Inhibition of A2780 Human Ovarian Carcinoma Cell Proliferation by a

Subscriber access provided by ORTA DOGU TEKNIK UNIVERSITESI KUTUPHANESI

Article

Inhibition of A2780 Human Ovarian Carcinoma Cell Proliferation by a Rubus Component, Sanguiin H-6 Dahae Lee, Hyeonseok Ko, Young-Joo Kim, Su-Nam Kim, Kyung-Chul Choi, Noriko Yamabe, Ki Hyun Kim, Ki Sung Kang, Hyun Young Kim, and Takayuki Shibamoto J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05461 • Publication Date (Web): 03 Jan 2016 Downloaded from http://pubs.acs.org on January 9, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20

Journal of Agricultural and Food Chemistry 1

1

Inhibition of the A2780 Human Ovarian Carcinoma Cell

2

Proliferation by a Rubus Component, Component, Sanguiin HH- 6

3 4

Dahae Lee,†,‡Hyeonseok Ko,§Young-Joo Kim,¶Su-Nam Kim,¶Kyung-Chul

5

Choi,# Noriko Yamabe,†Ki Hyun Kim,∮ Ki Sung Kang,†Hyun Young Kim,‡,*

6

Takayuki Shibamoto!,*

7 8

†

9

‡

College of Korean Medicine, Gachon University, Seongnam 13120, Korea Department of Food Science, Gyeongnam National University of Science and Technology,

10

Jinju 660-758, Korea

11

§

12

Dankook University College of Medicine, Seoul, Korea.

13

¶

14

Gangwon-do, Korea

15

#

16

Department of Pharmacology, University of Ulsan College of Medicine, Seoul, Korea; Cell

17

Dysfunction Research Center (CDRC), University of Ulsan College of Medicine, Seoul,

18

Korea

19

∮

20

Suwon 440-746, Korea

21

!

Laboratory of Molecular Oncology, Cheil General Hospital Women's Healthcare Center,

Natural Medicine Center, Korea Institute of Science and Technology, Gangneung,

Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul, Korea;

Natural Product Research Laboratory, School of Pharmacy, Sungkyunkwan University,

Department of Environmental Toxicology, University of California, Davis, California 95616,

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 20 2

22

United States

23 24 25

ABSTRACT: The effects of a red raspberry component, Sanguiin H-6 (SH-6), on the

26

induction of apoptosis and the related signaling pathways in A2780 human ovarian carcinoma

27

cells were investigated. SH-6 caused an anti-proliferative effect and a severe morphological

28

change resembling that of apoptotic cell death with dose dependent but no effect on the

29

cancer cell cycle arrest. In addition, SH-6 induced an early apoptotic effect and activation of

30

caspases as well as the cleavage of PARP, which is a hallmark of apoptosis. The early

31

apoptotic percentages of A2780 cells exposed to 20 µM and 40 µM SH-6 were 35.39 and

32

41.76, respectively. Also, SH-6 caused the activation of mitogen-activated protein kinases

33

(MAPKs), especially p38, and the increase of truncated p15/BID. These results in the present

34

study suggest that the apoptosis of A2780 human ovarian carcinoma cells by SH-6 is

35

mediated by the MAPK p38 and a caspase-8-dependent BID cleavage pathway.

36 37

KEYWORDS: apoptosis, A2780 ovarian carcinoma cells, caspase, MAPK p38, polyphenols,

38

raspberry; sanguiin H-6, truncated BID

39 40 41

■ INTRODUCTION

42 43

Sanguiin H-6 is one of polyphenol compounds, as shown in Figure 1, called ellagitannins

44

found in Rubus and berry species, such as strawberries,1,2 red raspberries, and cloudberries3

45

as well as in a crude drug Sanguisorbae Radix.1,4 Ellagitannins, including SH-6, have been


Page 3 of 20


46

reported to have various biological activities, including antioxidant,5 amylase inhibition6 and

47

resistance to powdery mildew.7

48

It has been reported that SH-6 inhibited not only the expression of iNOS mRNA, but also

49

iNOS activity with dose dependent. Namely, it acted not only as an NO scavenger, but it also

