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Curcumin Suppresses Phthalate-induced Metastasis and the Proportion of CSCs-like via the Inhibition of AhR/ERK/SK1 Signaling in Hepatocellular Carcinoma Cheng-Fang Tsai, Tsung-Hua Hsieh, Jau-Nan Lee, Chia-Yi Hsu, Yu-Chih Wang, Kung-Kai Kuo, Hua-Lin Wu, Chien-Chih Chiu, Eing-Mei Tsai, and Po-Lin Kuo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04415 • Publication Date (Web): 20 Nov 2015 Downloaded from http://pubs.acs.org on November 25, 2015

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Journal of Agricultural and Food Chemistry

Curcumin Suppresses Phthalate-induced Metastasis and the Proportion of CSCs-like via the Inhibition of AhR/ERK/SK1 Signaling in Hepatocellular Carcinoma

§

§

Cheng-Fang Tsai , Tsung-Hua Hsieh , Jau-Nan Lee , Chia-Yi Hsu , Yu-Chih Wang , ○ △ § Kung-Kai Kuo , Hua-Lin Wu , Chien-Chih Chiu , Eing-Mei Tsai * and Po-Lin Kuo

#*

Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University,

Kaohsiung City 807, Taiwan. §Department of Obstetrics and Gynecology Kaohsiung Medical University Hospital, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, City 807, Taiwan. ○ Division of Hepatobiliary Pancreatic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.

△

Department of Biochemistry and Molecular Biology, National

Cheng Kung University, Tainan, Taiwan. Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan. #Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. *Corresponding authors: Eing-Mei Tsai, Graduate Institute of Medicine and Department of Obstetrics and Gynecology, Kaohsiung Medical University, No. 100, Zihyou 1st Rd., Sanmin District, Kaohsiung City 807, Taiwan; E-mail: [email protected]; Tel: +886-7-3121101-6446; Fax: +886-7-321-2062 Po-Lin Kuo, Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical 1

ACS Paragon Plus Environment


University, No. 100, Zihyou 1st Rd., Sanmin District, Kaohsiung City 807, Taiwan; E-mail: [email protected]; Tel: +886-7-3121101-5528; Fax: 886-7-321-0701

2


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1

ABSTRACT

2

Recent evidence indicating that phthalates promote cancer development, including

3

cell proliferation, migration, and invasion, have raised public health concerns. Here,

4

we show that bis(2-ethylhexyl) phthalate promotes the migration, invasion, and

5

epithelial-mesenchymal transition of hepatocellular carcinoma cells. In addition,

6

bis(2-ethylhexyl) phthalate increased the proportion of cancer stem cell (CSC)-like

7

cells and stemness maintenance in vitro as well as tumor growth and metastasis in

8

vivo. The various activities of curcumin, including anti-cancer, anti-inflammation,

9

antioxidation and immunomodulation have been investigated extensively. Curcumin

10

suppressed phthalate-induced cell migration, invasion and epithelial-mesenchymal

11

transition, decreased the proportion of CSC-like cells in hepatocellular carcinoma

12

cell lines in vitro, and inhibited tumor growth and metastasis in vivo. We also

13

revealed that curcumin suppressed phthalate-induced migration, invasion, and

14

CSC-like cells maintenance through inhibition of the aryl hydrocarbon

15

receptor/ERK/SK1/S1P3 signaling pathway. Our results suggest that curcumin may

16

be a potential antidote for phthalate-induced cancer progression

17 18

KEYWORDS:

phthalate;

curcumin;

19

side-population; cancer stem cells

hepatocellular

3


carcinoma;

metastasis;


20

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INTRODUCTION

21

Curcumin, a phenolic compound extracted from Zingiberaceae turmeric, has shown

22

potent anti-inflammatory, antioxidant, and antitumor properties.1-4 Recent studies

23

have reported that curcumin inhibits the growth, migration, invasion, and metastasis

24

of a variety of tumor cells including those of thyroid, lung, and breast cancers as well

25

as hepatocellular carcinoma (HCC). 5-9 Several animal studies have demonstrated the

26

inhibitory effects of curcumin on HCC tumorigenesis and metastasis,10-12 and

27

curcumin can be applied as chemopreventive agent against HCC. 13

28

Phthalates have been widely used as plasticizers to increase the flexibility of

29

plastics for more than 50 years. 14 In addition to acting as an endocrine disruptor with

30

toxic effects on reproductive and developmental processes, 15,16 recent studies indicate

