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Oleanolic acid induces metabolic adaptation in cancer cells by activating AMP-activated protein kinase pathway Jia Liu, Lanhong Zheng, Ning Wu, Leina Ma, Jiateng Zhong, Ge Liu, and Xiukun Lin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf500622p • Publication Date (Web): 23 May 2014 Downloaded from http://pubs.acs.org on June 2, 2014

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Journal of Agricultural and Food Chemistry

Oleanolic acid induces metabolic adaptation in cancer cells by activating AMPactivated protein kinase pathway Jia Liu 1, 3, *, Lanhong Zheng *, Ning Wu 1, *, Leina Ma 4, Jiateng Zhong 5, Ge Liu 1, 3, Xiukun Lin, **, 1, 6 1. Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China 2. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China 3. Graduate School, University of Chinese Academy of Sciences, Beijing 100049, China 4. School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China 5. Department of Pathophysiology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China 6. Department of Pharmacology, Capital Medical University, Beijing 100069, China

AUTHOR INFORMATION * These authors contributed equally to this work. Corresponding Author ** (X. L.) Phone: 86-10-83911837. Fix: 86-10-83911837. Email: [email protected] Funding This work was supported by National innovative drug development projects of (2014ZX09102043-001) and 863 High Technology Project (No. 2014AA093503). The study was also supported in part by National Foundation of Natural Sci. of China (81302906,

ACS Paragon Plus Environment


81273550 and 41306157) and Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences (2013B01YQ01). Notes The authors declare no competing financial interest Key words Aerobic glycolysis; AMPK; Metabolism; Oleanolic acid 1

Abstract

2

Cancer cells are well known to require constant supply of protein, lipid, RNA and DNA

3

via altered metabolism for accelerated cell proliferation. Targeting metabolic pathway is,

4

therefore, a promising therapeutic strategy for cancers. Oleanolic acid (OA) is widely

5

distributed in dietary and medicinal plants and displays a selective cytotoxicity to cancer

6

cells, primarily by inducing apoptosis and cell cycle arrest. In this study, we investigated

7

if OA inhibited growth of tumor cells by affecting their metabolism. OA was found to

8

activate AMP-activated protein kinase (AMPK), the master regulator of metabolism, in

9

prostate cancer cell line, PC-3, and breast cancer cells line, MCF-7. AMPK activation is

10

required for the anti-tumor activity of OA on cancer cells. Lipogenesis, protein synthesis

11

and aerobic glycolysis were inhibited in cancer cells treated with OA, in an AMPK

12

activation-dependent manner. The metabolic alteration was shown to mediate the tumor

13

suppressor activity of OA on cancer cells. Collectively, we provided evidence that OA, as

14

a widely distributed nutritional component, is able to exert anti-tumor function by

15

interfering metabolic pathway in cancer cells. This finding encouraged researchers to

16

study if affecting cancer metabolism is a common mechanism by which nutritional

17

compounds suppress cancers.


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INTRODUCTION

25

Rapid and unlimited growth of cancer cells requires a constant supply of both energy

26

and structural materials that is essential for cell division. Transformed cells usually adjust

27

their metabolic machinery to these needs by modifying the activity and expression of key

28

metabolic enzymes in a concerted fashion.1 Metabolic switch is not only an adaptive

29

response of pre-existing malignant cells, but also is a driver of tumorigenicity.2 The

30

population that suffers from metabolic syndromes, such as diabetes, is more prone to

31

cancers,2 although the detailed mechanisms underlying increased tumor risk from altered

32

metabolism is still unknown. Interfering with metabolism has been shown to retard the

33

growth of tumor cells,2 indicating it as a promising therapeutic target for cancer treatment.

34

Multiple kinases have been shown to participate in the regulation of metabolic activity.