50

inhibited iNOS mRNA induction and enzyme activity.8 Moreover, in a previous report

51

suggested that SH-6 contributed to the inhibition of endothelial cell proliferation by reducing

52

the level of NO production via inhibition of iNOS activity and mRNA expression. In addition,

53

SH-6 efficiently blocked the VEGF induced HUVEC proliferation with dose dependent but

54

had no effect on the growth of HT1080 human fibrosarcoma cells, suggesting that it is a

55

potential anti-angiogenic agent.9 However, the effects of SH-6 on the apoptosis of A2780

56

ovarian cancer cells still have not been fully elucidated.

57

Ovarian cancer is the second most lethal gynecologic cancer among woman in developed

58

countries.10 There were estimated 22,000 new cases and 14,000 mortalities due to ovarian

59

cancer in 2013.11 Although many women may respond well to initial first-line treatment,

60

relapse frequently occurs with chemotherapy-resistant disease, presenting a major barrier to

61

improving the prognosis for ovarian cancer patients.12 Thus, searching novel chemicals from

62

natural food stuff for anticancer agents is pressing need for ovarian cancer treatment.

63 64

■ MATERIALS AND METHODS

65 66

Chemicals and Reagents. Reagents. Cleaved caspase-8, cleaved caspase-3, BID, poly(ADP-

67

ribose) polymerase (PARP), p38 MAP Kinase, phosphor-p38, p44/42 MAP Kinase (Erk1/2),

68

Phospho-p44/42 (Erk1/2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and



Page 4 of 20 4

69

horseradish peroxidase (HRP) conjugated anti-rabbit antibodies were bought from Cell

70

Signaling (Boston, MA, USA). Other chemicals and reagents in high quality were obtained

71

from reliable commercial sources.

72

SH-6 (Figure 1) was isolated from red raspberries by using Sephadex LH-20 column

73

chromatography and preparative HPLC. A structure of isolated SH-6 was confirmed by ESI-

74

MS method reported previously.1 Briefly, 50 mL of an aqueous red raspberry extract was

75

loaded on a Sephadex LH-20 column and polyphenols, such as anthocyanins, were eluted

76

with a 500 mL methanol/water (30/70 v/v) solution. The fraction containing SH-6 was eluted

77

with a 500 mL acetone/water (70/30 v/v) solution. The SH-6 -rich fraction was further

78

purified by preparative HPLC.

79

Measurement Measurement of Cytotoxic Effects on A2780 Human Ovarian Carcinoma Cells. Cells. To

80

investigate the anti-proliferative effects of SH-6 on A2780 cells, A2780 cells were exposed to

81

different concentrations (0, 10, 20 µM, and 40 µM) of SH-6 for 24 h, and then the inhibition

82

of proliferation was determined by a CCK8 assay kit. The proliferation of cells was measured

83

by a Cell Counting Kit-8 (CCK8; Dojindo Laboratories, Kumamoto, Japan) according to the

84

previously reported method.13 CCK8 allows sensitive detection of living cells by utilizing the

85

highly water-soluble tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-

86

5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] which produces a water-soluble

87

formazan dye upon reduction by dehydrogenases in cells.

88

The A2780 human ovarian carcinoma cells (American Type Culture Collection ATCC,

89

Manassas, VA, USA) were grown in an RPMI1640 medium (Cellgro, Manassas, VA, USA)

90

and then mixed with 10% fetal bovine serum (Gibco BRL, Carlsbad, MD, USA), 100

91

units/mL penicillin, and 100 µg/mL streptomycin. The cell solution was incubated at 37 °C in

92

a humidified atmosphere with 5 % CO2. The cells were seeded in 96-well culture plates at


Page 5 of 20


93

1×104 cells per well and treated with different concentrations of SH-6 (0, 10, 20, and 40 µM).

94

After incubated for 24 h, inhibition of proliferation was determined by a CCK8 assay kit. Cell

95

viability was measured by a microplate reader (PowerWave XS; Bio-Tek Instruments,

96

Winooski, VT, USA) at 450 nm.

