Isoliensinine, a Bioactive Alkaloid Derived from Embryos of Nelumbo

Subscriber access provided by UNIV OF ARIZONA

Article

Isoliensinine, a bioactive alkaloid derived from embryos of Nelumbo nucifera, induces hepatocellular carcinoma cell apoptosis through suppression of NF-#B signaling Guangwen Shu, Ling Yue, Wenhao Zhao, Chan Xu, Jing Yang, Shaobing Wang, and Xinzhou Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02993 • Publication Date (Web): 21 Sep 2015 Downloaded from http://pubs.acs.org on September 24, 2015

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 37

Journal of Agricultural and Food Chemistry

Isoliensinine, a bioactive alkaloid derived from embryos of Nelumbo nucifera, induces

1 2

hepatocellular carcinoma cell apoptosis through suppression of NF-κB signaling

3 †

†

†

†

†

4

Guangwen Shu, Ling Yue,§ Wenhao Zhao, Chan Xu, Jing Yang, Shaobing Wang, and

5

Xinzhou Yang*,

6

†

College of Pharmacy, South-Central University for Nationalities, Wuhan, PR China

7

§

8

Wuhan, PR China

9

*To whom correspondence can be addressed.

10

†

Department of Endocrinology, Wuhan General Hospital of Guangzhou Military Command,

Phone/Fax: + 86 27 6784 1196. E-mail address: [email protected]

11

1 ACS Paragon Plus Environment


12

ABSTRACT:

13

Isoliensinine (isolie) is an alkaloid produced by the edible plant Nelumbo nucifera. Here,

14

we unveiled that isolie was able to provoke HepG2, Huh-7 and H22 hepatocellular carcinoma

15

(HCC) cell apoptosis. Isolie decreased NF-κB activity and constitutive phosphorylation of

16

NF-κB p65 subunit at Ser536 in HCC cells. Overexpression of p65 Ser536 phosphorylation

17

mimics abrogated isolie-mediated HCC cell apoptosis. Furthermore, intraperitoneal injection

18

of isolie inhibited the growth of Huh-7 xenografts in nude mice. Additionally, isolie given by

19

both intraperitoneal injection and gavage diminished the proliferation of transplanted H22

20

cells in Kunming mice. Reduced tumor growth in vivo was associated with inhibited p65

21

phosphorylation at Ser536 and declined NF-κB activity in tumor tissues. Finally, we revealed

22

that isolie was bioavailable in the blood of mice and exhibited no detectable toxic effects on

23

tumor-bearing mice. Our data provided strong evidence for the anti-HCC effect of isolie.

24 25

KEYWORDS:

26

Nelumbo nucifera; Isoliensinine; Hepatocellular carcinoma; Apoptosis; NF-κB

27


Page 2 of 37

Page 3 of 37

28


INTRODUCTION

29

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies and is the third

30

most frequent cause of cancer-related mortality worldwide.1 Surgical resection is considered

31

to be the standard strategy for treatment of HCC. However, only a small proportion of HCC

32

patients are eligible for surgical intervention, since HCC is often diagnosed in later stages due

33

to the extended length of time it takes for HCC to begin and develop.2 Therefore, the majority

34

of HCC patients must rely on systemic chemotherapies. Though compounds such as sorafenib,

35

5-fluorouracil and cisplatin are approved for clinical HCC treatment, they only provide

36

limited benefit due to toxic side effects in patients and chemoresistance in HCC cells. Thus, it

37

is imperative to discover and develop new chemotherapy reagents that are capable of inducing

38

HCC cell death but have low or no toxic side effects in patients.

39

Nuclear factor κB (NF-κB) is a family of widely expressed and structurally related homo-

40

or heterodimeric DNA-binding proteins that control the transcription of NF-κB responsive

41

genes and participate in a multitude of important biological processes.3 Aberrant activation of

42

NF-κB is involved in the pathogenesis of HCC.4 The heterodimer p65/p50 is a representative

43

form of NF-κB. Phosphorylation of the p65 subunit of NF-κB is an important mechanism

44

modulating NF-κB activity. For instance, phosphorylation of p65 at Ser536 enhances NF-κB

45

transcriptional potential.5 In cancer cells of different origins, such as myeloma cells, stomach

46

cancer cells and embryonic carcinoma cells, reduction of p65 phosphorylation at Ser536 is

47

associated with cancer cell apoptosis induced by potential chemotherapeutic reagents.6-8

48

However, the role of p65 dephosphorylation at Ser536 in HCC cell apoptosis remains to be

49

fully elucidated.

50

In recent years, natural products have garnered a lot of attentions from pharmaceutical



51

researchers and clinicians owing to their efficiency, safety, and immediate availability.9-11

52

During the past 30 years, more than 70% of the drugs approved by the U.S. Food and Drug

53

Administration originated from natural products.12 Nelumbo nucifera (N. nucifera),

54

commonly known as lotus, is a well-known edible plant. Food related to N. nucifera is

55

popular in some areas of East Asia, especially China. Embryos of N. nucifera contain

56

structurally related bioactive alkaloids including liensinine, isoliensinine (isolie) and neferine

57

(chemical structures shown in Figure 1A). In previous pilot studies in our lab, liensinine,

58

isolie and neferine were prepared from embryos of N. nucifera and tested for cytotoxic

59

activity in a series of human HCC cell lines. Isolie displayed stronger cytotoxic activity on

60

HCC cells compared to liensinine or neferine. Interestingly, the cytotoxic effect of isolie on

61

untransformed hepatocytes, including human HL-7702 hepatocytes and primary mouse

62

hepatocytes, was much lower than the cytotoxic effects of neferine or liensinine (Table 1). In

63

the current study, we demonstrate that isolie provokes HCC cell apoptosis both in vitro and in

64

vivo. Mechanistically, dephosphorylation of p65 at Ser536 which inhibits NF-κB activity is a

65

crucial molecular event mediating isolie-induced HCC cell apoptosis. The bioavailability and

66

toxic impact of isolie were also assessed on tumor-bearing mice.

67 68

MATERIALS AND METHODS

69

Isolation and purification of isolie

70

The embryos of N. nucifera were purchased from Jointown Pharmaceutical Group Co. Ltd.

71

(Wuhan, Hubei Province, PR China) in September 2011 and identified by Prof. Dingrong

72

Wan from the College of Pharmacy, South-Central University for Nationalities, Wuhan,

73

China. Voucher specimens (2011009N) were deposited in the Herbarium of the College of


Page 4 of 37

Page 5 of 37


74

Pharmacy, South-Central University for Nationalities. Air-dried embryos of N. nucifera (1.0

75

kg) were ground into powder and extracted using 90% EtOH (4 × 3.0 L). After evaporation of

76

the collected percolate, the crude extract was acidified with diluted HCl (5%) to a pH of 1-2

77

and partitioned between hexane (3 × 1.5 L) and the acid water layer. The aqueous part was

78

basified with aqueous NH3 to a pH of 9-10 and extracted with CHCl3 to yield 14.5 g of crude

79

alkaloid. A portion of the crude alkaloid (14 g) was then subjected to column chromatography

80

over silica gel (300-400 mesh) and eluted with the isocratic gradient solvent system of CH2Cl2,

81

methanol and diethylamide (100: 5: 2) to yield five major fractions (F1-F5) (F1, 1.8 L; F2, 2.4

82

L; F3, 1. 7 L; F4, 2.6 L; F5, 3.5 L). After evaporation under vacuum, crude isolie (1.8 g) was

83

obtained from F4 and then dissolved in anhydrous acetone (50 mL). The solution was

84

acidified with drops of diluted HClO4 (10%) to a pH of 2-3. The acidized isolie solution was

85

kept in 4 °C to crystallize and yielded 1.9 g of pure isolie perchloride. The isolie perchloride

86

(1.8 g) was dissolved in 50 mL of water, and this solution was basified with aqueous NH3 to a

87

pH of 9-10. The solution was extracted with CHCl3 to give 1.3 g of the free isolie. 1H,

88

NMR and LC-ESIMS data were obtained to support the identification of isolie (Supporting

89

Information, Supplementary Figure 1). The isolie were analyzed by high-performance liquid

90

chromatography (HPLC) on a Waters 1525 HPLC instrument by a single injection of 20 µL

91

with an analytical C18 HPLC column (Betasil 150 × 4.6 mm, 5 µm) detected at 254 nm;

92

Gradient conditions: 90% H2O (0.1% TFA) + 10% MeCN (0.1% TFA) → 100% MeCN

93

(0.1% TFA), 20 min, 100% MeCN (0.1% TFA), 25 min; flow rate: 1.0 mL/min. The purity of

94

the isolie was above 95% as detected by HPLC and UV spectrum analysis (Figure 1A).