35

AMP-activated protein kinase (AMPK) is one of the most important kinases that trigger

36

metabolic adaption in cells. As an energy sensor, AMPK is an enzyme consisting of three

37

subunits (α, β and γ). α subunit is responsible for its catalytic activity and the other two

38

ones serves as regulatory parts. AMPK senses the increase in intracellular AMP/ATP

39

ratio and responds to the changes by phosphorylating and activating/inactivating some

40

key metabolic enzymes, thereby inducing metabolic switch from glycolysis and



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anabolism to oxidative phosphorylation and catabolism. This AMPK-mediated metabolic

42

alteration compromises the generation of molecular intermediates that is essential for

43

macromolecule synthesis, and hence, slows down cell proliferation. AMPK, evidenced by

44

recent studies, is closely implicated in the correlation between metabolism and cancer.

45

AMPK suppresses aerobic glycolysis in malignant cells and also hampers the growth of

46

tumors in vivo.3 In line with the tumor suppressor function of AMPK, the risk of cancer

47

has been found to be lowered in Type 2 diabetes patients who received the administration

48

of metformin, an AMPK activator.4 This observation raises the possibility that AMPK is

49

likely to be a promising pharmacological target for cancer therapy.

50

Activated AMPK suppresses the activity of acetyl-CoA carboxylase (ACC) by

51

phosphorylation at Ser218 and negatively regulated the expression of fatty acid synthase

52

(FASN), finally inhibiting lipid synthesis in cancer cells. In parallel, mammalian target of

53

the rapamycin (mTOR) is a downstream effector of AMPK, which promotes protein

54

synthesis by activating p70S6K and eIF-4E. Through phosphorylation-dependent

55

activation of TSC1/TSC2, a major upstream negative regulator of mTOR activity, AMPK

56

can abrogate the stimulatory effect of mTOR on mRNA translation in transformed cells.

57

AMPK also promotes glycolysis by phosphorylating and activating 6-phosphofructo-2-

58

kinase/fructose-2,6-biphosphatase 2 (PFKFB2), a key enzyme in glycolytic pathway.

59

Accumulated

60

intermediates required for the synthesis of macromolecules. Above all, the activation

61

status of AMPK determines the metabolism in cancer cells.1 Biosynthesis of

62

macromolecules, such as lipid, protein and nucleic acids, may be braked by reactivating

63

the kinase function of AMPK in cancer cells that has no or low AMPK activity.

evidences

have

documented

that

enhanced


glycolysis

produces

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Oleanolic acid (OA) belongs to naturally occurring triterpenoid that is widely

65

distributed in edible and medicinal plants. OA has been known, for a long time, to exert a

66

variety of bioactivity, including tumor-suppressing function.5 However, the molecular

67

mechanism by which OA was able to inhibit tumor growth has been not completely

68

explored. In this study, we found that OA was able to induce AMPK activation in cancer

69

cells. Sequentially, AMPK activation led to the metabolic alteration in OA-treated cancer

70

cells. Finally, we demonstrated that AMPK-dependent alteration in metabolic pattern is

71

associated with the anti-tumor function of OA.

72 73 74 75 76 77 78 79 80 81 82 83 84 85 86



87 88 89 90 91 92 93

MATERIALS and METHODS

94

Cell culture. Human prostate carcinoma cell line, PC-3, and human breast cancer cell line,

95

MCF-7, were purchased from American Type Culture Collection C (ATCC). Human normal

96

liver cell, L-02, was obtained from Shanghai Cell Collection (Shanghai, China). PC-3 was

97

cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS; GIBCO-BRL) at

98

37 ○C under a humidified 5% CO2 condition. MCF-7 cells were cultured in DMEM.

99

Chemical agents. Oleanolic acid (OA) was purchased from Sigma Aldrich (O5504). An

100

AMPK inhibitor, Compound C, was purchased from Millipore (#171260). The cells were

101

incubated with Compound C (20 µM) for 1 hr prior to OA treatment. An mTOR activator,

102

MHY1485, was purchased from Millipore (#5.00554.0001). The cells were incubated with

103

MHY1485 (2 µM) for 1 hr prior to OA treatment. The above substances were all dissolved

104

with DMSO for storing. PBS or culture media was added to dilute the agents when needed.