97

Flow Cytometric Assay Assay. ssay. A2780 Cells were exposed to 20 µM and 40 µM of SH-6 for 24

98

h, and stained with Annexin-V/PI for flow cytometry. Treatment of A2780 cells with SH-6

99

induced an increase in the fraction (Annexin V+/PI-) of early apoptotic cells from 4.17 % to

100

41.76 %. (B) Effects of SH-6 on cell cycle in A2780 human ovarian carcinoma cells. A2780

101

cells were exposed to 20 µM and 40 µM of SH-6 for 24 h, and lysated with RNase and

102

stained with PI; the DNA content of those cells was determined by flow cytometry.

103

Apoptotic cell death and changes in cell cycle distribution were analyzed by a FACS

104

Calibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). Cell cycle arrest was

105

assessed by PI staining by an Annexin-V-FLUOS Staining Kit (Roche, Penzberg, Germany).

106

After the treated cells were washed with Dulbecco`s phosphate buffered saline (DPBS), they

107

were fixed in a 70 % ethanol solution at 4 °C for 30 min. The cells were further washed with

108

DPBS and then incubated with 300 µL DPBS containing 10 µg/mL RNase A and a propidium

109

iodide (PI) staining solution containing 40 µg/mL PI at room temperature for 15 min in the

110

dark. Apoptotic cell death was assessed by annexin-V/PI double staining by an Annexin-V-

111

FLUOS Staining Kit (Roche, Penzberg, Germany) according to the manufacturer’s

112

recommendations. At least 20,000 events were evaluated for each experiment.

113

Western Blot Analysis. Analysis. Western blot analysis on the A2780 cells treated with SH-6 at

114

different concentrations (10 µM, 20 µM, and 40 µM) for 24 h was carried out using methods

115

previously reported.14 To confirm the expression of protein, whole-cell extracts were prepared

116

according to the manufacturer’s instructions using a RIPA buffer (Cell Signaling Technology,



Page 6 of 20 6

117

MA, USA) to which 1 mM phenylmethylsulfonyl fluoride (PMSF) was added with 1 ×

118

complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Each protein

119

concentration was determined by the QuantiPro BCA Assay Kit (Sigma-Aldrich Saint Louis,

120

MO, USA). Proteins (whole-cell extracts, 20 µg/lane) were separated by electrophoresis in a

121

precast 4 %–15 % Mini-PROTEAN TGX gel (Bio-Rad, CA, USA) blotted onto PVDF

122

transfer membranes (Millipore, MA, USA) and analyzed with epitope-specific primary and

123

secondary antibodies. Bound antibodies were visualized using ECL Advance Western

124

Blotting Detection Reagents (GE Healthcare, UK) and a LAS 4000 imaging system (Fujifilm,

125

Japan). Whole cell lysates (20 µg) were separated by sodium dodecyl sulfate—

126

polyacrylamide gel electrophoresis (SDS−PAGE)—and transferred onto PVDF transfer

127

membranes, and then were probed with the indicated antibodies. Proteins were visualized

128

using an ECL detection system. GAPDH was used as an internal control.

129

Statistical Analysis. The results were subjected to statistical analysis using SPSS software

130

version 16. The significance of differences between the mean values were calculated using

131

unpaired Student’s t-test. P-value < 0.05 was considered to be statistically significant.

132 133 134

■ RESULTS AND DISCUSSION

135 136

Figure 2 shows the inhibitory effect of SH-6 on the proliferation of A2780. Values are mean

137

± SD (n = 3). The effect occurred in a dose-dependent manner, suggesting that SH-6

138

significantly inhibits the proliferation of A2780 cells. The effects of SH-6 on normal cell

139

viability were assessed. We found that SH-6 did not affect cell viability in LLC-PK1 cells

140

(data not shown). Furthermore, as shown in Figure 3, it was observed that when A2780 cells


Page 7 of 20


141

were treated with various concentration of SH-6, these cells underwent severe morphological

142

changes in a dose-dependent manner, which were consistent with that of apoptotic cell death.