95 96

Cells, reagents, kits and antibodies


13

C


97

Human HL-7702 untransformed hepatocytes and murine H22 ascitic hepatoma cells were

98

supplied by the China Center for Type Culture Collection. HepG2 and Hep3B human HCC

99

cells were from the American Type Culture Collection. Huh-7 HCC cells, Panc-1 pancreatic

100

carcinoma cells and HT-29 colon carcinoma cells were got from the Cell Bank of Shanghai

101

Institute of Biological Sciences. Primary mouse hepatocytes were separated and cultured as

102

described previously.13 Dulbecco’s Modified Eagle Medium (DMEM) was got from Hyclone

103

(Logan, UT, USA). Fetal calf serum (FCS), penicillin and streptomycin for cell culture and

104

Trizol reagents for RNA extraction were purchased from Invitrogen (Carlsbad, CA, USA).

105

FuGENE reagents for cell transfection were purchased from Roche (Penzberg, Germany).

106

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliunbromide (MTT) and propidium iodine

107

were purchased from Wolsen Biotechnology (Xi’an, Shanxi Province, PR China). Caspase-3

108

activity assay kits, BCA protein quantification kits and JC-1 which is a fluorescent probe for

109

mitochondria membrane potential were purchased from Beyotime Biotechnology (Nantong,

110

Jiangsu Province, PR China). NF-κB-dependent luciferase reporters, pRL-TK plasmids and

111

Dual Reporter assay systems for the determination of intercellular luciferase activity were got

112

from Promega (Fitchburg, WI, USA). The pRL-TK plasmid encodes the Renilla luciferase

113

gene driven by the promoter of herpes simplex virus thymidine kinase gene and is used as an

114

internal control in luciferase activity assays. Reverse transcriptase ReverTra Ace-α were got

115

from Toyobo (Osaka, Japan). Primary antibodies against Bcl-2 (SC-492), Bcl-xL (SC-8392),

116

MMP9 (SC-6841), p65 (SC-372) and IκBα (SC-371), and secondary antibodies conjugated

117

with horseradish peroxidase were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

118

Antibodies against Ser536-phosphorylated p65 (No. 13346), myc tag (No. 2278) and β-actin

119

(No. 3700) were got from Cell Signaling Technology (Danvers, MA, USA).


Page 6 of 37

Page 7 of 37

120


Cells culture and transfection

121

Cells were maintained in DMEM containing 10% FCS, 100 U/mL penicillin and 100

122

µg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. To overexpress p65

123

phosphorylation mimics in which Ser536 is mutated into an Asp (p65-S536D), the encoding

124

gene of human p65-S536D was cloned into the vector pcDNA3.1A. Plasmids were transected

125

into HCC cells using FuGENE reagents, according to the manufacturer’s instructions.

126 127

Cell viability assay and calculation of IC50 value

128

Cells were incubated with different concentrations of the indicated compounds. After 48 h,

129

MTT cell viability assays were performed as described previously.14 The IC50 value of a

130

compound is defined as the concentration at which cell viability was reduced by 50% as

131

determined by MTT cell viability assays and calculated by using the Origin Software.

132 133

Flow cytometry analysis (FACS)

134

FACS was performed as described previously.15 Briefly, after undergoing treatment as

135

indicated in the text, the cells were trypsinized and fixed with cold 70% ethanol (stored at

136

-20 °C) for at least 2 h. Samples were then pelleted and washed with PBS containing 20 mM

137

EDTA. Intracellular RNA was removed by incubating samples with 1 mg/mL RNaseA at

138

37 °C for more than 2 h. Finally, cells were stained with 30 µg/mL propidium iodine and

139

subjected to FACS (Becton Dickinson FACSCalibur, Franklin Lakes, NJ, USA). Cells in Sub

140

G1 phase were considered to be apoptotic.

141 142



143 144 145

Caspase-3 activity assay Caspase-3 activity assay kits were used to measure caspase-3 activity in the total lysates of cultured cells or tumors tissues, according to the manufacturer’s instructions.

146 147

NF-κB luciferase reporter assay

148

Cells were cultured for 24 h in 24-well plates prior to transfection. Then, NF-κB luciferase

149

reporters and pRL-TK plasmids were co-transfected into HCC cells using FuGENE reagents.

150

After 24 h, the media was replaced and cells were incubated with various stimuli as indicated

151

in the text. The Dual Reporter assay system was used to measure luciferase activity in cell

152

lysates, according to the manufacturer’s instructions.

153 154

RNA isolation, reverse transcription and real-time polymerase chain reaction (PCR)

155

RNA was extracted from cultured HCC cells or in vivo tumors tissues by Trizol reagent,

156

according to the manufacturer’s instructions. To get cDNA from each sample, 3 µg of total

157

RNA was reverse transcribed at 42 °C for 30 min using ReverTra Ace-α. mRNA levels of

158

indicated genes were analyzed by real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase

159

(GAPDH) was used as the loading control.

160 161 162 163

Chromatin immunoprecipitation (ChIP) ChIP assay was performed as previously described.16 Relative binding of p65 to promoter regions of Bcl-2 and Bcl-xL encoding genes was measured by real-time PCR.

164 165


Page 8 of 37

Page 9 of 37

166


Assessment of mitochondria dysfunction by JC-1 staining

167

JC-1 is a fluorescent dye which forms aggregates in normal mitochondrias and emits red

168

fluorescence. However, JC-1 monomers emit green fluorescence in cells with mitochondria

169

dysfunction. Cells were seeded in 24-well plates and stimulated as indicated in the text. After

170

the treatment, the medium was discarded and replaced by 500 µL of fresh medium containing

171

5 µg/mL JC-1. After incubating for 1 h, cells were washed twice with PBS and photographed

172

at a magnification of 100 × under a fluorescence microscope.

173 174

Animals

175

Male athymic nude mice (6 weeks old) and Kunming mice, weighting between 18 and 22 g,

176

were obtained from the Center of Experimental Animals, Institute of Health and Epidemic

177

Prevention (Wuhan, PR China) and housed under standard specific pathogen-free conditions

178

with access to sterilized water and food ad libitum. All of the animal experimental procedures

179

were performed in accordance with the terms approved by the Experimental Animal Care and

180

Use Committee of South-Central University for Nationalities (Wuhan, PR China).

181 182

Huh-7 xenograft tumor growth in nude mice

183

Huh-7 cells (1 × 106) were suspended in 0.2 mL of serum-free DMEM with 50% Matrigel

184

(BD Biosciences, Bedford, MA, USA) and subcutaneously injected into the dorsal flanks of

185

nude mice. Mice were randomly divided into groups of 6 mice each. When the xenograft

186

tumor volume reached about 200 mm3, isolie was administrated at the indicated dosages once

187

daily, for 3 weeks, via intraperitoneal (i. p.) injection; normal saline (0.9% NaCl) was used as

188

the negative control. Tumor volumes were monitored every 3 days by the following formula:



189

tumor volume = length × width × width / 2. Blood samples were collected from animals

190

anesthetized with diethyl ether. All mice were then euthanized and the livers and xenograft

191

tumors were immediately resected for further analysis.