105

Immunoblotting assays. Total proteins were extracted from cells with M-PER®

106

Mammalian Protein Extraction Reagent (Thermo Scientific, IL), separated with

107

electrophoresis with 10-12 % SDS-PAGE gel and transferred onto 0.45 µm nitrocellulose

108

membranes. Membranes were incubated in blocking solution (PBS, 0.1 % Tween-20, and

109

5 % nonfat dry milk powder) for 2 hr at room temperature, and then incubated with


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primary antibodies (1:1000, listed below). After overnight incubation, the membranes

111

were washed with PBS containing 0.1 % Tween-20 for three times, and then were

112

incubated with horseradish peroxidase-conjugated secondary antibody for 1 hr at room

113

temperature. The involved antibodies were described as follows: Phospho-AMPKα

114

(Thr172), (Santa Cruz, sc-33524); AMPKα (CST, #2532); Phospho-HMG-CoA reductase

115

(Ser872) (Millipore, #09-356); HMG-CoA reductase (Millipore, #ABS229); Phospho-

116

Acetyl-CoA Carboxylase (Ser79) (CST, #3661); Acetyl-CoA Carboxylase, (CST, #3676);

117

Fatty Acid Synthase, (CST, #3180). The intensity of blots in some figure was determined

118

with ImageJ software.

119

Immunofluorescence. PC-3 and MCF-7 cells were incubated with or without OA

120

(100 µg/ml) for 6 hr. The cells were fixed with 4% Polyoxymethylene for 30 minutes and

121

then incubated with phospho-AMPKα antibody (1:200) for 2 hr at 4 ○C. Subsequently,

122

the cells were stained with FITC-conjugated secondary antibody and 4´,6´-diamidino-2-

123

phenylindole (DAPI). A confocal microscope (CLS-2SS, Thorlabs) was used to observe

124

the fluorescence.

125

Detection of fatty acid synthesis activity. A radioactive assay was employed to

126

detect the effect of OA on lipid synthesis in PC-3 and MCF-7 cancer cells. The cells were

127

incubated with [3H]-acetyl-CoA (2µCi) (Perkin Elmer, #NET290050UC) for 12 hr as

128

well as OA (50 or 100 µg/ml) for 6 or 12 hr. Lipid was isolated with a mixture of

129

Water/Methanol/Chloroform (1:1:1) according to a procedure previously described 6.

130

Organic fraction that contains radioactive lipid was evaporated with N2, followed by

131

detection with a MicroBeta Liquid Scintillation Counter (Perkin Elmer).



132

Protein synthesis detection assay. The effect of OA on nascent protein synthesis in

133

cancer cells were detected using Click-iT® HPG Alexa Fluor® Protein Synthesis Assay

134

Kit (Life technologies, #C10428). 5×103 PC-3 and MCF-7 cells were treated with 50 or

135

100 µg/ml of OA for 6 or 12 hr (Fig. 3C). For the experiments in Fig. 3E, 100 µg/ml of

136

OA was added to the culture of 5×103 PC-3 cells with or without 1 hr compound C (20

137

µM) pretreatment, and then, the cells were incubated for another 12 hr. The subsequent

138

experiments were performed according to the procedures provided by the manufacturer.

139

The fluorescence was detected and record with a confocal microscope (CLS-2SS,

140

Thorlabs). The signal intensities were determined with ImageJ and the relative values

141

normalized by those in untreated control groups were presented in Fig. 3C and 3E.

142

Glycolytic activity detection assays. Glucose consumption of cancer cell treated with

143

OA was assessed with Glucose Uptake Colorimetric Assay Kit (BioVision, #K676).

144

Briefly, 5×103 PC-3 and MCF-7 cells were treated with 50 or 100 µg/ml of OA for 6 or

145

12 hr (Fig. 4A). For the experiments shown in Fig. 4D, 100 µg/ml of OA was added to

146

the culture of 5×103 PC-3 cells with or without compound C (20 µM) pretreatment for 1

147

hr and the cells were incubated for 12 hr. Then, the cells were starved for glucose with

148

100 µl of Krebs-Ringer-Phosphate-HEPES (KRPH) buffer containing 2 % BSA for 30

149

min, followed by 20 min exposure with 100 nmol of 2-DG. Subsequent steps were done

150

according to the manufacturer’s instructions. The absorbance in each sample was

151

measured with a microplate reader at 412 nm. Glucose consumption was determined

152

based on the established standard curves.

