Ursolic Acid Triggers Apoptosis in Human Osteosarcoma Cells via

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Ursolic acid triggers apoptosis in human osteosarcoma cells via caspase activation and the ERK1/2 MAPK pathway Chia-Chieh Wu, Chun-Hsiang CHENG, Yi-Hui Lee, IngLin Chang, Hsin-Yao Chen, Chen-Pu Hsieh, and Pin Ju Chueh J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 12 May 2016 Downloaded from http://pubs.acs.org on May 12, 2016

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Journal of Agricultural and Food Chemistry

Ursolic acid triggers apoptosis in human osteosarcoma cells via caspase activation and the ERK1/2 MAPK pathway

Chia-Chieh Wu†,‡,§ Chun-Hsiang Cheng†, Yi-Hui Lee§, Ing-Lin Chang‡, Hsin-Yao Chen‡, Chen-Pu Hsieh*,†,‡, Pin-Ju Chueh*,§,⊥,∆,¶.

†

Orthopedics & Sports Medicine Laboratory, Changhua Christian Hospital, Changhua, Taiwan ‡ Orthopaedic Surgery Department, Changhua Christian Hospital, Changhua, Taiwan § Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan ⊥

Department of Biotechnology, Asia University, Taichung, 41354, Taiwan Graduate Institute of Basic Medicine, China Medical University, Taichung, 40402, Taiwan ¶ Department of Medical Research, China Medical University Hospital, Taichung, 40402, Taiwan ∆

*Corresponding authors: 1. Chen-Pu Hsieh, MD. Orthopaedic Surgery Department, Changhua Christian Hospital 135 Nansiao St., Changhua500-06, Taiwan Telephone number: 886-(4)-7238595; Fax number: 886-(4)-7228289 E-mail: [email protected] 2. Pin-Ju Chueh, Professor Institute of Biomedical Sciences, National Chung Hsing University, 145 Xingda Rd., South Dist., Taichung City 402, Taiwan Telephone number: 886-(4)-22840896; Fax number: 886-(4)-22853469 E-mail: [email protected] Running title: Wu et al: URSOLIC ACID INDUCES APOPTOSIS IN OSTEOSARCOMA CELLS

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1

Abstract

2

Ursolic acid (UA), a naturally occurring pentacyclic triterpene acid found in many

3

medicinal herbs and edible plants, has been shown to trigger apoptosis in several lines

4

of tumor cells in vitro. We found that treatment with UA suppressed the viability of

5

human osteosarcoma MG-63 cells and induced cell cycle arrest at sub-G1 and G2/M

6

phases. Furthermore, exposure to UA induced intracellular oxidative stress and

7

collapse of mitochondrial membrane permeability, resulting in the subsequent

8

activation of apoptotic caspases 8, 9 and 3 as well as PARP cleavage, ultimately to

9

apoptosis in MG-63 cells. Moreover, protein analysis of MAPK-related protein

10

expression showed an increase in activated ERK1/2, JNK, and p38 MAPK in

11

UA-treated MG-63 cells. In addition, UA-induced apoptosis was significantly

12

abolished in MG-63 cells that had been pre-treated with inhibitors of caspase 3, 8 and

13

9 and ERK1/2. Furthermore, UA-treated MG-63 cells also exhibited an enhancement

14

in Bax/Bcl-2 ratio whereas anti-apoptotic XIAP and survivin were downregulated.

15

Taken together, we provide evidence demonstrating that ursolic acid mediates

16

caspase-dependant and ERK1/2 MAPK-associated apoptosis in osteosarcoma MG-63

17

cells.

18 19

Key words: ursolic acid, MG-63 cells, apoptosis, MAPK, caspase. 1


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Introduction

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In Taiwan, the incidence of primary bone cancer was 6.78 per million people

22

during the years between 2003 and 2010, and osteosarcoma was the most common

23

bone cancer subtype (45%)1. Osteosarcoma is often associated with a high incidence

24

of metastasis and death. The vast majority of patients with osteosarcoma are children

25

and adolescents. The disease can occur in any bone in the body but most commonly

26

presents in metaphyseal locations of the appendicular skeleton2, 3. In over 90% of

27

patients with metastatic disease, lung is found to be the most common site of

28

metastasis.