192 193

H22 transplanted tumor establishment and isolie administration

194

H22 cells (1 × 106) were subcutaneously transplanted into the dorsal flanks of Kunming

195

mice. Mice were randomly divided into groups of 10 mice each. Drug treatment began 24

196

hours later. Drugs were administrated once daily, for 10 days, via i. p. injection or gavage as

197

indicated in the text. Isolie groups received different dosages (3 and 10 mg/kg). The vehicle

198

control group received normal saline. A group of mice without tumor transplantation was also

199

included. Tumor volumes were monitored every 2 days. At last, blood samples were collected

200

under diethyl ether anesthesia. Immediately after euthanizing all animals, transplanted tumors

201

and corresponding organs were resected for further analysis. The organ index was calculated

202

using the following formula: Organ index = organ weight (g) / body weight (g) × 1000.

203 204

Blood and serum physiochemical indexes determination

205

An automatic blood cell count apparatus (Mindray, Shenzhen, Guangdong Province, PR

206

China) was used to record the amount of white blood cells (WBC), red blood cells (RBC),

207

hemoglobins (HGB) and platelets (PLT). Serum was prepared by centrifuging blood samples

208

at 4500 × g for 10 min. Serum hepatic and renal function parameters, including aspartate

209

aminotransferace (AST), alanine aminotransferace (ALT), blood urea nitrogen (BUN), uric

210

acid (UA) and creatinine (CRE), were measured by an automatic biochemical analyzer

211

(Sysmex, Kobe, Japan) according to the manufacturer’s instructions.


Page 10 of 37

Page 11 of 37

212


Statistical analysis

213

Data shown in this study are representatives or statistics (mean value ± standard deviation)

214

of the results from at least three independent experiments or all mice of each group. One-way

215

analysis of variance (ANOVA) was performed to determine the statistical significance. If the

216

treatment involved more than one concentration or dosage, Dunnett’s t tests were further

217

conducted to determine the statistical significance of each concentration/dosage group

218

compared to the negative control. A p value of < 0.05 was considered to be statistically

219

significant and indicated by “*”. A p value of < 0.01 was considered to be statistically very

220

significant and indicated by “**”.

221 222 223

More detailed materials and methods are provided as Supplementary Materials and Methods in Supporting Information.

224 225

RESULTS

226

Isolie induced HCC cell apoptosis in vitro

227

To assess the ability of isolie to provoke HCC cell apoptosis, human HepG2 and Huh-7

228

HCC cells were treated with 3 and 10 µg/mL isolie. After 48 h, ratios of Sub-G1 DNA in the

229

cell populations were quantified by FACS. The result showed that 48-hour isolie treatment

230

dose-dependently increased the ratio of Sub-G1 DNA (Figure 1B, left and middle). In

231

addition, caspase-3 activity was dose-dependently elevated in HCC cells by isolie (Figure 1B,

232

right). Similarly, as shown by FACS and caspase-3 activity assay, isolie time-dependently

233

triggered HepG2 and Huh-7 cell apoptosis (Figure 1C). Moreover, isolie dose-dependently

234

induced murine H22 ascitic hepatoma cell apoptosis (Figure 1D). These data demonstrated



235

that isolie is capable of provoking HCC cell apoptosis in vitro.

236 237

Isolie suppressed NF-κB activity in HCC cells

238

To understand the molecular mechanisms underlying isolie-mediated HCC cell apoptosis,

239

we firstly evaluated the impacts of isolie on an array of signaling pathways that regulate cell

240

apoptosis including p53, STAT3, Foxo3a and NF-κB using corresponding luciferase reporter

241

assays. Isolie showed few effects on the activity of p53, STAT3 or Foxo3a (data not shown),

242

but substantially decreased the basal level of NF-κB activity in both HepG2 and Huh-7 HCC

243

cells (Figure 2A). Consistently, mRNA and protein levels of typical NF-κB target genes, such

244

as Bcl-2, Bcl-xL and MMP9, in HCC cells were also downregulated by isolie (Figure 2B and

245

C). ChIP assay confirmed that isolie suppressed the interaction between NF-κB p65 subunit

246

and promoter regions of Bcl-2 and Bcl-xL encoding genes (Figure 2D). In HepG2 and Huh-7

247

cells without isolie treatment, mitochondrias were intact and emitted red fluorescence after

248

JC-1 staining. Isolie reduced the red fluorescence signal emitted by JC-1, but elevated the

249

green fluorescence signal, indicating that isolie induced mitochondria dysfunction in HCC

250

cells (Figure 2E). In murine H22 HCC cells, isolie dose-dependently inhibited transcription

251

and translation of NF-κB responsive genes (Figure 2F and G). Interactions between p65

252

proteins and promoter regions of Bcl-2 and Bcl-xL were also reduced by isolie (Figure 2H).

253

These data suggested that isolie suppresses NF-κB activity in HCC cells.

254 255

Isolie provoked dephosphorylation of p65 protein at Ser536

256

Degradation of IκBα protein is an important molecular event that activates NF-κB signaling.

257

However, IκBα remained unchanged in response to isolie (Figure 3A). Phosphorylation of


Page 12 of 37

Page 13 of 37


258

NF-κB p65 subunit at Ser536 plays important roles in promoting NF-κB-dependent gene

259

transcription. Isolie dose-dependently suppressed p65 Ser536 phosphorylation in HCC cells

260

(Figure 3A). To confirm the role of p65 dephosphorylation at Ser536 in isolie-induced HCC

261

cell apoptosis, plasmids encoding myc-tagged p65-S536D which is a p65 phosphorylation

262

mimic at Ser536 were transfected into HCC cells (Figure 3B). Overexpressing p65-S536D

263

dramatically alleviated isolie-induced mitochondria dysfunction (Figure 3C). Accordingly,

264

isolie-induced HCC cell apoptosis was also considerably abrogated (Figure 3D and E). These

265

findings suggested that p65 dephosphorylation at Ser536 is a critical molecular event

266

accounting for isolie-induced NF-κB inhibition and apoptosis in HCC cells.

267 268

Isolie suppressed Huh-7 xenograft tumor growth in nude mice

269

To explore whether the pro-apoptotic activity of isolie on HCC cells lines has any potential

270

clinical implications, we evaluated the in vivo impacts of isolie on the proliferation of Huh-7

271

HCC xenograft tumors. Isolie substantially attenuated the proliferation of xenograft tumors as

272

compared to vehicle (Figure 4A). Isolie-induced HCC cell apoptosis in vivo was verified by

273

caspase-3 activity assay (Figure 4B). To assess side impacts of isolie on normal hepatocytes

274

of tumor-bearing mice, its effects on host liver indexes and serum makers of liver function

275

including ALT and AST were tested immediately at the end of the treatment. There was no

276

significant change in any of these parameters in response to i. p. injection of isolie (Figure

277

4C). To correlate the tumor-suppressing activity in vivo with mechanisms of action identified

278

in vitro, effects of isolie on p65 phosphorylation and NF-κB activity in Huh-7 xenograft

279

tumors were detected. Isolie dose-dependently decreased p65 Ser536 phosphorylation (Figure

280

4E, left and middle). Moreover, mRNA and protein levels of NF-κB responsive genes were



281

dose-dependently declined in xenograft tumors as shown by real-time PCR (Figure 4D) and

282

immunoblotting (Figure 4E, left and right). These data suggested that in Huh-7 xenograft

283

HCC tumors, isolie provokes p65 dephosphorylation at Ser536 that reduces NF-κB activity

284

and leads to cell apoptosis. Toxic impacts of isolie on normal livers were undetectable.