31

that phthalates promote the progression of several types of cancers. 17-19 Thus, public

32

health concerns about phthalate exposure have increased. In animal studies,

33

bis(2-ethylhexyl)

34

N-nitrosodiethylamine20 and induces tumors in the liver and testes after long-term

35

treatment in rats. 21

36

HCC is the third most common cause of cancer mortality worldwide. 22 The long-term

37

survival rate remains low because of a high (75%-100%) 5-year recurrence rate and

38

frequent metastasis after hepatic resectioning. 23 Recent evidence indicates that HCC

phthalate

(DEHP)

promotes

4


HCC

initiated

by

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39

progression is driven by a subpopulation of cancer cells exhibiting stem cell

40

called cancer stem cells (CSCs). CSCs are involved in tumor initiation, progression,

41

recurrence, and metastasis owing to their ability to self-renew, differentiate, and give

42

rise to new tumors in local and distant organs. CSCs are characterized by several

43

markers including CD133, CD44, CD90, aldehyde dehydrogenase, OV6, epithelial

44

adhesion molecule, and CD13, which have been identified in various human HCC cell

45

lines and specimens. 24-27 Our previous study showed that phthalates induce HCC

46

angiogenesis and metastasis. 28 Despite knowing the effect on tumor progression, the

47

mechanisms underlying the effects of phthalates on HCC metastasis and CSCs

48

maintenance remain unclear.

49

Therefore, we investigated whether phthalate exposure induces HCC metastasis and

50

CSCs maintenance, elucidated the signaling pathways involved in these processes,

51

and determined the mechanism of curcumin as the anti-dote for phthalate-induced

52

tumor progression. This report presents the first evidence that curcumin suppresses

53

phthalate-induced increases in the proportion of CSC-like cells (CSCs-like) as well as

54

metastasis by inhibiting epithelial-mesenchymal transition (EMT) through aryl

55

hydrocarbon receptor (AhR)/ERK/SK1/ sphingosine 1-phosphate receptor (S1PR3)

56

signaling pathways. Our results reveal that the application of curcumin may be an

57

effective strategy for the treatment of HCC progression induced by phthalate. 5



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MATERIALS AND METHODS

59

Reagents. DEHP, di-(n-butyl) phthalate (DBP), butyl benzyl phthalate (BBP),

60

curcumin

61

butylidenephthalide, Hoechst 33342, DAPI, and verapamil were purchased from

62

Sigma-Aldrich

63

Calciochem-Novabiochem (San Diego, CA, USA). Antibodies against AhR and

64

vimentin were purchased from Santa Cruz Biotechnology (CA, USA), and antibodies

65

against E-cadherin, N-cadherin, phospho-p44/42 MAPK (Erk1/2), p44/42 MAPK

66

(Erk1/2), SPHK1, CD44, and β-actin were purchased from Cell Signaling (Danvers,

67

MA, USA). An antibody against CD133 was obtained from Abcam (Cambridge, UK),

68

and an antibody against sphingosine kinase 1 (SK1; Ser-225) was purchased ECM

69

Biosciences (Versailles, KY, USA).

70

Cell culture. Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium (Life

71

Technologies, Grand Island, NY, USA), and PLC/PRF/5 cells were cultured in

72

minimum essential medium (Life Technologies, Grand Ialand, NY, USA)

73

supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 1%

74

penicillin (100 U/mL), streptomycin (10 µg/mL), and amphotericin-B (250 µg/mL)

75

(Sigma-Aldrich, St. Louis, MO) at 37°C with 5% CO2.

76

Trans-well Migration and Invasion Assays. Cell migration and invasion assays

(analytical

(St.

standard,

Louis,

≥98.0%

MO,

USA).

purity,

HPLC

PD98059

6


grade),

was

glabridin,

obtained

from

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77

were performed in 24-well inserts (BD Biosciences, Franklin Lakes, NJ, USA). Cells

78

(2 × 104) in serum-free medium were seeded in the upper chamber and treated with

79

phthalate or curcumin. Medium containing 10% fetal bovine serum was added to the

80

lower chamber. The cells were incubated for 24 h to assess migration and 48 h to

81

assess invasion. Cells were removed from the upper chamber using cotton swabs.

82

Cells underneath the membrane were fixed with 4% paraformaldehyde for 30 min,

83

stained with 0.5% crystal violet for 2 h, and counted under a microscope.