29

Surgery and chemotherapy, including doxorubicin, cisplatin, and high-dose

30

methotrexate, are highly efficacious against early-stage osteosarcoma; 4 however,

31

those strategies are less effective in patients with late-stage disease and in those with

32

early metastasis2, 3. Thus, novel drugs and treatment strategies are urgently needed,

33

especially for patients with late-stage and metastatic osteosarcoma 4. An array of

34

chemotherapeutic agents are derived from natural products, including aspaclitaxel

35

(from Taxus brevifolia L., the Pacific yew), vincristine (from Catharanthus roseus G.

36

Don, rosy periwinkle), podophyllotoxin (from Podophyllum peltatum L., Mayapple),

37

and camptothecin (from Camptotheca acuminate, Happy tree)5. Additionally, many

38

other biologically active compounds extracted from medicinal herbs also have 2



39

demonstrated their anti-tumor effects in vitro, such as Tanshinone IIA, Dodecyl

40

gallate and ursolic acid6-8.

41

Ursolic acid (UA)(3β-hydroxy-urs-12-en-28-oic acid) is a naturally occurring

42

pentacyclic triterpenoid compound found in large quantity in countless plants and

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medicinal herbssuchas cranberries (Vaccinium oxycoccus), rosemary (Rosemarinus

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officinalis), snake-needle grass (Oldenlandia diffusa), and apples (Malus

45

domestica)9-11. Many studies have shown that UA exerts several pharmacological

46

effects including antioxidant, antiviral, anti-metastatic, anti-inflammatory, anticancer,

47

and hepatoprotective effects 9, 12, 13. More importantly, UA exerts its inhibitory effect

48

on cell growth and induces apoptosis in an array of lines of cancer cells, such as lung

49

cancer, melanoma, endometrial cancer, colon carcinoma, and cervical cancer cells 12,

50

14-16

51

primary bone cancer cells.

. Until now, to our knowledge, there is no study focus on the effect of UA on

52

Here, we intended to investigate the antiproliferative and potential apoptotic effects

53

of UA on human MG-63 osteosarcoma cells and to elucidate the underlying signaling

54

pathways.

3


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Materials and Methods

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Cell culture and reagents

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The human osteosarcoma MG-63 cells were purchased from the Bioresource

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Collection and Research Center (Hsinchu, Taiwan). MRC-5 (human lung tissue) cells

59

and MG-63 were maintained in Minimum Essential Medium (MEM; #11095-080;

60

Gibco, Carlsbad, CA, USA). NIH3T3 (mouse embryonic fibroblast) cells were grown

61

in Dulbecco’s Modified Eagle Medium (DMEM). All medium contained 10% FBS

62

(#10437-028; Gibco), 1.0 mM sodium pyruvate (#11360070; Gibco), 0.1 mM

63

non-essential amino acids (#11140050; Gibco), and 1% penicillin/streptomycin

64

(#15140-122; Gibco). Cells were cultured at 37°C in a humidified atmosphere of 5%

65

CO2 in air, and the media were replaced every 2-3 days. Cells were treated with

66

different concentrations of ursolic acid dissolved in DMSO (#sc-200383A; Santa Cruz

67

Biotechnology, CA, USA). The inhibitors for ERK1/2 (U0126), JNK (SP600125),

68

and P38 (SB203580) were purchased from Sigma-Aldrich.

69

Measurement of MG-63 cell viability

70

Cells were seeded onto 96-well plates at 5 × 103 cells/well, allowed to grow

71

overnight at 37°C, and exposed to different concentrations of ursolic acid for 24 or 48

72

h.

73

3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyl-tetrazolium

After

incubation,

cells

were

exposed

to

a

0.5

mg/ml

bromide

4


solution (MTT;

of 100


74

µl/well)(#0793; Amresco, St. Louis, MO, USA) for 2 hours at 37°C. The number of

75

viable cells was determined by uptake and reduction of MTT, assayed at 590 nm

76

using a microplate reader (Thermo Multiskan SPECTRUM Thermo Fisher Scientific,

77

Waltham, MA, USA) as previously reported 7.

78

Cell impedance measurements

79

Cell impedance measurements were used to continuously monitor MG-63 cell

80

growth and 104 cells per well were plated onto E-plates, then set down onto the RTCA

81

station (Roche, Germany). Cells were allowed to grow overnight before treated with

82

ursolic acid and cell impedance was recorded hourly as reported before 17.