153

Lactate production was determined with Lactate Colorimetric Assay Kit II (BioVision,

154

#K627) following the manufacturer’s instructions. Briefly, 5 µl of culture media were


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harvested from PC-3 and MCF-7 cells incubated with 50 or 100 µg/ml of OA for 6 or 12

156

hr (Fig. 4B) and mixed with 45 µl of Lactate Assay Buffer. Compound C (20 µM) was

157

simultaneously added to PC-3 cells in some experiments (Fig. 4E). A 10kDa MW spin

158

filter (BioVision, #1997-25) was used to remove the lactate dehydrogenase in serum.

159

After 50 µl of Reaction Mix was added, the reaction was performed for 30 min at room

160

temperature. The absorbance in each sample was measured with a microplate reader at

161

450nm. The concentration of lactic acid in each samples were determined with the

162

established standard curve. The values in each group relative to that untreated control

163

group were shown in Fig. 4B and Fig. 4E.

164

Oxygen consumption rate was determined with Oxygen Consumption Rate Assay Kit

165

(Cayman Chemical Company, #600800). PC-3 and MCF7 cells were treated with OA

166

and/or compound C according to the procedures used to evaluate the glucose uptake and

167

lactic acid production. After 12 hr OA stimulation, the cells were processed following the

168

manufacturer’s instruction. The signals in each sample were read at 650 nm with

169

VICTOR™ X3 Multilabel Plate Reader (Perkin Elmer). The oxygen consumption rate

170

was presented as lifetime versus time (µs/hr). Their values was normalized by untreated

171

control samples and shown in Fig. 4C and 4F.

172

Cell cycle analysis. Cell cycle analysis was performed by fluorescence-activated cell

173

sorter (FACS)-based evaluation of DNA content with Propidium Iodide (PI) staining.

174

2×105 PC-3 cells were treated with OA for 12 hr (indicated groups were pretreated with

175

20 µM compound c for 1 hr prior to OA stimulation). The cells were fixed with cold 70%

176

ethanol for 1 hr. Subsequently, the cells were processed with RNase (20 µg/ml) at 37○C



177

for 1 hr and then stained with PI (50 µg/ml), immediately followed by FACS-based cell

178

cycle analysis (Aria II, BD Biosciences). 5×104 cells were counted for each sample.

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Colony formation assay. 3×103 PC-3 cells were plated in a 6-well plate and then cultured

180

in media with or without 100 µg/ml OA at 37 ○C under a humidified 5 % CO2 condition for

181

10 days. Colonies consisting of more than 50 cells were counted after staining with crystal

182

violet solution.

183

Animal experiments. The animal experimental procedures were approved by

184

Committee on Animal Care in Jilin University (Permission No. 2013032, Changchun,

185

China). 5×106 PC-3 cells were injected into the flanks of 4-6 week old male nude

186

BALB/c mice (n=18). OA (40mg/kg, low dose or 120 mg/kg, high dose) was orally

187

administrated every day for 4 weeks. So the mice were divided into three groups: Control

188

(n=6), low dose OA (n=6) and high dose OA (n=6). The diameters of tumors were

189

periodically determined with calipers. The volumes were calculated using the following

190

formula. Volume (mm3) = length × (width)2 / 2. The expression of indicated proteins was

191

detected in the dissected tumors after the mice were sacrificed by immunoblot assay.

192

Statistical analysis. The experiments except immunoblot assays were performed for

193

at least three times. All values were reported as means ± SD, and compared at a given

194

time point by unpaired, two-tailed t test. Data were considered to be statistically

195

significant when p < 0.05 (*) and p < 0.01 (**).

196 197 198 199


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200 201 202 203 204 205 206 207 208 209 210

RESULTS AND DISCUSSION

211

OA induces AMPK activation in cancer cells in a dose- and time-dependent fashion.