84

Cell Viability Assay. Cells were seeded in 96-well plates at 3000 cells per well in

85

complete medium and cultured for 24 h. The medium was removed, and the treatment

86

medium was added. After 24 h of treatment, cell viability was assessed by a WST-1

87

assay (Clontech, Mountain View, CA, USA) that quantifies mitochondrial metabolic

88

activity.

89

Western Blotting. After treatments, cells were lysed using RIPA lysis buffer

90

(Millipore, Temecula, CA, USA). After boiling for 10 min in SDS- polyacrylamide

91

gel electrophoresis (PAGE) sample buffer, equal amounts of protein were subjected to

92

SDS-PAGE. The separated proteins were transferred to a polyvinylidene difluoride

93

membrane. The membrane was blocked with 5% nonfat dried milk in TBST (50 mM

94

Tris-HCl, 150 mM NaCl, and 0.1% Tween-20, pH 7.6) at room temperature for 1 h

95

and subsequently incubated with primary antibodies overnight at 4°C. The membrane 7



96

was washed three times (10 min each) with TBST and then incubated with a

97

horseradish peroxide–conjugated secondary antibody for 1 h at room temperature,

98

followed by three 10-min washes with TBST. Immunoreactive bands in each blot

99

were visualized using ECL western blotting substrate (Millipore, Billerica, MA,

100

USA).

101

Immunofluorescence Microscopy. Cells grown on coverslips were fixed and

102

permeabilized for 10 min in ice-cold methanol. After blocking in 5% bovine serum

103

albumin, the cells were incubated with the primary antibody (1:200) overnight at 4°C.

104

The cells were washed in PBS, incubated with Alexa Fluor 488 or Alexa Fluor 594

105

(Invitrogen, Carlsbad, CA) as the secondary antibody (1:100) for 2 h and then

106

mounted with DAPI (1 µg/mL). Images were obtained by fluorescence microscopy.

107

Side Population (SP) Analysis and Purification by Flow Cytometry. Cells were

108

detached from dishes using trypsin-EDTA (Gibco, Grand Island, NY, USA) and

109

suspended at 1 × 106 cells/mL in Hank’s balanced salt solution containing 3% fetal

110

bovine serum. The cells were incubated at 37°C for 90 min with 20 µg/mL Hoechst

111

33342 either alone or in the presence of 50 µM verapamil, an inhibitor of the

112

verapamil-sensitive ABC transporter. Then, the cells were immediately centrifuged at

113

4°C for 5 min at 300 × g and resuspended in ice-cold Hank’s balanced salt solution.

114

The cells were kept on ice to inhibit efflux of the Hoechst dye. Finally, the cells were 8


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115

filtered through a 40-µm cell strainer (Falcon; BD Biosciences, Mississauga, Canada)

116

to obtain a single-cell suspension. Dual-wavelength analysis and cell purification

117

were performed on a dual-laser fluorescence-activated cell sorter (Vantage SE; BD

118

Biosciences, San Jose, CA, USA). Hoechst 33342 was excited with 355 nm UV light.

119

Blue and red fluorescences were measured with a 450/20 band-pass filter and 675-nm

120

edge filter (long pass). A 610-nm dichroic mirror (short pass) was used to separate the

121

emission wavelengths.

122

Flow Cytometry. To determine the CD44+/CD133+ phenotype, cells were

123

trypsinized, washed, and suspended at 1 × 106 cells/mL in phosphate-buffered saline

124

(PBS)

125

(phycoerythrin-conjugated) ; BD Pharmingen, San Jose, CA, USA) and CD133

126

(Abcam) antibodies. Alexa Fluor 488–conjugated goat anti-human IgG (Invitrogen,

127

Carlsbad, CA, USA) was used as a the secondary antibody. The cells were analyzed

128

using an LSR II flow cytometer (Becton Dickinson, San Jose CA, USA) and

129

CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA). Three independent

130

experiments were performed.

131

Quantitative Polymerase Chain Reaction (qPCR). Total RNA was isolated from

132

Huh7 cells using TRIzol reagent (Invitrogen). RNA (2 μg) was used to synthesize

133

cDNA in a Reverse Transcription System (Promega, Madison, WI, USA), and cDNAs

containing

0.5%

bovine

serum

albumin

and

9


anti-human

CD44


134

were amplified with the following primers:

135

S1PR1 (forward, 5′-GAGGGAGGAAGGGGGAGAAT-3′, reverse,

136

5′-AGAGACGCTTTCACATGGGG-3′); S1PR2 (forward,

137

5′-TGCTGCACTCTCACCTTCTG-3′, reverse 5′-ATCCACCTGGGGGTGACTC-3′);

138

S1PR3 (forward, 5′-TTGCCTTCCCACACACAAGT-3′, reverse

139

5′-ACCCAGATATGGAGGCTGT-3′); and 18S (forward,

140

5′-GTAACCCGTTGAACCCCATT-3′, reverse

141

5′-CCATCCAATCGGTAGTAGCG-3′). Reactions were performed using TaqMan

142

Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA) and an

143

ABI 7500 system (Applied Biosystems).