83

Cell cycle analysis

84

Briefly, 105 cells were exposed to ursolic acid for 24 h, and then collected and

85

washed in PBS, fixed in 70% ethanol, and kept at -20°C for 24 h. The cell pellet was

86

then washed again with PBS, and centrifuged at 400 × g for 10 minutes. The pellet

87

was resuspended in 200 µl cold PBS and nuclear DNA was stained in the dark with a

88

propidium iodide (PI) solution (0.1% Triton X-100, 0.2 µg/ml Ribonuclease A, 40

89

µg/ml PI)(PI; #P4170; Sigma-Aldrich) for 60 minutes on ice. Phases of cell cycle

90

were determined by FC500 flow cytometer (Beckman Coulter Inc., Brea, CA, USA).

91

Measurement of ROS production

92

At the end of ursolic acid treatments, 2 × 105 cells were rinsed and collected by 5


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centrifugation at 400 × g for 5 min, followed by staining the cell pellets with Muse™

94

Oxidative Stress Kit (#MCH100111; Merck Millipore, Darmstadt, Germany) for 30

95

min at 37◦C. Oxidative stress generation by ursolic acid was analyzed with the Muse™

96

Cell Analyzer (Merck Millipore, Darmstadt, Germany).

97

Annexin V/ Propidium Iodide staining to detect apoptosis

98

Cells were cultured on 12-well plates (105 cells per well) overnight and then treated

99

with different concentrations of ursolic acid for 24 h. After the end of treatment, cell

100

were collected, rinsed with PBS, and stained with Muse™ Annexin V & Dead Cell

101

Assay Kit (#MCH100105; Merck Millipore, Darmstadt, Germany) for 25 min at 37◦C.

102

Apoptosis was analyzed by the Muse™ Cell Analyzer (Merck Millipore, Darmstadt,

103

Germany).

104

Mitochondrial membrane potential Assay

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MG-63 cells were exposed to various concentrations of ursolic acid for 24 h and

106

then washed with PBS, after which cell pellets were harvested by centrifugation at

107

400 × g for 5 min. Cell pellets were then stained with Muse™ MitoPotential Kit

108

(#MCH100110; Merck Millipore, Darmstadt, Germany) for 25 min at 37◦C and data

109

were analyzed by the Muse™ Cell Analyzer (Merck Millipore, Darmstadt, Germany).

110

Western blot assay

111

MG-63 cell extracts were prepared in lysis RIPA buffer on ice for 30 min 6



112

containing protease inhibitors. Cells were scraped from the dishes and centrifuged at

113

15900 × g at 4°C for 10 min. Volumes of extract containing equal amounts of proteins

114

(20 µg) were separated by 8-12% SDS-PAGE and transferred onto PVDF membranes

115

(Millipore, Bedford, MA). The membranes were blocked, washed and then probed

116

with primary antibodies, including p-AKT (#4060; 1:1000 dilution), AKT (#9272;

117

1:1000 dilution), p-ERK (#4370; 1:1000 dilution), ERK (#4695; 1:1000 dilution),

118

p-SAPK/JNK (#4668; 1:1000 dilution), SAPK/JNK (#9258; 1:1000 dilution), p-P38

119

(#9211; 1:1000 dilution), P38 (#9212; 1:1000 dilution), Bcl-2 (#9258; 1:1000

120

dilution), XIAP (#2042; 1:1000 dilution), Survivin (#2808; 1:1000 dilution), cleaved

121

caspase-3 (#9661; 1:1000 dilution), Caspase-9 (#9508; 1:1000 dilution), PARP

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(#9542; 1:1000 dilution), GAPDH (#2118; 1:1000 dilution) (above are all from Cell

123

Signaling Technology), bax (sc-493; 1:1000 dilution; Santa Cruz Biotechnology, CA,

124

USA), or Caspase-8 (NB100-56116; 1:1000 dilution; Novus Biologicals, Littleton,

125

CO, USA). After washing to remove primary antibody, membranes were incubated

126

with HRP-conjugated secondary antibody (Cell Signaling Technology) for one hour.

127

The blots were washed again, and detected by an enhanced chemiluminescence

128

detection system (#WBKLS0500; Millipore, Darmstadt, Germany). Density of protein

129

bands was quantified using Image J software (http://rsb.info.nih.gov/ij/; 1.42q;

130

National Institutes of Health, Bethesda, MD, USA, 2009). 7


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Statistical analysis

132

All data are expressed as the mean ± SD of no less than three independent trials.

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The differences between groups were calculated using a Student's t-test provided by

134

the GraphPad Prism (Version 4.0, GraphPad Software; San Diego, CA, USA).