212

The chemical structure of OA was shown here (Fig. 1A). First of all, AMPK activation

213

was examined in OA-treated cancer cells by immunoblotting analysis of its

214

phosphorylation at Thr172. AMPK phosphorylation was found to be increased in all the

215

tested cells (Fig.1B). The increase in phosphorylated AMPK expression was correlated

216

with the dose of OA administered to cancer cells (Fig.1B). Also, the phosphorylation of

217

AMPK was progressively elevated during the time when cancer cells were exposed with

218

OA (Fig.1C). OA-induced phosphorylation of AMPK was also further confirmed in

219

cancer cells by immunofluorescent staining (Fig. 1D). These data showed that OA

220

induced the phosphorylation and activation of AMPK in cancer cells in a concentration-

221

and time-dependent manner.



222

OA has been demonstrated to exert multiple bioactivities, including anti-inflammation,

223

anti-cancer, anticoagulation7 and hepatoprotection,8 by affecting specific molecular

224

pathways in cells. These signaling pathways were associated with apoptosis, cell cycle

225

regulation, metastasis, angiogenesis and inflammation. AMPK, the central modulator of

226

metabolism, has been reported to be activated in mouse cardiomyocytes under OA

227

treatment. In turn, AMPK activation protected cardiomyocytes from contractile

228

dysfunction induced by hypoxia in a FOXO3-dependent manner.9 This finding shed light

229

on the mechanism of OA cardioprotective activity. However, it is still unclear that OA is

230

able to induce AMPK activation in cancer cells. In this study, we revealed that OA

231

treatment resulted in the phosphorylation and activation of AMPK in cancer cells both in

232

dose- and time-dependent fashion.

233

AMPK activation mediated the anti-tumor activity of OA on cancer cells.

234

Subsequently, we aimed to establish the role of AMPK activation in tumor suppressor

235

property of OA on cancer cells. Cell proliferation was determined by counting the cells at

236

the different time points. An AMPK inhibitor, Compound C, was used to block the

237

AMPK activation in PC-3 cancer cells treated with OA (Fig. 2A). OA was able to

238

decelerate the proliferation of PC-3 cells, whereas Compound C-mediated AMPK

239

inactivation resulted in a partial recovery of the growth of PC-3 cells (Fig. 2B). Cell cycle

240

progression was also retarded by OA treatment. However, this effect was reversed by

241

compound C (Fig. 2C). Furthermore, compound C was also able to abolish the inhibitory

242

activity of OA on colony forming ability of PC-3 cells (Fig. 2D).

243

Subsequently, we aimed to establish the role of AMPK activation in anti-tumor activity

244

of OA. We found that AMPK inhibitor, compound C, was able to rescue the effect of OA


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245

on PC-3 cells, suggesting that AMPK activation is required for OA to suppress tumor

246

growth. AMPK activation has been also found to be essential for anti-tumor activity of

247

other natural compounds. Curcumin induced apoptosis in CaOV3 ovarian cancer cells in

248

an AMPK-dependent way. Blocking AMPK activation with compound c was able to

249

abolish curcumin induction of apoptotic event in cancer cells.10 However, compound C-

250

dependent AMPK inactivation in OA-treated cancer cells partially, not completely,

251

abolished OA suppression of the proliferation, cell cycle progression and colonigenecity

252

of PC-3 cells, implying some molecular mechanisms other than AMPK activation are

253

responsible for OA anti-tumor activity.

254

OA-stimulated AMPK activation suppressed lipid biosynthesis in cancer cells.

255

AMPK is a master kinase that regulates the cellular lipid metabolism. Its activation

256

leads to a metabolic switch from anabolism to catabolism by phosphorylating several

257

enzymes that play key roles in the biosynthesis of macromolecule, such as lipids.