144

Microinjection of Huh7-GFP Cells into Zebrafish. After treatment with DEHP for

145

24 h, Huh7-GFP cells were washed, resuspended in PBS, and then subjected to

146

fluorescence-activated cell sorting to separate side population (SP) and non-SP cells.

147

For microinjection of tumor cells into embryos, Tg (fli1: EGFP)y1 zebrafish embryos

148

at 2 days post-fertilization were dechorionated and anesthetized with tricaine

149

(Sigma-Aldrich). The desired number of Huh7-GFP cells was injected into the middle

150

of the embryonic yolk sac region using a Pneumatic Pico-Pump Injector (Harvard

151

Apparatus, Holliston, MA, USA) with an injection needle (World Precision

152

Instruments Inc., Sarasota, FL, USA) pulled by a P-97 Flam/Brown Micropipette 10


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Puller (Sutter Instruments Co., Novato, CA, USA). After injection, embryos with

154

fluorescent cells outside of the desired injection region were excluded from further

155

analyses. Tumor growth in zebrafish was measured by the fluorescence intensity with

156

MetaMorph software (Molecular Devices, Sunnyvale, CA, USA).

157

Short Hairpin RNA (shRNA) Transfection. The following shRNAs were obtained

158

from the National RNAi Core Facility at Academic Sinica: control shRNA (shGFP;

159

TRCN0000072178),

160

(TRCN00000245285), S1PR1 shRNA (TRCN0000011382, TRCN0000221136 and

161

TRCN0000221138), and S1PR3 shRNA (TRCN0000221126, TRCN0000221128 and

162

TRCN0000356946). Cells were transfected with shRNA (2 μ g) using LT1

163

transfection reagent (Mirrus Bio, Madison, WI, USA).

164

Animals. Male 6-week-old nude mice (BALB/cA-nu nu/nu) were purchased from the

165

National Laboratory Animal Center (Taipei, Taiwan). All animal experiments were

166

performed according to a protocol approved by the Institutional Animal Care and Use

167

Committee of Kaohsiung Medical University Hospital (IACUC Approval No: IACUC

168

Approval No: 101060).

169

In vivo orthotopic HCCs model. Huh7 cells were stably transfected with green

170

fluorescent protein (GFP). Lentivirus-containing medium (200 µL) was mixed with

171

800 µL Dulbecco’s modified Eagle’s medium containing 8 µg/mL polybrene and then

AhR

shRNA-1

(TRCN0000021255),

11


AhR

shRNA-2


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172

added to each well. The cells were incubated for 1 day. A stable clone was selected

173

with puromycin (2 µg/mL) for 14 days. The HCC model of direct intrahepatic

174

injection was established according to a previous study29 with some modifications.

175

Briefly, after a small incision was made in each nude mouse to access the liver,

176

Huh7-GFP cells (1 × 106) suspended in PBS were slowly injected into the upper-left

177

lobe of the liver using a 28 G needle. A transparent bleb of cells was formed through

178

the liver capsule after injection. To prevent bleeding, a small piece of sterile gauze

179

was applied with light pressure at the injection site. After cell implantation, the mice

180

were placed on a heating pad or below a heat lamp until fully motile. The mice were

181

randomly divided into four groups (vehicle control, DEHP, curcumin, and curcumin

182

plus DEHP) with six mice per group. After 14 days, DEHP (60 mg/kg) and curcumin

183

(50 mg/kg) were administered via intraperitoneal injection every 2 days. After 1

184

month of treatment, the mice were sacrificed and their livers were removed and

185

viewed using a Non Invasive In Vivo Imaging System (IVIS) (Caliper Life Sciences,

186

Hopkinton, MA, USA).

187

Immunohistochemistry.