143

A previous report indicates that the externalization of phosphatidylserine (PS) preceded

144

the loss of membrane integrity, which accompanied the later stages of cell death induced by

145

either apoptosis or necrosis.15 Staining with fluorescein isothiocyanate (FITC) Annexin V,

146

which detects PS, is typically used with a vital dye such as propidium iodide (PI). Figure 4

147

shows the results of analysis by flow cytometry performed to assess apoptosis in the present

148

study. Viable cells with intact membranes exclude PI, whereas death cells with damaged

149

membranes are permeable to PI. Therefore, viable cells are negative for both FITC Annexin

150

V and PI. The early apoptotic cells are FITC Annexin V positive and PI negative and the late

151

apoptotic or necrotic cells are both FITC Annexin V and PI positive. The present study

152

clearly showed that there was a dose-dependent apoptotic effect of SH-6 on A2780 cells.

153

Moreover, the early apoptotic percentages of A2780 cells exposed to 20 µM and 40 µM SH-6

154

were 35.39 % and 41.76 %, respectively as shown in Figure 4A. Figure 4B shows the effect

155

of SH-6 on cell cycle profiles obtained by flow cytometric analysis after treating A2780

156

cancer cells with SH-6 for 24 h. It was observed that a cell cycle arrest was not altered by

157

either 20 µM or 40 µM SH-6 treatment. These results indicate that SH-6 did not affect A2780

158

cancer cell cycle arrest.

159

Figure 5 shows the effect of SH-6 on pro-apoptotic proteins expressions in A2780 human

160

ovarian carcinoma cells determined by the western blot analysis. To investigate the

161

mechanism of SH-6-induced apoptosis, it was examined whether SH-6 treatment could

162

induce poly (ADP-ribose) polymerase (PARP) cleavage or not as a first step. In the present

163

study, the PARP protein (116 kDa) was cleaved into its characteristic 85 kDa fragment in a

164

dose dependent manner upon SH-6 treatment as shown in Figure 5A—PARP is a 116 kDa

165

protein that is cleaved into 85 kDa fragments during apoptotic cell death.16 It has been



Page 8 of 20 8

166

reported that caspases are aspartate-specific cysteine proteases that play an important role in

167

mediating apoptotic response. Furthermore, caspases are an evolutionarily conserved family

168

of cysteine proteases that are responsible for diverse cellular functions, such as inflammation.

169

Moreover, caspases are responsible for many biochemical and morphological changes,

170

including chromatin condensation, nuclear membrane breakdown, and the formation of

171

apoptotic bodies that occur during apoptosis.17 The present study showed that the expression

172

levels of cleaved caspase-3 (effector caspase) and caspase-8 (initiator caspase) proteins were

173

upregulated by SH-6 in a dose-dependent manner after a 24 h exposure as shown in Figure

174

5A. It has been reported that activated caspase-8 stimulates apoptosis via two parallel

175

apoptotic signaling pathways—directly cleaves and activates caspase-3 or cleaves the pro-

176

apoptotic Bcl-2 family protein BID. Once cleaved (truncated) BID (tBID) moved into the

177

mitochondria, it releases cytochrome C which sequentially activates caspase-3.18 Therefore,

178

was investigated. In the present study, a western blot assay showed that tBID was increased

179

by SH-6 after a 24 h exposure with a dose-dependent indicating that a possible induction of

180

tBID upregulation by SH-6 was occurred.

181

In the present study, when A2780 cells were treated with 40 µM SH-6, p38 activity

182

increased markedly and reached a maximum after exposure to SH-6 for 24 h. In contrast, no

183

significant alterations in ERK activity were observed (Figure 5B). MAPKs have been

184

proposed to play an important role in the regulation of apoptosis and the activation of p38

185

promotes apoptosis, whereas the activation of ERK prevents apoptosis.19 The results also

186

indicate that p38 was significantly activated by SH-6 but not ERK. Therefore, it is proposed

187

that activation of p38, but not ERK, might promote SH-6-induced apoptosis. The activation

188

of caspases is known to be a general mechanism in the induction of apoptosis.20

189

It has been shown that MAPK family members have a role in activating caspase cascades.