285 286

Effects of isolie on transplanted H22 HCC cell growth in Kunming mice

287

To explore the anti-HCC activity of isolie in hosts with intact immune systems that is closer

288

to clinical situations, effects of isolie on the proliferation of H22 HCC cells subcutaneously

289

transplanted into the dorsal flanks of Kunming mice were evaluated. Two routes of drug

290

administration, including i. p. injection and gavage, were both tested in this model. As shown

291

in Figure 5A and B, isolie given by both routes dose-dependently reduced the growth of H22

292

transplanted tumors and elevated caspase-3 activity in tumor tissues. Additionally, gavage of

293

isolie dose-dependently reduced constitutive phosphorylation of p65 at Ser536 in transplanted

294

tumor tissues (Figure 5D, left and middle). There were also dramatic decreases in mRNA and

295

protein levels of NF-κB target genes (Figure 5C and D, left and right). To understand isolie

296

uptake by mice, the concentration of isolie in the blood was quantified by ultra-performance

297

liquid chromatography tandem mass spectrometry (UPLC-MS/MS). After i. p. injection of

298

isolie at the dosage of 10 mg/kg, the maximum absorption peak of isolie appeared at 15 min

299

with the concentration of 1356.3 ng/mL in plasma. The concentration of isolie in plasma

300

declined to 469.1 ng/mL at 30 min, and further to 95.5 ng/mL at 2 h (Supporting Information,

301

Supplementary Figure 2). These data provided more evidences to support the capacity of

302

isolie to inhibit NF-κB signaling and induce apoptosis in HCC cells in vivo.

303


Page 14 of 37

Page 15 of 37

304


Effects of isolie on tumor-bearing mice

305

Finally, we evaluated toxic impacts of i. p. injection of isolie on Kunming mice. We found

306

that i. p. injection of isolie (10 mg/kg) showed few effects on body weights of tumor-bearing

307

mice (data not shown). Moreover, amounts of WBC, RBC, HGB and PLT were comparable

308

among all experimental groups (Table 2). In addition, effects of isolie on serum levels of liver

309

function parameters including ALT and AST were unobvious. BUN, UA, and CRE are serum

310

renal function indicators. These parameters remained almost unchanged after i. p. injection of

311

isolie (Table 3). Moreover, isolie showed little influence on indexes of thymus, spleens, and

312

livers of tumor-bearing mice (Table 4). These data suggested that toxic effects of isolie at the

313

dosage of 10 mg/kg on vital organs of tumor-bearing mice are undetectable.

314 315 316

DISSCUSSION In this study, we prepared isolie from embryos of N. nucifera. The 1H,

13

C NMR and

317

LC-ESIMS data of isolie we isolated are as followed. 1H NMR data (CDCl3, 500 MHz): δH

318

3.65 (1H, m, H-1), 3.73 (1H, m, H-1'), 2.46 (1H, s, 2-NCH3), 2.56 (1H, s, 2'-NCH3), 3.12 (1H,

319

m, H1-3), 2.76 (1H, m, H1-3), 3.18 (1H, m, H1-3'), 3.00 (1H, m, H1-3'), 2.73 (2H, m, H-4),

320

2.74 (1H, m, H1-4'), 2.72 (1H, m, H1-4'), 6.65 (1H, s, H-5), 6.48 (1H, s, H-5'), 3.80 (3H, s,

321

6-OCH3), 3.82 (3H, s, 6'-OCH3), 6.25 (1H, s, H-8), 6.29 (1H, s, H-8'), 2.89 (1H, dd, J = 12.0,

322

8.7 Hz, H1-9), 2.81 (1H, J = 12.0, 5.1 Hz, H2-9), 3.12 (1H, m, H1-9'), 2.72 (1H, J = 13.8, 6.9

323

Hz, H2-9'), 6.90 (1H, d, J = 8.6 Hz, H-11, H-15), 6.43 (1H, d, J =1.8 Hz, H-15'), 6.70 (1H, d, J

324

=8.6 Hz, H-12, H-14), 3.73 (3H, s, 13-OCH3), 6.83 (1H, d, J =8.2 Hz, H-14'), 6.76 (1H, 1H,

325

dd, J =8.2, 1.8 Hz, H-11'). 13C NMR data (CDCl3, 125 MHz): δ 64.3 (C-1), 64.5 (C-1'), 42.3

326

(2-NCH3, 2'-NCH3), 47.1 (C-3), 46.6 (C-3'), 25.7 (C-4), 24.9 (C-4'), 130.1 (C-4a), 130.5



327

(C-4a'), 112.4 (C-5), 110.6 (C-5'), 149.2 (C-6), 145.4 (C-6'), 55.8 (6-OCH3), 56.0 (6'-OCH3),

328

142.9 (C-7), 143.7 (C-7'), 120.0 (C-8), 113.9 (C-8'), 130.1 (C-8a), 125.4 (C-8a'), 40.4 (C-9),

329

39.1 (C-9'), 130.8 (C-10), 131.0 (C-10'), 130.5 (C-11, C-15), 125.4 (C-11'), 113.6 (C-12,

330

C-14), 144.5 (C-12'), 157.9 (C-13), 145.4 (C-13'), 55.2 (13-OCH3), 115.5 (C-14'), 119.1

331

(C-15'). ESI-MS: m/z 611 [M + H]+. Compared with the spectroscopic data of isolie reported

332

in the previous literature,17 this compound we purified from embryos of N. nucifera could be

333

unambiguously determined as isolie. Although isolie has been recognized for decades, it still

334

remains poorly understood whether it possesses any pro-apoptotic activity against HCC cells.

335

Here, we showed that isolie time- and dose-dependently induced apoptosis in HepG2, Huh-7

336

and H22 HCC cells in vitro. Additionally, isolie also displayed strong cytotoxic effects on

337

HT-29 colon carcinoma cells and Panc-1 pancreatic carcinoma cells (Supporting Information,

338

Supplementary Figure 3). These data suggested that isolie is able to inhibit other cancer cells

339

such as colon and pancreatic cancer cells.

340

On the basis of its favorable in vitro pro-apoptotic activity against HCC cells, effects of

341

isolie on Huh-7 xenograft tumors in nude mice were assessed. It was revealed that i. p.

342

injection of isolie dramatically decreased growth of xenografted Hun-7 tumors. Caspase-3

343

activity assay further demonstrated that isolie has the capacity of provoking apoptosis in

344

xenografted HCC tissues in vivo. Similarly, i. p. injection of isolie inhibited transplanted H22

345

tumor growth and induced H22 HCC cell apoptosis in Kunming mice. Gavage is a safer and

346

more convenient route than i. p. injection. We thus also tested the anti-HCC activity of isolie

347

given by gavage. Excitingly, isolie gavage achieved a similar tumor-suppressing effect as

348

compared to that of i. p. injection, implying a possibility that this compound is stable in the

349

digestive tract of animals and can be efficiently absorbed into the circulatory system.


Page 16 of 37

Page 17 of 37


350

An important part of cancer chemotherapy is that the agent used should induce malignant

351

cell death with low toxic effects on their normal counterparts. Cisplatin is a conventional

352

chemotherapeutic anticancer drug with a high tumor-suppressing activity. However, it exhibits

353

severe hepatotoxicity and nephrotoxicity in patients, leading to a very poor prognosis.18

354

Sorafenib is one of few targeted therapeutic reagents for treating advanced HCC approved by

355

the US Food and Drug Administration. Unfortunately, sorafenib has also been shown to exert

356

side effects on some vital organs including the liver itself.19, 20 In this study, isolie exerted its

357

in vivo anti-HCC effects at the dosage of 10 mg/kg. At this dosage, i. p. injection of isolie

358

showed few changes in liver indexes and serum parameters of liver function in nude mice.

359

Toxic effects of i. p. injection of isolie at 10 mg/kg on Kunming mice were unobvious as well.