188

paraformaldehyde, embedded in paraffin, and cut into 4-µm-thick sections. The

189

sections were deparaffinized in xylene, rehydrated with a graded series of

190

ethanol/water solutions (100% and 95% ethanol), and then washed with water. The

Liver

and

lung

tissues

were

12


fixed

with

4%

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191

sections were treated with 10 mM sodium citrate buffer (pH 6.5) at 95°C to retrieve

192

antigens and then blocked with 5% bovine serum albumin in PBS. Primary antibodies

193

against CD44 (1:200), CD133 (1:200), and AhR (1:200) were applied to the sections

194

at 4°C overnight. The sections were incubated with secondary antibodies and

195

3,3’-diaminobenzidine

196

(TissueGnostics USA, Ltd.).

197

Wound Healing Assay. Cells were cultured in 12-well plates to a near confluent

198

monolayer. A scratch test was performed by scratching the cell monolayer using a

199

pipette tip. The cells were washed twice with PBS to remove debris and detached

200

cells from the monolayer before photographing the scratched area. Then, glabridin,

201

butylidenephthalide, or curcumin were added at various concentrations (1 and 10 µM)

202

with or without phthalate. The control well contained DMSO as the solvent

203

control. Cells were incubated for 12 h before the same areas were re-photographed.

204

Statistical Analysis. The student’s t-test was used for comparisons of more than two

205

mean values. The means ± standard deviation (SD) of three independent experiments

206

are shown. Results with p values less than 0.05 were considered statistically

207

significant.

208 209

RESULTS

210

DEHP Increases HCC Migration, Invasion, SP cells and CSCs-like in vitro. To

and

then

analyzed

using

Tissue

13


Quest

software


211

examine the effects of phthalates on cell migration and invasion, trans-well migration

212

and invasion assays were performed. DEHP at 0.1 µM concentration significantly

213

increased the migration and invasion of Huh7 and PLC cells. BBP increased

214

migration and DBP increased invasion in PLC cells (Figure 1A). Because it had the

215

most potent effects on cell migration and invasion, DEHP was used in subsequent

216

experiments.

217

Next, the effect of DEHP on cell viability was examined using a WST-1 assay.

218

DEHP did not affect cell viability at concentrations from 10–9 M to 10–6 M (Figure

219

1B). We examined the ability of DEHP to promote EMT that plays a critical role in

220

promotion of metastasis in cancer cells30. E-cadherin (epithelial marker) was

221

significantly downregulated, while N-cadherin and vimentin (mesenchymal markers)

222

were upregulated after DEHP treatment (0.1 µM, 24 h; Figure 1C). The

223

downregulation of E-cadherin and upregulation of vimentin were confirmed by

224

immunofluorescence which corroborated the western blot results (Figure 1D).

225

Accumulating evidence shows that hepatic CSCs can be isolated based on

226

several markers, such as CD133 and CD44 or selected through functional assays such

227

as isolation of

228

with DEHP (0.1 µM) for 24 h, and the proportion of CSCs-like were evaluated by

229

flow cytometry. DEHP (0.1 µM) significantly increased the percentage of SP cells

SP cells based on Hoechst dye staining.31 Huh7 cells were treated

14


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230

stained with Hoechst 33342 (Figure 1E) and the percentage of CD44+/CD133+ cells

231

(Figure 1F). These data suggested that DEHP increases HCC cell migration, invasion,

232

and EMT, and might play a role in HCC CSCs maintenance in vitro.

233

DEHP Regulates EMT through AhR/ERK/SK1/S1PR3 signaling. To examine the

234

effects of DEHP on AhR, Huh7 cells were treated with various concentrations of

235

DEHP, and then AhR protein levels were assessed by western blotting. AhR levels

236

increased by treatment with 0.1 or 1 µM DEHP (Figure 2A). Therefore the lower

237

concentration was used in subsequent experiments. To investigate whether DEHP

238

increases cell migration, invasion, and the SP fraction through an AhR-dependent

239

mechanism, Huh7 cells were transfected with two different AhR shRNAs or a control

240

shRNA. The two AhR shRNAs decreased DEHP-induced cell migration (Figure 2B),

241

invasion (Figure 2C), and the SP fraction (Figure 2D). SK1 activity is regulated by

242

ERK,32 that is activated by phthalates through AhR signaling.28 Knockdown of AhR

243

with shRNA inhibited the phosphorylation of ERK and downregulated DEHP-induced

244

SK1 activity, (Figure 2E). Pretreatment with the ERK inhibitor PD98059 reduced the

245

DEHP-induced phosphorylation of SK1. Additionally, E-cadherin (epithelial marker)