190

Also, it has been reported that p38 MAPK signaling was linked to the activation of caspase-8,


Page 9 of 20


191

which results in the activation of downstream caspases and the cleavage of cytosolic

192

substances such as BID, a BH3 domain-only protein18,21 In the present study, it was observed

193

that the intensity of full-length BID decreased over time upon SH-6 treatment. The change

194

seems to be a result of the cleavage of BID to tBID. Moreover, the cleavage of BID might be

195

induced by the activation of caspase-8 and p38, which was associated with the mitochondria

196

pathway triggered by caspase-8-dependent BID cleavage in SH-6-induced apoptosis. In

197

addition, a mechanism of SH-6-induced apoptosis was associated with the activation of p38

198

MAPK, p38 MAPK signaling. It may regulate caspase-8 activity by phosphorylating caspase-

199

8 itself and caspase-8-mediated cleavage of BID followed by its translocation to the

200

mitochondria. These phenomena may be associated with the release of cytochrome C, which

201

promotes the formation of an apoptosome.

202

It is the first observation that the contribution of the p38 MAPK and mitochondrial

203

pathway on apoptosis was induced by SH-6. These results from the present study suggest that

204

SH-6 is a useful anticancer drug which enhances a therapeutic efficacy. However, further

205

studies to clarify the molecular mechanisms of SH-6-induced apoptosis is in order.

206 207

■ AUTHOR INFORMATION

208 209

Corresponding Authors

210

*Phone: 1 (530) 752-4523. Fax: 1 (530) 752-3394. E-mail: [email protected]

211

*Phone: 82 (55) 751-3277. Fax: 82 (55) 751-3279. E-mail: [email protected]

212 213

Author Contributions Contributions



Page 10 of 20 10

214

D.L and H. K. contributed equally to this study.

215 216

Funding

217

This work was supported in part by the Korea Institute of Science and Technology

218

Institutional Program (2Z04371). This research was also supported by a grant of the Korea

219

Health technology R&D Project through the Korea Health Industry Development

220

Institute(KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea. (Grant

221

Number : HI15C0089).

222 223

Note

224

The authors declare there are no conflicts of interest.

225 226 227

■ REFERENCES

228 229

(1) Gasperotti, M.; Masuero, D.; Vrhovsek, U.; Guella, G.; Mattivil, F. Profiling and

230

accurate quantification of Rubus ellagitannins and ellagic acid conjugates using direct

231

UPLC-Q-TOFHDMS and HPLC-DAD analysis. J. Agric. Food Chem. 2010, 58, 4602–

232

4612.

233

(2) Vrhovsek, U.; Guella, G.; Gasparotti, M.; Pojert, E.; Zancato, M.; Mattivit, F. Clarifying

234

the identity of the main ellagitannin in the fruit of the strawberry, Fragaria vesca and

235

Fragaria ananassa Duch. J. Agric. Food Chem. 2012, 60, 2507–2518.

236

(3) Rao, A. V.; Snyder, D. M. Raspberries and human health: A review. J. Agric. Food


Page 11 of 20


237 238 239 240

Chem. 2010, 58, 3871–3883. (4) Nishioka, I. Chemistry and biological activities of tannins. Yakugaku Zasshi 1983, 103, 125–142. (5) Prior, R. L.; Wu, X.; Schaich, K. Standardized methods for the determination of

241

antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food

242

Chem. 2005, 5, 4290–4302.

243

(6) Grussu, D.; Stewart, D.; McDougall, G. J. Berry polyphenols inhibit α-amylase in vitro:

244

Identifying active components in rowanberry and raspberry. J. Agric. Food Chem. 2011,

245

59, 2324–2331.

246

(7) Hukkanen, A.; Kokko, H.; Buchala, A. J.; McDougall, G. J.; Stewart, D.; Karenlampl, S.

247

O.; Karjalainen, R. O. Benzothiadiazole induces the accumulation of phenolics and

248

improves resistance to powdery mildew in strawberries. J. Agric. Food Chem. 2007, 55,

249

1862–1870.