258

As a rate-limiting enzyme for fatty acid synthesis, acetyl-CoA carboxylase 1 (ACC1)

259

produces malonyl-CoA, an intermediate essential for fatty acid elongation, by catalyzing

260

the carboxylation of acetyl-CoA. ACC1 is overexpressed in a variety of human cancers

261

and highly contributes to the enhanced lipogenesis in cancer cells.11 AMPK inhibits

262

ACC1 activity by phosphorylating it at Ser79.12 In OA-stimulated cancer cells, ACC1

263

phosphorylation was found to be augmented in both dose- and time-dependent manners

264

(Fig. 3A and 3B) and appeared to be closely correlated with OA-induced AMPK

265

activation (Fig. 1B and 1C). Compound C abolished ACC1 phosphorylation at Ser79

266

(Fig. 3C), indicating the requirement of AMPK activation in OA-stimulated ACC1

267

phosphorylation.



268

Fatty acid synthase (FASN) is also a critical enzyme in lipogenic pathway and is

269

frequently found to be overexpressed in cancer cells.13 Its transcription level is negatively

270

regulated by AMPK-dependent SREBP1c phosphorylation.14 As shown in Fig. 3A and

271

3B, OA reduced the expression of FASN protein in cancer cells. This effect was

272

associated with both OA concentration and the time of OA treatment (Fig. 3A and 3B).

273

After the treatment of OA, the protein levels of FASN were decreased. However, the

274

protein levels of FASN were restored in the presence of compound C (Fig. 3C).

275

In addition to the fatty acid, sterol is also required for the growth of cancer cells. 3-

276

hydroxy-3-methylglutaryl-CoA reductase (HMGR) is the enzyme responsible for

277

generating mevalonic acid, an essential intermediate for the synthesis of cholesterol and

278

isoprenoids. AMPK has been reported to phosphorylate and inactivate HMGR.1 OA was

279

found to increase the phosphorylation of HMGR in cancer cells, both in dose- and time-

280

dependent fashion (Fig. 3A and 3B). Compound C treatment was able to block the OA-

281

induced phosphorylation of HMGR at Ser872 (Fig. 3C).

282

Reactivation of AMPK has been documented to suppress the production of lipid in

283

cancer cells. Radioactive assays revealed that OA was able to diminish the de novo lipid

284

synthesis, shown as a reduced incorporation rate of [3H] acetyl-CoA into lipid product of

285

PC-3 cells (Fig. 3D). OA suppression of lipid synthesis was rescued by compound C (Fig.

286

3E), implying that AMPK activation is essential for its effect against fatty acid

287

production.

288

Next, we investigated if OA was able to affect the lipogenesis of cancer cells by

289

detecting the phosphorylation or expression of some lipid synthesis-associated genes,

290

which have been confirmed as AMPK downstream effectors, and performing [3H] acetyl-


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291

CoA incorporation assay. The results showed that OA was able to suppress the lipid

292

synthesis in a dose- and time-dependent manner. Compound C abolished the inhibitory

293

effect of OA on lipogenic process in cancer cells, supporting the role of AMPK activation

294

in OA suppression of lipid synthesis (Fig. 3). Pomolic acid, a natural anti-tumor

295

compound, was also found to suppress the lipid synthesis in MCF-7 breast cancer cells in

296

an AMPK-dependent manner.15 There is a possibility that all the compounds identified as

297

AMPK activators may exert an inhibitory activity on lipogenic pathways.

298

Next, we investigated if OA was able to affect the lipogenesis of cancer cells by

299

detecting the phosphorylation or expression of some lipid synthesis-associated genes,

300

which have been confirmed as AMPK downstream effectors, and performing [3H] acetyl-

301

CoA incorporation assay. The results showed that OA was able to suppress the lipid

302

synthesis in a dose- and time-dependent manner. Compound C abolished the inhibitory

303

effect of OA on lipogenic process in cancer cells, supporting the role of AMPK activation

304

in OA suppression of lipid synthesis. Pomolic acid, a natural anti-tumor compound, was

305

also found to suppress the lipid synthesis in MCF-7 breast cancer cells in an AMPK-

306

dependent manner.15 There is a possibility that all the compounds identified as AMPK

307

activators may exert an inhibitory activity on lipogenic pathways.

308

Protein synthesis was inhibited in cancer cells treated with OA in an AMPK-

309

activation-dependent way.