246

downregulation and N-cadherin (mesenchymal marker) upregulation by DEHP were

247

reversed (Figure 2F). These results suggest that DEHP induces EMT through AhR,

248

which is supported by previous studies showing that AhR regulates cell motility by 15



249

promoting EMT through induction of Slug and extracellular matrix remodeling via

250

indirect induction of matrix metalloproteinases.33,34 DEHP-induced migration (Figure

251

2G) and invasion (Figure 2H) were reduced by PD98059 pretreatment. Evidence has

252

shown that S1P, a product of SK1, triggers signaling pathways that mediate

253

pro-survival processes by engaging S1P receptors 1–5 (S1PR)1-5.35 To further

254

understand the mechanisms, the cells were treated with DEHP, and mRNA levels were

255

detected by qPCR. S1PR1 and S1PR3 mRNA levels increased significantly after

256

DEHP treatment (Figure 2I). Thus, Huh7 cells were transfected with S1P1 and S1PR3

257

shRNAs after DEHP treatment. The S1PR3 shRNA transfection reduced the

258

percentage of CD44+/CD133+ cells induced by DEHP, suggesting that DEHP

259

regulates HCC CSCs-like through S1PR3 rather than S1PR1 (Figure 2J). Thus, DEHP

260

regulates migration, invasion, and CSCs-like populations through AhR/ERK/SK1

261

activation via S1PR3 signaling.

262

DEHP Increases HCC Stemness Maintenance, Tumor Growth, and Metastasis in

263

vivo. To identify the stemness properties of SP cells, the SP and non-SP of Huh7-GFP

264

cells were sorted and treated with DEHP (0.1 µM) for 24 h. The cells were then

265

injected into the yolk of zebrafish eggs, and the GFP fluorescence intensity was

266

measured using MetaMorph software. At 5 days post-injection, the tumor size in the

267

DEHP-treated group increased was significantly compared with the control group as 16


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represented by the numbers of non-SP (1.8-fold) and SP (3.7-fold) cells (Figure 3A).

269

These results suggested that DEHP promotes the maintenance of CSCs stemness that

270

is correlated to tumor growth. To examine the effect of DEHP on tumor growth in

271

vivo, we established an orthotopic HCCs model. An IVIS was used to measure the

272

fluorescence intensity that reflected the tumor size. DEHP-treated groups had

273

significantly greater tumor growth compared with the control group (Figure 3B). To

274

examine the potential effect of DEHP on metastasis in vivo, mice were sacrificed at

275

the end of experiment, and hematoxylin and eosin staining was applied. The number

276

of metastatic lung nodules was significantly higher in the DEHP-treated group

277

compared with the control group. Figure 3C (right panel) shows the largest nodule

278

(~400 µm diameter) in the DEHP-treated group. Based on flow cytometry, the

279

proportion of CD44+/CD133+ cells in metastatic tumors was larger than that in the

280

primary tumor (Figure 3D). These results suggest that DEHP promotion of lung

281

metastasis may be related to the increase in CSCs-like. To confirm the importance of

282

AhR signaling in tumor progression and metastasis in vivo, immunohistochemical

283

staining was performed. AhR expression appeared to be higher in the DEHP-treated

284

group compared with the control group, which corroborated the in vitro results

285

(Figure 3E). Moreover, the levels of AhR, CD44, and CD133 (CSC marker) were

286

higher at metastatic sites than in primary tumor sites (Figure 3F). These results 17



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287

suggested that DEHP promotes HCC tumor growth and metastasis, and may regulate

288

the maintenance of HCC CSCs in vivo.

289

Curcumin Blocks the Effect of DEHP in vitro. Three herbal drugs, which have

290

anti-cancer potentials,36,37 were screened for their ability to inhibit phthalate-induced

291

Huh7

292

phthalate-induced cell migration, whereas glabridin and butylidenephthalide did not

293

(Figure 4A). Therefore, curcumin was used in subsequent experiments. Moreover,

294

curcumin treatment at 1.25 to 5 µΜ with or without DEHP (0.1 µΜ) did not affect

295

Huh7 or PLC cell viability (Figure 4B). To investigate whether curcumin inhibits

296

DEHP-induced cell migration and invasion, trans-well migration and invasion assays

297

were performed. Curcumin at various concentrations inhibited the DEHP-induced

298

migration and invasion of Huh7 cells (Figure 4C) and PLC cells (Figure 4D). Huh7

299

cells were treated with DEHP (0.1 µM) or curcumin (5 µM) for 24 h, and the

300

proportion of CSCs-like was evaluated by flow cytometry. Curcumin significantly

301

inhibited the percentage of DEHP-induced SP cells stained with Hoechst 33342

302

(Figure 4E). Additionally, the sphere-formation assay was performed to validate that

303

the ability of the inhibitory effect of curcumin on DEHP-induced proportion of

304

CSCs-like (Supplementary Fig.1). To elucidate the mechanisms, Huh7 cells were

305

treated with or without curcumin (5 µΜ) or DEHP (0.1 µΜ) and then analyzed by

cell

migration

using

wound

healing

assays.