360

Consistently, our unpublished data showed that the Median Lethal Dosage of isolie given by i.

361

p. injection is greater than 1.2 g/kg for mice. These observations can be supported by the fact

362

that the plant N. nucifera is widely consumed as food. Pervious studies showed that the mass

363

fraction of isolie in the crude extract of embryos of N. nucifera was 45.7 mg/g. In their report,

364

the yield of the crude extract from embryos of N. nucifera was 593 g / 10 kg = 5.93%.21 The

365

mass fraction of isolie in the raw plant material was thereby 2.71 mg/g. Hence, the dosage of

366

isolie (10 mg/kg) in our in vivo experiment should be equivalent to an intake of about 3.69

367

g/kg embryos of N. nucifera for mice. According to the notion of animal surface area, this

368

dosage is equivalent to 0.3 g/kg for human.22 Thus, a person weighting 65 kg would need to

369

consume about 20 g of embryos of N. nucifera every day, which is in fact a feasible dosage.

370

Among three alkaloids including liensinine, isolie and neferine, isolie exhibited the most

371

prominent cytotoxic activity on HCC cells. Cytotoxic activity of neferine on HCC cells was

372

less potent than that of isolie, but stronger than that of liensinine. Similar to our observations,



373

a very recent study reported that, among these three alkaloids, cytotoxic activity of isolie on

374

MDA-MB-231 breast cancer cells was the most potent and liensinine was the least potent.23

375

Based on these data, structure-activity relationships of these three alkaloids can be summed

376

up as follows (Positions R1 and R2 are indicated in Figure 1A). Replacing the hydrogen atom

377

at the R2 position by a methyl group is conducive to enhancing the cytotoxic potency of the

378

compound. But the same substitution at the R1 position can potentially decline its cytotoxic

379

potency. Mechanistically, neferine-mediated malignant cell death is associated with the

380

inhibition of Akt signaling,24,

381

through DNM1L-mediated mitochondrial fission.26 Here, we reported that suppression of

382

NF-κB signaling is the key molecular event mediating isolie-induced HCC cell apoptosis.

25

while liensinine sensitizes cancer cells to chemotherapy

383

Epidemiological studies revealed that viral infection and steatohepatitis are significant risk

384

factors of HCC. Both of them have the potential to activate hepatic NF-κB, suggesting the

385

important role of persistently activated NF-κB in hepatocarcinogenesis.27, 28 At molecular

386

level, activation of NF-κB has the potential to promote transcription of genes antagonizing

387

apoptosis such as Bcl-2 and Bcl-xL as well as enzymes degrading extracellular matrix such as

388

MMP9.29, 30 These NF-κB-responsive genes are important contributors to the survival and

389

metastasis of HCC. Our current study clearly displayed that in HCC cells, isolie dramatically

390

declined their transcription and translation both in vitro and in vivo. These data suggested that

391

isolie inhibited NF-κB in HCC cells. Similarly, previous studies reported that an array of

392

reagent with the capacity of suppressing NF-κB signaling induced HCC cell apoptosis.31-34

393

These findings not only supported the feasibility of considering NF-κB as a potential target in

394

HCC chemotherapy, but also implied a possible participation of hyperactivated NF-κB for

395

maintaining HCC cell survival.


Page 18 of 37

Page 19 of 37


396

Declined p65 phosphorylation at Ser536 was implicated in isolie-mediated inhibition of

397

NF-κB, which could be supported by lines of evidences in this study. First, isolie suppressed

398

p65 phosphorylation at Ser536 both in vitro and in vivo, and p65 dephosphorylation at Ser536

399

was associated with HCC cell apoptosis. Second, transfection of p65 Ser536 phosphorylation

400

mimics abrogated isolie-induced mitochondria injury and apoptosis in HCC cells. It has been

401

reported by previous studies that p65 phosphorylation at Ser536 plays critical roles in the

402

proliferation and invasion of cancer cells of different origins.35-37 In addition, aberrant p65

403

phosphorylation at Ser536 has been detected in some other malignant tissues dissected from

404

human cancers and correlates with cancer progression.38-41 A series of kinases are involved in

405

the phosphorylation of p65 protein at Ser536. For example, activation of kinases, such as Akt

406

and IKK, is positively associated with elevated p65 protein phosphorylation at Ser536.42-44

407

Dysregulation of Akt and IKK are increasingly implicated in the pathogenesis of HCC.45, 46 In

408

addition, Akt- and IKK-related signaling pathways have been considered as potential targets

409

for the clinical management of HCC.47, 48 It is thus reasonable to speculate that the possible

410

interaction between isolie and these pathways in HCC cells plays roles in isolie-mediated

411

dephosphorylation of p65 at Ser536.

412

For a brief summary, in this study, pro-apoptotic effects of isolie on HCC cells were tested

413

and possible molecular mechanisms underlying isolie-induced HCC cell apoptosis were

414

explored. It was unveiled that isolie dose- and time-dependently provoked HepG2, Huh-7 and

415

H22 HCC cell apoptosis. Mechanistically, isolie gave rise to a substantial decrease in NF-κB

416

activity in HCC cells. Isolie dramatically declined constitutive phosphorylation of NF-κB p65

417

subunit at Ser536. Overexpressing p65 Ser536 phosphorylation mimics abrogated HCC cell

418

apoptosis induced by isolie. Moreover, i. p. injection of isolie inhibited the growth of Huh-7



419

xenografts in nude mice. In Huh-7 xenografts, isolie triggered p65 Ser536 dephosphorylation

420

and inhibited NF-κB activity. In addition, isolie given by both i. p. injection and gavage had

421

the capacity of diminishing in vivo proliferation of transplanted H22 cells in Kunming mice.

422

Toxic effects of isolie on tumor-bearing mice were undetectable. Our findings provided strong

423

evidence for the potential anti-HCC capacity of isolie. Suppression of NF-κB via p65

424

dephosphorylation at Ser536 is implicated in isolie-induced HCC cell apoptosis.

425 426

ASSOCIATED CONTENT

427

Supporting Information

428

Supporting Information associated with this article includes Supplementary Materials and

429

Methods, Supplementary Figure 1, 2 and 3 and their captions. This material is available free

430

of charge via the Internet at http://pbs.acs.org.

431 432

AUTHOR INFORMATION

433

Corresponding Author

434

Tel./Fax: + 86 27 6784 1196. E-mail: [email protected] (Xinzhou Yang)

435

Funding

436 437 438

This work is supported by the Natural Science Foundation of China (31301147, 81573561). Notes The authors declare no conflicts of interest.

439 440 441

ABBREVIATIONS USED Isolie, Isoliensinine; HCC, hepatocellular carcinoma; NF-κB, nuclear factor κB


Page 20 of 37

Page 21 of 37


442

REFERENCES

443

(1) Siegel, R.; Naishadham, D.; Jemal, A. Cancer statistics, 2012. CA Cancer J. Clin. 2012,

444

62, 10-29.

445

(2) Verslype, C.; Libbrecht, L. The multidisciplinary management of gastrointestinal cancer.

446

The diagnostic and therapeutic approach for primary solid liver tumours in adults. Best Pract.

447

Res. Clin. Gastroenterol. 2007, 21, 983-996.

448

(3) Verstrepen, L.; Beyaert, R. Receptor proximal kinases in NF-kappaB signaling as

449

potential therapeutic targets in cancer and inflammation. Biochem. Pharmacol. 2014, 92,

450

519-529.

451

(4) Glauert, H. P. Role of NF-kappaB in hepatocarcinogenesis and its potential inhibition by

452

dietary antioxidants. Curr. Cancer Drug Targets 2012, 12, 1160-1172.

453

(5) Oeckinghaus, A.; Ghosh, S. The NF-kappaB family of transcription factors and its

454

regulation. Cold Spring Harb. Perspect. Biol. 2009, 1, a000034.

