #P < 0.05 vs. Andro.

782 783 784

Figure 9. Hematoxylin and eosin staining of major organs in mice treated with saline,

785

Andro and AGS-30.

786 45