310

AMPK reduces the activity of the mammalian target of rapamycin complex 1

311

(mTORC1) by phosphorylating its upstream inhibitory regulator, TSC2,16 and its

312

regulatory subunit, Raptor.17 In OA-treated cancer cells, mTOR was shown to be less

313

phosphorylated at Ser2448 (Fig. 4A and 4B). The expression of phosphorylated p70S6K,



314

a downstream effector of mTOR that hampers protein synthesis by inactivating S6

315

ribosomal protein, was consequently reduced in cancer cells under OA treatment (Fig. 4A

316

and 4B). This mTOR-dependent phosphorylation is required for the kinase activity of

317

p70S6K.18

318

eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), was also found to

319

be decreased in OA-stimulated cancer cells (Fig. 4A and 4B). The Thr37 and Thr46

320

phosphorylations prime 4E-BP1 for subsequent phosphorylations that abolish the

321

inhibitory activity of 4E-BP1 on mRNA translation.19 Therefore, OA-induced

322

dephosphorylation of 4E-BP1 probably suppresses protein synthesis in cancer cells. The

323

changes in the phosphorylation level of these protein synthesis-related factors were all

324

abolished by Compound C (Fig. 4C), indicating that AMPK activation is required for OA

325

inhibition of mTOR signaling pathway.

The phosphorylation of another important mTOR downstream substrate,

326

A fluorescence-based protein synthesis assay kit was used to determine the effect of

327

OA on protein synthesis in cancer cells. Microscopic observation revealed that the

328

intensity of fluorescence was reduced in cells 3 and 12 hr after the treatment of different

329

dose of OA (Fig. 4D), suggesting that OA compromised the nascent synthesis of protein.

330

Compound C suppression of AMPK was able to partially prevent the reduction in protein

331

synthesis in cancer cells treated with OA (Fig. 4E).

332

We also studied the changes in protein synthesis in cancer cells treated with OA. The

333

data showed that protein synthesis in PC-3 and MCF-7 cells was significantly

334

compromised under OA stimulation. Consistently, the promoters of protein synthesis

335

were also inactivated under OA treatment, evidenced by decreased phosphorylation levels.


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336

Compound C-mediated AMPK inhibition revealed that AMPK activation is required for

337

the inhibitory effect of OA on protein synthesis in cancer cells.

338

OA suppressed aerobic glycolysis in cancer cells in an AMPK-dependent manner.

339

AMPK has been recently demonstrated as a negative regulator of the Warburg effect in

340

cancer cells 3. The inhibitory effect of AMPK on glycolysis may be associated with its

341

suppression of PKM2 activity via affecting mTOR activation.20 Herein, we investigated if

342

OA treatment affected the glycolytic metabolism in cancer cells. Glucose uptake was

343

significantly reduced in cancer cells treated with OA (Fig. 5A). Furthermore, cancer cells

344

produced less lactic acid, which is the end product of glycolysis, under the treatment of

345

OA (Fig. 5B). The changes in glucose consumption and lactate generation indicated that

346

OA suppressed the glycolytic metabolism in cancer cells.

347

Cancer cells

are also featured

with compromised mitochondrial oxidative

348

phosphorylation that contributes to the maintenance of malignant phenotypes. Oxygen

349

consumption, an indicator for the efficiency of oxidative phosphorylation, was

350

subsequently measured in OA-stimulated cancer cells. An increase in O2 consumption

351

was observed in cancer cells exposed with OA (Fig. 5C).

352

Similar to the role of AMPK activation in lipid and protein synthesis, compound C was

353

also able to partially inhibit OA-induced metabolic switch in cancer cells, evidenced by

354

increases in glucose uptake, lactate production, and O2 consumption in the cells treated

355

with both OA and compound C (Fig. 5D, 5E and 5F).