18


Curcumin

inhibited

Page 19 of 45


306

western

blotting.

Curcumin

blocked

DEHP-induced

AhR

upregulation,

307

phosphorylation of ERK and SK1, and EMT (Fig 4F). These results suggest that

308

curcumin blocks DEHP-induced cell migration, invasion, and EMT as well as the

309

expansion of HCC CSCs through AhR/ERK/SK1-related pathways.

310

DEHP Enhances Metastasis and Curcumin Blocks the Effect of DEHP in vivo. To

311

determine the effect of curcumin on metastasis in vivo, we established an orthotopic

312

HCCs model. DEHP significantly increased the tumor size, whereas curcumin

313

inhibited the growth induced by DEHP, which was measured using the IVIS (Figure

314

5A). We calculated the number of nodules in hematoxylin and eosin-stained lung

315

sections. Curcumin significantly reduced the number of DEHP-induced nodules in the

316

lungs (Figure 5B), suggesting that curcumin inhibited DEHP-induced metastasis in

317

vivo.

318

We propose a signaling pathway for DEHP-stimulated HCC metastasis and

319

CSCs-like maintenance through the AhR/ERK/S1P/S1P3 pathway, which can be

320

inhibited by curcumin (Figure 6).

321

DISCUSSION

322

Curcumin has been extensively investigated for its anti-cancer properties. It

323

inhibits EMT, migration, invasion, and metastasis of cancer cells. 1,5-6, 38 Moreover,

324

curcumin targets CSCs by reducing the SP size, decreasing sphere formation, 19



325

downregulating CSC markers, and suppressing tumorigenicity. It also enhances the

326

effectiveness of cisplatin by suppressing CD133+ CSCs in the treatment of laryngeal

327

carcinoma.39-41

328

HCC is a cancer that tends to undergo vascular invasion and metastasis, and most

329

patients do not survive the metastatic disease.42 Our previous studies have shown that

330

phthalate induces HCC metastasis in vitro and in vivo. Huh7 is well-differentiated and

331

PLC is poorly-differentiated HCC. Moreover, HCC cell lines Huh7 and PLC show

332

activation of AhR upon phthalate treatment.28 Therefore, we used these two HCC cell

333

lines in the present study and demonstrated that DEHP induced HCC cell migration,

334

invasion, and EMT. We also showed that DEHP increased tumor growth and

335

metastasis by in vivo orthotopic HCCs model.

336

An increasing body of evidence has shown that CSCs are the cause of

337

carcinogenesis and are involved in metastasis and recurrence.43 Targeting CSCs is a

338

fundamental treatment strategy for cancer. However, limited reports investigate

339

whether phthalates are involved in CSC maintenance. A previous study has shown

340

that a CD133+CD44high subpopulation of tumor cells is responsible for hematogenous

341

metastasis of liver cancers.44 The present study showed that DEHP increased the

342

CSCs-like growth in vitro and in vivo. Furthermore, metastatic lung section had

343

higher levels of HCC CSCs markers (CD44 and CD133) than the primary tumor in 20


Page 20 of 45

Page 21 of 45


344

orthotopic HCCs model, suggesting that phthalate affects CSCs-like maintenance and

345

may be related to metastasis.

346

Moreover, we investigated the molecular mechanisms of phthalate. We

347

previously shown tthat phthalate activates AhR in HCC to enhance progression.28

348

AhR is a ligand-activated transcription factor that influences the major stages of

349

tumorigenesis, initiation, promotion, progression and metastasis.45 Our current study

350

demonstrated that DEHP induced cell migration, invasion, and EMT and affected the

351

percentages of SP cells and CSCs marker expression (CD44 and CD133) through

352

AhR. The involvement of the AhR/ERK/SK1/S1P/S1P3 signaling pathway, when

353

stimulated with DEHP, was confirmed by AhR shRNA transfection experiments and

354

the treatment with ERK inhibitor pd98059. Animal experiments showed that AhR

355

signaling may lead to phthalate-induced CSCs-like maintenance, which is supported

356

by a previous study showing that AhR is involved in the maintenance of

357

hematopoietic stem and progenitor cells during development and adult life. 46 S1P, a

358

sphingolipid metabolite that regulates many physiological processes such as cell

359

survival, growth and migration as well as angiogenesis is an extracellular ligand that

360

binds to G protein–coupled receptors (S1PR1-5). S1P is produced by the actions of

361

sphingosine kinases, and two isoforms of this enzyme have been identified and cloned.