135

8



136

Results

137

Effect of ursolic acid on viability of MG-63cells

138

To understand the effects of ursolic acid on human osteosarcoma MG-63 cell

139

viability, cells were treated with UA at diverse concentrations (0 – 25µg/ml) for 24 or

140

48 h and then viability was measured by MTT assay. As shown in Fig 1, UA

141

significantly decreased the viability of MG-63 cells. The estimated half-maximum

142

inhibitory concentration (IC50) values of UA were calculated to be 11.64 µg/ml at 24 h

143

and 8.35 µg/ml at 48 h, indicating that ursolic acid reduces MG-63 cell viability in a

144

dose- and time-dependent fashion. Cells derived from human lung tissue MRC-5 cells

145

were also utilized to determine the cytotoxic effects of UA. The dynamic effects of

146

UA on cell growth were continuously monitored using cell impedance technology

147

displaying the results as cell index values. Using this approach, we found that MRC-5

148

cell growth was decreased by 10 µg/ml of UA (Fig 2). The lesser responsiveness of

149

MRC-5 cells to UA is reflected in IC50 values, which was determined to be 14.34

150

µg/ml at 24 h and 12.04 µg/ml at 48 h.

151

Effects of ursolic acid on cell-cycle distribution in MG-63

152

cells

153

MG-63 cells were treated with UA for 24 h and flow cytometry was then used to

154

investigate whether UA alters cell cycle phases. As shown in Fig 3, treatment with 9


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various concentrations (10 – 20 µg/ml) of UA for 24 h induced G2/M phase

156

accumulation in a dose-dependent fashion. Furthermore, a significant accumulation of

157

sub-G1 was induced by 10 and 20 µg/ml UA, indicating that UA at those

158

concentrations induced apoptosis.

159

Ursolic acid induces apoptosis in MG-63 cells but not

160

non-cancerous cells

161

To further dissect the inhibition of UA on cell growth is associated with apoptosis

162

induction, we exposed MG-63 cells to UA (0 – 20 µg/ml) for 24 h and then

163

determined apoptosis by the Muse™ Cell Analyzer assays. Results from those assays

164

demonstrated that a dose-dependent increase in populations of apoptotic cells was

165

mediated by UA at 24 h in MG-63 cells (Fig 4). In contrast, UA did not induce

166

significant apoptosis in non-cancerous MRC-5 and NIH3T3 (mouse embryonic

167

fibroblast) cells even at as high as 20 µg/ml (Fig 5).

168

Ursolic acid induces the generation of reactive oxygen

169

species in MG-63 cells

170

Next, we investigated whether UA-induced cell death is linked to reactive oxygen

171

species (ROS) generation. Our results from the Muse™ Cell Analyzer assays

172

illustrated that exposed MG-63 cells to 0 – 20 µg/ml UA for 12 h initiated a

10



173

dose-dependent rise in intracellular ROS level (Fig 6), suggesting that UA-induced

174

apoptosis may proceed via a ROS-mediated pathway.

175

Ursolic acid induces changes in mitochondrial membrane

176

potential

177

To assess the involvement of the mitochondrial pathway, we used the Muse™

178

MitoPotential Kit to measure the integrity of the mitochondrial membrane. In Fig 7,

179

treatment of MG-63 cells with 0 – 20 µg/ml UA for 12 h caused a noticeable increase

180

in population of depolarized cells (including live and dead groups), indicating UA

181

triggered collapse of mitochondrial membrane potential thereby induced apoptosis.

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Effects of ursolic acid on MAPK signaling in MG-63 cells

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Mitogen-activated protein kinase (MAPK) signaling is crucial for cell maintenance

184

18

185

migration and apoptosis 19. Therefore, we investigated whether UA-induced apoptosis

186

involved MAPK signaling. As shown in Fig 8A, exposure of MG-63 cells to 0 – 20

187

µg/ml UA for 24 h amplified expressions of phospho-JNK, phospho-p38 and

188

phospho-ERK. To confirm whether UA-induced apoptosis is linked to the activation

189

of these MAP kinases, we pretreated MG-63 cells with 40 µM U0126 (an ERK1/2

190

inhibitor), SP600125 (a JNK inhibitor), or SB203580 (a p38 inhibitor) for 2 h

191

followed by exposure to 20 µg/ml UA for additional 24 h. As shown in Fig 8B,

. The MAPK family of proteins regulates cell proliferation, growth, differentiation,

11


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pretreatment with U0126 markedly reversed UA-induced apoptosis, indicating that

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UA mediates apoptosis at least partially through the ERK1/2 MAPK pathway.