455

(6) Shu, G.; Tang, Y.; Zhou, Y.; Wang, C.; Song, J. G. Zac1 is a histone acetylation-regulated

456

NF-kappaB suppressor that mediates histone deacetylase inhibitor-induced apoptosis. Cell

457

Death Differ. 2011, 18, 1825-1135.

458

(7) Chen, G.; Han, K.; Xu, X.; Du, X.; Zhang, Z.; Tang, J.; Shi, M.; Wang, M.; Li, J.; Cao, B.;

459

Mao, X. An anti-leishmanial thiadiazine agent induces multiple myeloma cell apoptosis by

460

suppressing the nuclear factor kappaB signalling pathway. Br. J. Cancer 2014, 110, 63-70.

461

(8) Xu, X. X.; Jiang, H. R.; Li, H. B.; Zhang, T. N.; Zhou, Q.; Liu, N. Apoptosis of stomach

462

cancer cell SGC-7901 and regulation of Akt signaling way induced by bovine lactoferrin. J.

463

Dairy Sci. 2010, 93, 2344-2350.

464

(9) Wu, C. H.; Hong, B. H.; Ho, C. T.; Yen, G. C. Targeting cancer stem cells in breast cancer:



465

potential anticancer properties of 6-shogaol and pterostilbene. J. Agric. Food Chem. 2015, 63,

466

2432-2441.

467

(10) Yuan, L.; Wei, S.; Wang, J.; Liu, X. Isoorientin induces apoptosis and autophagy

468

simultaneously by reactive oxygen species (ROS)-related p53, PI3K/Akt, JNK, and p38

469

signaling pathways in HepG2 cancer cells. J. Agric. Food Chem. 2014, 62, 5390-5400.

470

(11) Kim, S. P.; Nam, S. H.; Friedman, M. The tomato glycoalkaloid α-tomatine induces

471

caspase-independent cell death in mouse colon cancer CT-26 cells and transplanted tumors in

472

mice. J. Agric. Food Chem. 2015, 63, 1142-1150.

473

(12) Newman, D. J.; Cragg, G. M. Natural products as sources of new drugs over the 30 years

474

from 1981 to 2010. J. Nat. Prod. 2012, 75, 311-335.

475

(13) Pan, X.; Wang, X.; Lei, W.; Min, L.; Yang, Y.; Song, J. Nitric oxide suppresses

476

transforming growth factor-beta1-induced epithelial-to-mesenchymal transition and apoptosis

477

in mouse hepatocytes. Hepatology 2009, 50, 1577-1587.

478

(14) Liu, C.; Lin, J.-J.; Yang, Z.-Y.; Tsai, C.-C.; Hsu, J.-L.; Wu, Y.-J. Proteomic Study Reveals

479

a Co-occurrence of Gallic Acid-Induced Apoptosis and Glycolysis in B16F10 Melanoma

480

Cells. J. Agric. Food Chem. 2014, 62, 11672-11680.

481

(15) Shu, G.; Yang, J.; Zhao, W.; Xu, C.; Hong, Z.; Mei, Z.; Yang, X. Kurarinol induces

482

hepatocellular carcinoma cell apoptosis through suppressing cellular signal transducer and

483

activator of transcription 3 signaling. Toxicol. Appl. Pharmacol. 2014, 281, 157-165.

484

(16) Shu, G.; Yang, T.; Wang, C.; Su, H.; Xiang, M. Gastrodin stimulates anticancer immune

485

response and represses transplanted H22 hepatic ascitic tumor cell growth: Involvement of

486

NF-kappaB signaling activation in CD4+ T cells. Toxicol. Appl. Pharmacol. 2013, 269,

487

270-279.


Page 22 of 37

Page 23 of 37


488

(17) Yang, J.; Zhou, K. NMR spectroscopic analysis of neferine and isoliensinine. Magn.

489

Reson. Chem. 2004, 42, 994-997.

490

(18) Cayir, K.; Karadeniz, A.; Simsek, N.; Yildirim, S.; Karakus, E.; Kara, A.; Akkoyun, H. T.;

491

Sengul, E. Pomegranate seed extract attenuates chemotherapy-induced acute nephrotoxicity

492

and hepatotoxicity in rats. J. Med. Food. 2011, 14, 1254-1262.

493

(19) Llovet, J. M.; Ricci, S.; Mazzaferro, V.; Hilgard, P.; Gane, E.; Blanc, J. F.; de Oliveira, A.

494

C.; Santoro, A.; Raoul, J. L.; Forner, A.; Schwartz, M.; Porta, C.; Zeuzem, S.; Bolondi, L.;

495

Greten, T. F.; Galle, P. R.; Seitz, J. F.; Borbath, I.; Haussinger, D.; Giannaris, T.; Shan, M.;

496

Moscovici, M.; Voliotis, D.; Bruix, J. Sorafenib in advanced hepatocellular carcinoma. N.

497

Engl. J. Med. 2008, 359, 378-390.

498

(20) Shah, R. R.; Morganroth, J.; Shah, D. R. Hepatotoxicity of tyrosine kinase inhibitors:

499

clinical and regulatory perspectives. Drug Saf. 2013, 36, 491-503.

500

(21) Chen, Y.; Fan, G.; Wu, H.; Wu, Y.; Mitchell, A. Separation, identification and rapid

501

determination of liensine, isoliensinine and neferine from embryo of the seed of Nelumbo

502

nucifera GAERTN. by liquid chromatography coupled to diode array detector and tandem

503

mass spectrometry. J. Pharm. Biomed. Anal. 2007, 43, 99-104.

504

(22) Reagan-Shaw, S.; Nihal, M.; Ahmad, N. Dose translation from animal to human studies

505

revisited. FASEB J. 2008, 22, 659-661.

506

(23) Zhang, X.; Wang, X.; Wu, T.; Li, B.; Liu, T.; Wang, R.; Liu, Q.; Liu, Z.; Gong, Y.; Shao,

507

C. Isoliensinine induces apoptosis in triple-negative human breast cancer cells through ROS

508

generation and p38 MAPK/JNK activation. Sci. Rep. 2015, 5, 12579.

509

(24) Poornima, P.; Quency, R. S.; Padma, V. V. Neferine induces reactive oxygen species

510

mediated intrinsic pathway of apoptosis in HepG2 cells. Food Chem. 2013, 136, 659-667.



511

(25) Poornima, P.; Weng, C. F.; Padma, V. V. Neferine from Nelumbo nucifera induces

512

autophagy through the inhibition of PI3K/Akt/mTOR pathway and ROS hyper generation in

513

A549 cells. Food Chem. 2013, 141, 3598-3605.

514

(26) Zhou, J.; Li, G.; Zheng, Y.; Shen, H. M.; Hu, X.; Ming, Q. L.; Huang, C.; Li, P.; Gao, N.

515

A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to

516

chemotherapy through DNM1L-mediated mitochondrial fission. Autophagy 2015, 11,

517

1259-1279.

518

(27) Kao, J. H.; Chen, P. J.; Chen, D. S. Recent advances in the research of hepatitis B

519

virus-related hepatocellular carcinoma: epidemiologic and molecular biological aspects. Adv.

520

Cancer Res. 2010, 108, 21-72.

521

(28) Park, E. J.; Lee, J. H.; Yu, G. Y.; He, G.; Ali, S. R.; Holzer, R. G.; Osterreicher, C. H.;

522

Takahashi, H.; Karin, M. Dietary and genetic obesity promote liver inflammation and

523

tumorigenesis by enhancing IL-6 and TNF expression. Cell 2010, 140, 197-208.

524

(29) Ni, H.; Zhao, W.; Kong, X.; Li, H.; Ouyang, J. NF-kappa B modulation is involved in

525

celastrol induced human multiple myeloma cell apoptosis. PLoS One 2014, 9, e95846.