250

(8) Yokozawa, T.; Chen, C. P.; Tanaka, T.; Kitani, K. (). Effects of sanguiin H-6, a

251

component of Sanguisorbae Radix, on lipopolysaccharide-stimulated nitric oxide

252

production. Biochem. Pharmacol. 2002, 63, 853–858.

253

(9) Lee, S. J.; Lee, H. K. Sanguiin H-6 blocks endothelial cell growth through inhibition of

254

VEGF binding to VEGF receptor. Arch Pharm. Res. 2005, 28, 1270–1274.

255

(10) Jemal, A; Bray, F; Center, M. M; Ferlay, J; Ward, E.; Forman, D. Global cancer

256 257 258 259 260 261

statistics. CA Cancer J. Clin. 2011, 61, 69–90. (11) Siegel, R.; Naishadham, D.; Jemal, A. Cancer statistics, 2013. CA Cancer J. Clin. 2013, 63, 11–30. (12) Kigawa, J. New strategy for overcoming resistance to chemotherapy of ovarian cancer. Yonago Acta Med. 2013, 56, 43–50. (13) Kim, Y. J.; Choi, W. I.; Jeon, B. N.; Choi, K. C.; Kim, K.; Kim, T. J.; Kim, Y. J.; Choi,



Page 12 of 20 12

262

W. I.; Jeon, B. N.; Choi, K. C.; Kim, K.; Kim, T. J.; Ko, H. Stereospecific effects of

263

ginsenoside 20-Rg3 inhibits TGF-beta1-induced epithelial-mesenchymal transition and

264

suppresses lung cancer migration, invasion and anoikis resistance. Toxicology 2014,

265

322, 23–33.

266

(14) Ko, H., Jeong, M. H.; Jeon, H.; Sung, G. J.; So, Y.; Kim, I.; Son, J.; Lee, S. W.; Yoon, H.

267

G.; Choi, K. C. Delphinidin sensitizes prostate cancer cells to TRAIL-induced apoptosis,

268

by inducing DR5 and causing caspase-mediated HDAC3 cleavage. Oncotarget 2015, 6,

269

9970–9984.

270

(15) Luo, J.; Hu, Y.; Kong, W.; Yang, M. Evaluation and structure-activity relationship

271

analysis of a new series of arylnaphthalene lignans as potential anti-tumor agents. PLoS

272

One. 2014, 9, e93516.

273

(16) Morales, J.; Li, L.; Fattah, F. J.; Dong, Y.; Bey, E. A.; Patel, M.; Gao, J.; Boothman, D.

274

A. Review of poly (ADP-ribose) polymerase (PARP) mechanisms of action and

275

rationale for targeting in cancer and other diseases. Crit. Rev. Eukaryot. Gene Expr.

276

2014, 24, 15–28.

277 278

(17) Rupinder, S. K.; Gurpreet, A. K.; Manjeet, S. Cell suicide and caspases. Vascul. Pharmacol. 2007, 46, 383–393.

279

(18) Muzio, M.; Chinnaiyan, A. M.; Kischkel, F. C.; O'Rourke, K.; Shevchenko, A.; Ni, J.;

280

Scaffidi, C.; Bretz, J. D.; Zhang, M.; Gentz, R.; Mann, M.; Krammer, P. H.; Peter, M.

281

E.; Dixit, V. M. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is

282

recruited to the CD95 (Fas/APO-1) death--inducing signaling complex. Cell 1996, 85,

283

817–827.

284 285 286

(19) Xia, Z.; Dickens, M.; Raingeaud, J.; Davis, R. J.; Greenberg, M. E. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 1995, 270, 1326-1331. (20) Thornberry, N. A.; Lazebnik, Y. Caspases: enemies within. Science 1998, 281, 1312–


Page 13 of 20


287 288

1316. (21) Bhattacharyya, A.; Pathak, S.; Basak, C.; Law, S.; Kundu, M.; Basu, J. Execution of

289

macrophage apoptosis by Mycobacterium avium through apoptosis signal-regulating

290

kinase 1/p38 mitogen-activated protein kinase signaling and caspase 8 activation. J.

291

Biol Chem. 2003, 278, 26517–26525.

292

Figure Legends

293 294

Figure 1. Chemical structures of SH-6.