356

Increasing evidences indicated that Warburg effect is critical for the unlimited growth

357

of cancers and targeting it seems to be an effective strategy for cancer treatment. We

358

therefore examined if OA interfere with the metabolic pathways in cancer cells. The



359

results on glucose uptake, lactate production and oxygen consumption rates showed that

360

OA switch metabolic patterns from aerobic glycolysis to mitochondrial oxidative

361

phosphorylaion. Similarly, AMPK inhibition protected PC-3 cells from this metabolic

362

change. However, it is unknown how OA-induced AMPK lead to the reversion of

363

Warburg effect.

364

AMPK-dependent metabolic adaptation contributes to the tumor suppressor

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activity of OA on cancer cells.

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Finally, we aimed to study the contribution of metabolic alteration to the anti-tumor

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activity of OA on cancer cells. Therefore, we used a small molecule activator, MHY1485,

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to interfere with the changes in metabolic pathway without affecting AMPK activation.21

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mTOR inhibition was rescued by MHY1485 in OA-stimulated cancer cells and AMPK

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activation was not affected under this treatment (Fig. 6A). MHY1485 was able to

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counteract the inhibitory effect of OA on protein synthesis in cancer cells and to stop the

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metabolic switch from aerobic glycolysis to oxidative phosphorylation (data not known),

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through mTOR pathway. Cell proliferation assay showed that MHY1485 could speed up

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the growth of PC-3 cells under the treatment of OA (Fig. 6B). The number of colonies in

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PC-3 cells was also increased by MHY1485 (Fig. 6C).

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We also determined the contribution of metabolic alteration to the anti-tumor activity of

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OA. Restoring protein synthesis and aerobic glycolysis in cancer cells partially abolish

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AMPK effect on cancer cells (Fig. 6), confirming the change in metabolic pattern is, at

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least partially, responsible for anti-tumor activity of OA. The reason mTOR activators

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recovered proliferation and colony formation ability of PC-3 cells only in a partial

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manner may be that lipid synthesis was still not restored in OA-treated cells, or that there


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Page 19 of 36


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are still some mechanisms underlying OA anti-tumor function, other than interfering

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metabolism. For example, there is a possibility that OA may induce G0/G1 cell cycle

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arrest via AMPK/p53/p21 pathway. Previous studies showed that AMPK phosphorylates

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p53 at Ser15, facilitates its accumulation and enhances its transcription activity of p21CIP,

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a CDK inhibitor, in both normal and cancer cells.22, 23 p27KIP is also reported to be

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substrate of AMPK kinase and phosphyralted at Thr-198. This phosphorylation event

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enhances the inhibitory function of p27KIP on cell cycle progression.24 In addition,

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AMPK activation-induced FASN downregulation may directly affect cell cycle

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progression in tumor cells.25 Furthermore, p53-associated apoptosis is another possible

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mechanism by which OA exerts AMPK-dependent anti-tumor activity, based on a recent

392

publication reporting AMPK overexpression lead to increased p53 nuclear accumulation

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and apoptosis. 26

394

Another question about OA induction of AMPK pathway is the mechanism by which

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OA initiates AMPK activation. Recently, H2O2 was found to induce AMPK activation in

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insulin-secreting cells.27 AMPK signaling can be also activated in a p38 MAPK-

397

dependent manner in cancer cells treated with anti-tumor compound.10 Thus, further

398

studies should be focused on these molecular pathways.

399

Another question about OA induction of AMPK pathway is the mechanism by which

400

OA initiates AMPK activation. Recently, H2O2 was found to induce AMPK activation in

401

insulin-secreting cells.27 AMPK signaling can be also activated in a p38 MAPK-

402

dependent manner in cancer cells treated with anti-tumor compound.10 Thus, further

403

studies should be focused on these molecular pathways.

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OA reduced tumor growth and activated AMPK pathway in mice.



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To further confirm the antitumor activity of OA and its stimulatory effect on AMPK

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pathway, we established a tumor xenograft mouse model by injecting PC-3 cells into the

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flanks of mice. OA was orally administrated every day at two different doses, followed

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by periodically measuring the diameters of tumors in mice. The results showed that tumor

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growth was significantly retarded by the administration of high dose OA (P

Oleanolic Acid Induces Metabolic Adaptation in Cancer Cells by

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