526

(30) Chen, Y. Y.; Lu, H. F.; Hsu, S. C.; Kuo, C. L.; Chang, S. J.; Lin, J. J.; Wu, P. P.; Liu, J. Y.;

527

Lee, C. H.; Chung, J. G. Bufalin inhibits migration and invasion in human hepatocellular

528

carcinoma SK‐Hep1 cells through the inhibitions of NF‐kB and matrix metalloproteinase

529

‐2/‐9‐signaling pathways. Environ. Toxicol. 2015, 30, 74-82.

530

(31) Zhao, B.; Ma, Y.; Xu, Z.; Wang, J.; Wang, F.; Wang, D.; Pan, S.; Wu, Y.; Pan, H.; Xu, D.;

531

Liu, L.; Jiang, H. Hydroxytyrosol, a natural molecule from olive oil, suppresses the growth of

532

human hepatocellular carcinoma cells via inactivating AKT and nuclear factor-kappa B

533

pathways. Cancer Lett. 2014, 347, 79-87.


Page 24 of 37

Page 25 of 37


534

(32) Xiang, M.; Su, H.; Hu, Y.; Yang, T.; Shu, G. Chemical composition of total flavonoids

535

from Salvia chinensia Benth and their pro-apoptotic effect on hepatocellular carcinoma cells:

536

potential roles of suppressing cellular NF-kappaB signaling. Food Chem. Toxicol. 2013, 62,

537

420-426.

538

(33) Yu, W.; Gu, K.; Yu, Z.; Yuan, D.; He, M.; Ma, N.; Lai, S.; Zhao, J.; Ren, Z.; Zhang, X.;

539

Shao, C.; Jiang, G. L. Sorafenib potentiates irradiation effect in hepatocellular carcinoma in

540

vitro and in vivo. Cancer Lett. 2013, 329, 109-117.

541

(34) Wong, L. L.; Lam, I. P.; Wong, T. Y.; Lai, W. L.; Liu, H. F.; Yeung, L. L.; Ching, Y. P.

542

IPA-3 inhibits the growth of liver cancer cells by suppressing PAK1 and NF-kappaB

543

activation. PLoS One 2013, 8, e68843.

544

(35) Zhang, L.; Shao, L.; Creighton, C. J.; Zhang, Y.; Xin, L.; Ittmann, M.; Wang, J. Function

545

of phosphorylation of NF-kB p65 ser536 in prostate cancer oncogenesis. Oncotarget 2015, 6,

546

6281-6294.

547

(36) Zou, Z.; Zeng, F.; Xu, W.; Wang, C.; Ke, Z.; Wang, Q. J.; Deng, F. PKD2 and PKD3

548

promote prostate cancer cell invasion by modulating NF-kappaB- and HDAC1-mediated

549

expression and activation of uPA. J. Cell Sci. 2012, 125, 4800-4811.

550

(37)Yang, Z. Y.; Qu, Y.; Zhang, Q.; Wei, M.; Liu, C. X.; Chen, X. H.; Yan, M.; Zhu, Z. G.; Liu,

551

B. Y.; Chen, G. Q.; Wu, Y. L.; Gu, Q. L. Knockdown of metallopanstimulin-1 inhibits

552

NF-kappaB signaling at different levels: the role of apoptosis induction of gastric cancer cells.

553

Int. J. Cancer 2012, 130, 2761-2770.

554

(38) MacKenzie, L.; McCall, P.; Hatziieremia, S.; Catlow, J.; Adams, C.; McArdle, P.;

555

Seywright, M.; Tanahill, C.; Paul, A.; Underwood, M.; Mackay, S.; Plevin, R.; Edwards, J.

556

Nuclear factor kappaB predicts poor outcome in patients with hormone-naive prostate cancer



557

with high nuclear androgen receptor. Hum. Pathol. 2012, 43, 1491-1500.

558

(39) Lewander, A.; Gao, J.; Carstensen, J.; Arbman, G.; Zhang, H.; Sun, X. F. NF-kappaB p65

559

phosphorylated at serine-536 is an independent prognostic factor in Swedish colorectal cancer

560

patients. Int. J. Colorectal Dis. 2012, 27, 447-452.

561

(40) Wang, Y. P.; Liu, I. J.; Chiang, C. P.; Wu, H. C. Astrocyte elevated gene-1 is associated

562

with metastasis in head and neck squamous cell carcinoma through p65 phosphorylation and

563

upregulation of MMP1. Mol. Cancer 2013, 12, 109.

564

(41) McCall, P.; Bennett, L.; Ahmad, I.; Mackenzie, L. M.; Forbes, I. W.; Leung, H. Y.;

565

Sansom, O. J.; Orange, C.; Seywright, M.; Underwood, M. A.; Edwards, J. NFkappaB

566

signalling is upregulated in a subset of castrate-resistant prostate cancer patients and

567

correlates with disease progression. Br. J. Cancer 2012, 107, 1554-1563.

568

(42) Bhattacharyya, S.; Sen, P.; Wallet, M.; Long, B.; Baldwin, A. S., Jr.; Tisch, R.

569

Immunoregulation of dendritic cells by IL-10 is mediated through suppression of the

570

PI3K/Akt pathway and of IkappaB kinase activity. Blood 2004, 104, 1100-1109.

571

(43) Adli, M.; Baldwin, A. S. IKK-i/IKKepsilon controls constitutive, cancer cell-associated

572

NF-kappaB activity via regulation of Ser-536 p65/RelA phosphorylation. J. Biol. Chem. 2006,

573

281, 26976-26984.

574

(44) Sakurai, H.; Chiba, H.; Miyoshi, H.; Sugita, T.; Toriumi, W. IkappaB kinases

575

phosphorylate NF-kappaB p65 subunit on serine 536 in the transactivation domain. J. Biol.

576

Chem. 1999, 274, 30353-30356.

577

(45) Hu, T. H.; Huang, C. C.; Lin, P. R.; Chang, H. W.; Ger, L. P.; Lin, Y. W.; Changchien, C.

578

S.; Lee, C. M.; Tai, M. H. Expression and prognostic role of tumor suppressor gene

579

PTEN/MMAC1/TEP1 in hepatocellular carcinoma. Cancer 2003, 97, 1929-1940.


Page 26 of 37

Page 27 of 37


580

(46) Jiang, R.; Xia, Y.; Li, J.; Deng, L.; Zhao, L.; Shi, J.; Wang, X.; Sun, B. High expression

581

levels of IKKalpha and IKKbeta are necessary for the malignant properties of liver cancer. Int.

582

J. Cancer 2010, 126, 1263-1274.

583

(47) Zhou, Q.; Lui, V. W.; Yeo, W. Targeting the PI3K/Akt/mTOR pathway in hepatocellular

584

carcinoma. Future Oncol. 2011, 7, 1149-1167.

585

(48) Huang, J. J.; Chu, H. X.; Jiang, Z. Y.; Zhang, X. J.; Sun, H. P.; You, Q. D. Recent

586

advances in the structure-based and ligand-based design of IKKbeta inhibitors as

587

anti-inflammation and anti-cancer agents. Curr. Med. Chem. 2014, 21, 3893-3917.

588 589

Figure captions

590

Figure 1. Isolie dose- and time-dependently provoked human and murine HCC cell apoptosis

591

in vitro. (A) Chemical structures of liensinine, isolie and neferine are shown. The purity of

592

isolie was determined by both HPLC and UV spectrum analysis. (B) HCC cells were treated

593

with indicated concentrations of isolie. After 48 h, cell apoptosis was examined by FACS (left

594

and middle, both representative FACS diagrams and statistics are shown) and caspase-3

595

activity assay (right). (C) HepG2 and Huh-7 cells were treated with 10 µg/mL isolie for

596

indicated times. Then, cell apoptosis was examined by FACS (left and middle) and caspase-3

597

activity assay (right). (D) H22 cells were incubated with indicated concentrations of isolie.