295 296

Figure 2. The results of CCK8 assay. Cytotoxic effect of SH-6 on A2780 human ovarian

297

carcinoma cells.

298 299

Figure 3. Morphological changes in A2780 human ovarian carcinoma cells induced by

300

different amounts of SH-6. The morphology of cells was confirmed using phase-contrast

301

microscopy and photographed (original magnification, X 200).

302 303

Figure 4. The results of analysis by flow cytometry performed to assess apoptosis

304

Apoptotic cell death and changes in cell cycle distribution involvement of apoptosis and cell

305

cycle arrest in A2780 human ovarian carcinoma cells induced by SH-6.

306 307

Figure 5. Effect of SH-6 on pro-apoptotic proteins expressions in A2780 human ovarian

308

carcinoma cells determined by the western blot analysis. (A) BID and tBID, cleaved caspase-

309

8, cleaved caspase-3, PARP and cleaved PARP (B) p-P38 and P38, p-ERK and ERK in

310

A2780 cells treated with Sanguiin H-6 at different concentrations (10 µM, 20 µM, and 40



Page 14 of 20 14

311

µM) for 24 h. (C) Quantitative data for the PARP, cleaved caspase-8, cleaved caspase-3, BID,

312

p-p38 and p-ERK Western blot analysis.

313 314 315 316 317 318 319 320


Page 15 of 20


Fig. 1



Page 16 of 20

Fig. 2

Cell viability (% of control)

120 100 80 60

40 20 0 0 Control

10

20

Amount of SH-6 (mM) ACS Paragon Plus Environment

40

Page 17 of 20

Fig. 3


Control

10 μM

20 μM

40 μM

X 200 ACS Paragon Plus Environment


Figure 4

A

103

FL2-H

102 Live

101

Early Apoptosis

100

101

102

FL1-H

103

104

103

103

102

102

101

4.17 %

100

104

FL2-H

Late Apoptosis or Necrosis

Necrosis

FL2-H

104

40 μM

20 μM

Control

Propidium iodide (PI)

Page 18 of 20

35.39 %

100 100

104

102

101

FL1-H

103

104

101

41.76%

100 100

102

101

FL1-H

103

104

Annexin V

B Control

493

# of cells

S

20 μM

427

G1

119

G1

S G2/M

Fluorescence (RFU)

S G2/M

2,500

0

0

40 μM G1

Fluorescence (RFU)

G2/M

2,500

Propidium iodide (PI) ACS Paragon Plus Environment

0

Fluorescence (RFU)

2,500

Page 19 of 20


Figure 5

B

A Amount of SH-6 (mM) 0

10

20

Amount of SH-6 (mM)

40

0

PARP

10

20

40

p-p38

Cleaved PARP Cleaved caspase-8

p38

Cleaved caspase-3

p-ERK

BID tBID

ERK

GAPDH

GAPDH

C

14 12 10 8 6 4 2 0 0 10 20 40

Amount of SH-6 (mM)

3 2.5 2 1.5 1 0.5 0 0 10 20 40

Amount of SH-6 (mM)

1

0.5

0

0.8 0.6 0.4 0.2 0

ACS Paragon Plus Environment 0 10 20 40 0 10 20 40 Amount of SH-6 (mM)

120

80

1

2

1.5

90

Amount of SH-6 (mM)

100

70

Ratio of p-ERK/GAPDH

16

3.5

1.2

Ratio of p-p38/GAPDH

Ratio of Cleaved caspase-8/GAPDH

Ratio of PARP/GAPDH

18

2.5

Ratio of BID/GAPDH

4

Ratio of Cleaved caspase 3/GAPDH

20

60 50 40 30 20

80 60 40 20

10 0

0 0 10 20 40

0 10 20 40

Amount of SH-6 (mM)

Amount of SH-6 (mM)


Page 20 of 20

TOC

Sanguiin H-6 Caspase-8

Z-VAD tBID

BID

Bax Bcl-2 Caspase-3

PARP

Caspase-9

Apoptosis


Inhibition of A2780 Human Ovarian Carcinoma Cell Proliferation by a

Recommend Documents