362

SK1 is mainly cytosolic and mediates pro-survival functions, whereas SK2 is 21



363

predominantly localized in the nucleus where it inhibits growth and enhances

364

apoptosis. 47 SK1 activity is reported to be regulated by ERK.48 Previous studies have

365

shown that S1P promotes CSCs expansion via S1PR349, and high cytoplasmic S1PR1,

366

S1PR3, SK1 and ERK-1/2 expression levels are associated with shorter survival and

367

recurrence times in breast cancer patients49,50, which concur with our results. However,

368

our findings are the first to elucidate the effects of phthalate on HCC metastasis by

369

regulating CSCs-like through AhR/ERK/SK1/S1P/S1PR3 signaling pathways. Thus,

370

the present study opens new avenues for therapeutic strategies to treat

371

phthalate-related HCC progression.

372

Elimination of exposure to phthalates may be impossible because of their

373

widespread use in every aspect of modern life. Therefore, finding an agent that can

374

counteract the effects of phthalates is important. This study is the first to show that

375

curcumin inhibits the carcinogenic effects of DEHP. Our results showed that curcumin

376

inhibited DEHP-induced AhR expression, migration, invasion, and EMT in cells

377

through ERK/SK1 signaling in vitro. Activation of SK1/S1P signaling is significantly

378

inhibited by curcumin in diabetic rats 51, which supports our results. Moreover, we

379

found curcumin regulate CSCs induced by phthalates. Previous studies have shown

380

that high concentrations of curcumin inhibit cell growth (20–108 µM)52-54, induce

381

apoptosis (10–40 µM)55,56, and cause cell cycle arrest (25–50 µM)57 of HCC. The 22


Page 22 of 45

Page 23 of 45


382

present study showed that a low concentration of curcumin (5 µM) significantly

383

blocked DEHP-induced migration, invasion, and CSC maintenance of human HCC

384

cells.

385

In conclusion, our study showed that curcumin inhibits DEHP-induced

386

metastasis and CSCs-like maintenance through AhR/ERK/SK1/S1PR3 signaling

387

pathways and may thus constitute a potential therapeutic agent for HCC.

388

ACKNOWLEDGMENT

389

The authors thank the Center for Resources, Research and Development of Kaohsiung

390

Medical University for its support with instrumentation.

391

FUNDING SOURCES

392

This work was supported by the Ministry of Science and Technology, Taiwan

393

[102-2628-B-037- 011-MY3 and 102-2632-B-037-001-MY3] and the Kaohsiung

394

Medical University Research Fund, Aim for the Top Universities Grant,

395

[KMU-TP104A02, KMU-TP104E22, ], and Kaohsiung Medical University Hospital

396

Research Fund [KMUH103-10V07, KMUH 103-3R26].

397

ABBREVIATIONS USED

398

HCC, hepatocellular carcinoma; DEHP, bis(2-ethylhexyl) phthalate; CSCs, cancer

399

stem cells; EMT, epithelial mesenchymal transition; DBP, di-(n-butyl) phthalate; BBP,

400

butyl benzyl phthalate; GFP, green fluorescent protein; SP, side population; AhR, aryl 23



Page 24 of 45

401

hydrocarbon receptor; S1P, sphingosine 1-phosphate; SK1, sphingosine kinase 1; SK2,

402

sphingosine kinase 2

403

Supporting Information

404

Supplementary Fig.1 Curcumin significantly inhibited DEHP-induced sphere

405

formation.

406

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600 601

Figure legends

602

Figure 1. (A) Huh7 and PLC cells were treated with DMSO (control) or 0.1 µΜ of

603

individual phthalates (DBP, BBP and DEHP). Cell migration (24 h) and invasion (48

604

h) were measured by trans-well migration and invasion assays, respectively. Data are

605

the means ± SD of three independent experiments. *p < 0.05; **p

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