598

Cell apoptosis was examined by FACS (left and middle) and caspase-3 activity assay (right)

599

48 h later. * p < 0.05 and ** p < 0.01 versus HCC cells without isolie treatment.

600

Figure 2. Isolie inhibited NF-κB activity in HCC cells. (A) Plasmids encoding Renilla

601

luciferase and NF-κB-dependent luciferase reporter were cotransfected into HepG2 and

602

Huh-7 cells. After 24 h, these cells were treated with indicated concentrations of isolie, using



603

vehicle as the negative control. Cells were subjected to luciferase activity assay 24 h later. (B,

604

C and D) HCC cells were treated with different concentrations of isolie. After 24 h, mRNA

605

and protein levels of NF-κB target genes were determined by real-time PCR (B) and

606

immunoblotting (C, left). Density values of immunoreactive bands of Bcl-2, Bcl-xL and

607

MMP9 were normalized to those of β-actin (C, middle and right). Association between p65

608

protein and promoter regions of indicated genes was detected by ChIP (D). (E) HepG2 and

609

Huh-7 cells were treated with indicated concentrations of isolie for 24 h, and then subjected to

610

JC-1 staining assays. (F, G and H) H22 cells were treated with indicated concentrations of

611

isolie for 24 h. Then, mRNA and protein levels of Bcl-2, Bcl-xL and MMP9 were determined

612

by real-time PCR (F) and immunoblotting (G). Association between p65 and promoter

613

regions of Bcl-2 and Bcl-xL were quantified by ChIP (H). * p < 0.05 and ** p < 0.01 versus

614

HCC cells without isolie treatment.

615

Figure 3. Isolie decreased constitutive phosphorylation of p65 protein at Ser536. (A) Effects

616

of 24-hour isolie treatment on protein levels of phosphorylated p65 (Ser536), p65 and IκBα in

617

HCC cells were detected by immunoblotting (left). Density values of immunoreactive bands

618

of Ser536-phosphorylated p65 were normalized to those of total p65 (right) (B) Myc-tagged

619

p65-S536D was transfected into indicated HCC cells. After 24 h, exogenous p65-S536D

620

proteins were determined by immunoblotting using the primary antibody against myc tag. (C)

621

Cells transfected with indicated plasmids were incubated with or without 10 µg/mL isolie, and

622

subjected to JC-1 staining assays after 24 h. (D and E) Empty or p65-S536D encoding

623

plasmids were transfected into HepG2 and Huh-7 cells. After 24 h, HCC cells were incubated

624

with or without 10 µg/mL isolie. Cell apoptosis was examined 48 h later by FACS (D) and

625

caspase-3 activity assay (E). * p < 0.05 and ** p < 0.01 versus the indicated control.


Page 28 of 37

Page 29 of 37


626

Figure 4. Isolie suppressed the growth of Huh-7 xenografts in nude mice. (A) Vehicle or

627

indicated dosages of isolie were intraperitoneally injected into tumor-bearing nude mice for 3

628

weeks, and tumor growth was monitored every 3 days. (B) At the end of the study, tumor

629

lysates were prepared and subjected to caspase-3 activity assay. (C) Serum and livers were

630

collected from tumor-bearing mice. Serum activities of ALT and AST were detected, and liver

631

indexes were calculated. Values were normalized to those from the vehicle control group. (D

632

and E) At the end of isolie treatment, transcription levels of indicated genes were detected by

633

real-time PCR (D). Levels of indicated proteins were detected by immunoblotting (E, left).

634

Densitometric values of phosphorylated p65 at Ser536 were normalized to those of total p65

635

(E, middle). Densitometric values of Bcl-2, Bcl-xL and MMP9 were normalized to those of

636

β-actin (E, right). ** p < 0.01 versus tumors dissected from mice treated with saline.

637

Figure 5. Effects of isolie on transplanted H22 HCC cells in Kunming mice. (A and B)

638

Kunming mice bearing H22 tumors were treated with indicated dosages of isolie by i. p.

639

injection (left) or gavage (right) for 10 d, and tumor growth was monitored every day (A). At

640

the end of isolie treatment, tumor lysates were prepared and subjected to caspase-3 activity

641

assay (B). (C and D) Tumor-bearing mice were treated with indicated dosages of isolie by

642

gavage for 10 d. At the end of the study, mRNA levels of Bcl-2, Bcl-xL and MMP9 were

643

detected by reverse transcription real-time PCR (C). Indicated proteins were examined by

644

immunoblotting (D). Densitometric values of phosphorylated p65 at Ser536 were normalized

645

to those of total p65 (D, middle). Densitometric values of Bcl-2, Bcl-xL and MMP9 were

646

normalized to those of β-actin (D, right). ** p < 0.01 versus tumors dissected from mice

647

treated with saline.



Page 30 of 37

Tables Table 1 Cytotoxic effects of liensinine, isolie and neferine on HCC cells and untransformed hepatocytes IC50 (µg/mL) a Compound PMH b

HL-7702

Huh-7

HepG2

Hep3B

Bel-7402

liensinine

53.7

49.3

9.7

11.7

14.1

15.5

isolie

72.9

70.1

4.5

4.2

6.0

5.7

neferine

23.2

20.8

6.2

8.3

10.1

9.8

a

IC50, the concentration of a compound at which cell viability was reduced by 50%

b

PMH, primary mouse hepatocytes

Table 2 Effects of i. p. injection of isolie on WBC, RBC, HGB, and PLT in Kunming mice Dosage

Concentrations of blood cells (L -1)

Group (mg/kg)

WBC (109)

RBC (1012)

HGB (g)

PLT (109)

Non-tumor

-

6.62 ± 0.47

5.57 ± 0.73

109 ± 11

666 ± 63

vehicle

-

6.35 ± 0.55

6.48 ± 0.66

121 ± 12

680 ± 70

isolie

3

7.07 ± 0.58

5.91 ± 0.73

115 ± 12

662 ± 69

isolie

10

6.83 ± 0.62

6.61 ± 0.69

117 ± 13

692 ± 66

30

ACS Paragon Plus Environment

Page 31 of 37


Table 3 Effects of i. p. injection of isolie on serum parameters of liver and kidney functions in Kunming mice Dosage

Serum parameters of hepatic and renal functions (U/L)

Group (mg/kg)

BUN

UA

CRE

ALT

AST

non-tumor

-

6.5 ± 0.49

140 ± 8.8

45.8 ± 5.4

121 ± 13

589 ± 48

vehicle

-

6.7 ± 0.53

139 ± 9.4

44.7 ± 4.7

133 ± 17

611 ± 52

isolie

3

6.6 ± 0.55

135 ± 8.9

46.4 ± 5.1

118 ± 16

618 ± 46

isolie

10

6.6 ± 0.47

136 ± 9.5

47.1 ± 4.9

126 ± 15

577 ± 49

Table 4 Effects of i. p. injection of isolie on organ indexes of Kunming mice Organ indexes (mg/g)

Dosage Group (mg/kg)

Thymus

Spleen

Liver

non-tumor

-

2.72 ± 0.12

8.60 ± 0.40

48.2 ± 5.2

vehicle

-

2.70 ± 0.09

8.95 ± 0.35

47.9 ± 4.2

isolie

3

2.82 ± 0.11

8.90 ± 0.37

48.6 ± 4.8

isolie

10

2.84 ± 0.11

8.97 ± 0.42

48.1 ± 5.1

31



Figures

32


Page 32 of 37

Page 33 of 37


33



34


Page 34 of 37

Page 35 of 37


35



36


Page 36 of 37

Page 37 of 37


TOC graphic

37


Isoliensinine, a Bioactive Alkaloid Derived from Embryos of Nelumbo

Recommend Documents