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Effects of ursolic acid on caspase in MG-63 cells

195

To confirm the involvement of caspase activation in UA-induced apoptosis, protein

196

analysis was performed to understand the levels of cleaved/activated caspases and

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PARP. As shown in Fig 9A, MG-63 cells exposed to 15 – 20 µg/ml UA instigated an

198

enhancement in the levels of cleaved caspase-9, caspase-8, caspase-3 and PARP

199

dose-dependently. To further identify the importance of caspase activation,

200

ZVAD-FMK, a pan-caspase inhibitor, was used to block the activation of capsaicin

201

and we showed that the pretreatment with inhibitor effectively alleviated UA-induced

202

apoptosis, implying that UA possibly activates both the extrinsic and intrinsic

203

apoptotic pathways (Fig 9).

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Ursolic acid-induced apoptosis involves the expression of

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Bcl-2 and XIAP family proteins in MG-63 cells

206

To gain insight into the mechanism underlying UA-induced apoptosis, protein

207

analysis was utilized to detect changes in expression of Bcl-2 family. As demonstrated

208

in Fig 10, MG-63 cells exposed to UA (0 – 20 µg/ml) for 24 h were associated with an

209

upregulation of apoptotic Bax and a downregulation of pro-survival Bcl-2 protein.

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Given that XIAP and survivin in the IAP family play an important role in regulating 12



211

apoptosis20, 21, we also examined the changes in expression of these two protein in this

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system and revealed that both XIAP and survivin were downregulated by UA

213

exposure dose-dependently (Fig 10).

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Discussion A number of bioactive compounds found in traditional herbal medicines have been

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shown to be potential chemotherapeutic agents against various types of cancers.

217

Herein, we investigated whether UA exerts anti-cancer properties in human

218

osteosarcoma MG-63 cells, and elucidated the apoptotic signaling pathways involved.

219

Our data demonstrated that UA significantly inhibits cancer cell growth and induces

220

apoptosis dose- and time-dependently in MG-63 cells (Fig 4), but not non-cancerous

221

MRC-5 and NIH3T3 cells (Fig 5). Moreover, the IC50 value of UA for MG-63 cells

222

was 8.35 µg/ml at 48 h whereas that of MRC-5 cells was 12.04 µg/ml, suggesting that

223

UA exhibits lesser cytotoxicity in non-cancerous cells. Consistent with our findings in

224

this study, Liu also pointed out that UA exhibits relatively minimal cytotoxicity and

225

has been used in health products and cosmetics22.

226

Apoptosis assists in maintaining homeostasis of cell numbers by removing

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damaged or unwanted cells, thus, it is important in cancer therapy as previously

228

reported23. Here, we found that UA provokes apoptosis in osteosarcoma cells but not

229

non-cancerous cells. The results from PI-stained cells analyzed by flow cytometer

230

revealed that MG-63 cells exposed to UA had a significantly increased percentage of

231

cells with hypo-diploid DNA content (Fig 3). Moreover, a considerable increase in

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Annexin V-stained positive cells demonstrating the flipped membrane, a hallmark of 14



233

apoptosis, was also observed in MG-63 cells that had been exposed to UA for 24 h

234

(Fig4).

235

Targeting cancer-related defects in apoptosis is a major focus in cancer research9.

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Apoptosis is regulated by two major pathways. The extrinsic pathway is mediated by

237

the transduction of extracellular death ligand signaling, and the intrinsic pathway, also

238

referred to as the mitochondrial pathway, which is governed by a caspase cascade. In

239

this study, we found that treatment with UA activated the apoptotic initiator caspase-8,

240

which subsequently activated the apoptotic effector caspase-3, resulting in the cleaved

241

form of PARP and ultimately to apoptosis (Fig 9A). As demonstrated in Fig 9B,

242

treatment with the caspase-specific inhibitor ZVAD-FMK remarkably reduced the

243

apoptotic effect of UA, implying that UA-mediated apoptosis, partially, is through the

244

activation of caspases.

245

Lee et al. have reported that natural phytochemicals such as Morinda citrifolia

246

(Noni) enhanced apoptosis in human cervical cancer cells through mitochondrial

247

pathways24, 25. Overproduction of ROS is known to result in disruption of the outer

248

membrane of mitochondria, which leads to reduced membrane potential and

249

activation of the mitochondrial apoptotic pathway26. In this study, we found evidence

250

that UA may induce apoptosis, at least in part; by promoting ROS-mediated

251

disruption of mitochondrial membrane potential in osteosarcoma MG-63 cells (Fig 6 15


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and 7). It is documented that the downregulation of pro-survival Bcl-2 protein by

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phytochemicals could result in the alterations of mitochondrial membrane potential 24.

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In addition, inhibitors of apoptosis proteins (IAPs) suppress apoptosis by directly

255

binding to activated caspases, for example caspase 3 and caspase 7 27. We found here

256

that exposure of MG-63 cells to UA also caused an increase in Bax/Bcl-2 ratio

257

whereas the pro-survival XIAP and survivin were downregulated (Fig 10).

258

MAP kinases such as JNKs, ERK1/2, and p38MAPK play vital roles in controlling

259

of cell proliferation, differentiation, and apoptosis28, 29. Here, we also illustrated that

260

UA exposure stimulated a substantial increase in the expression of phospho-JNK,

261

phospho-p38 and phospho-ERK, and that pre-treatment of the ERK-specific inhibitor

262

U0126 attenuated UA-induced apoptosis (Fig 8B). Furthermore, we also confirmed

263

that treatment with UA in MG-63 cells significantly reduced the expression of

264

phospho/activated Akt (Fig 8), a serine/threonine-specific protein kinase vital for cell

265

survival30.

266

In conclusion, we report that UA inhibits cell growth by apoptosis induction in

267

MG-63 cells but not in non-cancerous MRC-5 and NIH3T3 cells. The lesser

268

cytotoxicity of UA on non-cancerous MRC-5 cells is reflected in the higher IC50

269

values and little apoptosis. Our data indicate that the anticancer effects of UA are

270

associated with the activation of ERK1/2 and caspases whereas the pro-survival Bcl-2 16



271

and XIAP are downregulated and Akt is inhibited in human osteosarcoma MG-63

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cells.

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13. Jin, Y. R.; Jin, J. L.; Li, C. H.; Piao, X. X.; Jin, N. G., Ursolic acid enhances mouse liver regeneration after partial hepatectomy. Pharm Biol. 2012, 50, 523-8. 14. Manu, K. A.; Kuttan, G., Ursolic acid induces apoptosis by activating p53 and

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caspase-3 gene expressions and suppressing NF-kappaB mediated activation of bcl-2 in B16F-10 melanoma cells. Int Immunopharmacol. 2008, 8, 974-81. 15. Xavier, C. P.; Lima, C. F.; Preto, A.; Seruca, R.; Fernandes-Ferreira, M.;

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Pereira-Wilson, C., Luteolin, quercetin and ursolic acid are potent inhibitors of proliferation and inducers of apoptosis in both KRAS and BRAF mutated human colorectal cancer cells. Cancer Lett. 2009, 281, 162-70. 16. Achiwa, Y.; Hasegawa, K.; Komiya, T.; Udagawa, Y., Ursolic acid induces Bax-dependent apoptosis through the caspase-3 pathway in endometrial cancer

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Figure legends Figure 1. Effect of ursolic acid on viability of MG-63 cells. The results of the MTT assay showed that cell viability decreased in a dose- and time-dependent manner.

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Each point is the mean ± SD of three experiments. **P˂0.001 as compared with the control group.

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Figure 2. Effect of ursolic acid on cell growth in MRC-5 cells. Cell growth with or without UA was dynamically monitored using impedance technology in MRC-5 cells. Normalized cell index values measured over 50hours are shown.

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Figure 3. Effects of ursolic acid on cell-cycle distribution in MG-63 cells. (A) Cells

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were treated with 5, 10 or 20 µg/ml of ursolic acid for 24 h, after which cells were stained with propidium iodide (PI) and then analyzed by flow cytometry. The peaks in the illustration correspond to the sub-G1, G0/G1, S and G2/M phases of the cell cycle. (B) Histogram showing the percentages of cells in each phase of the cell cycle. Data

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are representative of three independent experiments with similar results. **P˂0.001 as compared with the control group.

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Figure 4. Apoptotic effect of ursolic acid treatment on MG-63 cells. (A) Cells were

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treated with 5, 10 or 20 µg/ml of ursolic acid for 24 h, after which cells were stained with Muse™ Annexin V for 24 h. The results were then evaluated by the Muse Cell

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Analyzer. (B) Histogram shows the percentages of apoptotic cells for each concentration of ursolic acid. Data are representative of three independent experiments with similar results. **P < 0.001 as compared with the control group.

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Figure 5. Apoptotic effect of ursolic acid treatment on MRC-5 and NIH3T3 cells.

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Cells were treated with 5, 10 or 20 µg/ml of ursolic acid for 24 h. The percentage of apoptotic cells was determined by flow-cytometry, and the results are expressed as a

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percentage relative to the control group. Values (mean ± S. E.) are from three independent experiments.

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with 5, 10 or 20 µg/ml of ursolic acid for 12 h after which cells were stained with a Muse Oxidative Stress Kit and the results were determined by Muse Cell Analyzer analysis assay (B) Histogram shows the percentage of depolarized cells for each concentration of ursolic acid. Data are representative of three independent experiments with similar results. *P < 0.05, **P < 0.001 as compared with the control

Figure 6. Ursolic acid increases ROS levels inMG-63 cells. (A) Cells were treated

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group.

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Figure 7. Effect of ursolic acid on mitochondrial depolarization in MG-63 cells. (A)

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Cells were treated with 5, 10 or 20 µg/ml of ursolic acid for 12 h after which cells were stained with a Muse™ MitoPotential Kit and the results were determined using a

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Muse Cell Analyzer analysis assay (B) Histogram shows the percentage of depolarized cells for each concentration of ursolic acid. Data are representative of

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three independent experiments with similar results. *P < 0.05, **P < 0.001 as

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compared with the control group.

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Cells were treated with 5, 10 , 15 or 20 µg/ml of ursolic acid for 24 h. Total cell lysates were prepared, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies to detect the protein levels of phosphorylated ERK1/2, p-JNK, p-p38, and p-Akt, which were adjusted with total ERK1/2, t-JNK, t-p38, and t-Akt protein level.

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GAPDH served as a loading control. GAPDH protein levels indicated that an equal amount of protein was loaded into each lane. Changes in the levels of p/t-ERK1/2, p/t-JNK, p/t-p38, and p/t-Akt after being normalized to the levels of GADPH are

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shown below each blot.(B, upper panel)Cells were pretreated with 40µM U0126, SP600125, or SB203580 for 2 hour and then incubated with or without 20µg/ml ursolic acid for another 24 hours, after which cells were stained with a Muse™ Annexin V & Dead Cell Assay Kit the results were determined using a Muse Cell

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Analyzer analysis assay. (B, lower panel) Histogram shows the percentages of

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apoptotic cells for U0126, SP600125, or SB203580 with or without 20µg/ml ursolic acid.*P < 0.05as compared with the control group.

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Figure 9. Activation of caspases in ursolic acid-treatedMG-63 cells. (A) Cells were

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treated with 5, 10, 15 or 20 µg/ml of ursolic acid for 24 h. Total cell lysates were prepared, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies to detect the cleaved forms of caspase-8, caspase-9, caspase-3, and PARP. GAPDH

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served as a loading control.(B, upper panel) Cells were pretreated with 40 µM ZVAD-FMK for 2 hours and then incubated with or without 20µg/ml ursolic acid for another 24 hours, after which cells were stained with a Muse™ Annexin V & Dead Cell Assay Kit and analyzed using a Muse Cell Analyzer analysis assay. (B, lower panel) Histogram shows the percentages of apoptotic cells for ZVAD-FMK with or

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without 20µg/ml ursolic acid.*P < 0.05 as compared with the control group.

Figure 8. Effects of ursolic acid on MAPK signaling pathways in MG-63 cells. (A)

Figure 10. Decreased Bcl-2 and XIAP protein expression in MG-63 cells. The cells 22



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were treated with 5, 10, or 20 µg/ml of ursolic acid for 24 h. Total cell lysates were prepared, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies

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to detect the cleaved forms of Bax, Bc1-2, XIAP, and survivin. GAPDH served as a loading control. GAPDH protein levels indicate that an equal amount of protein was

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loaded into each lane. The protein expression levels were quantified using Image J software. Changes in the levels of Bax, Bcl-2, XIAP, and survivin after being normalized to the levels of GADPH are shown below each blot.

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Ursolic Acid Triggers Apoptosis in Human Osteosarcoma Cells via

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