Nutritional Available Selenocysteine Derivative Antagonizes Cisplatin

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Bioactive Constituents, Metabolites, and Functions

Nutritional Available Seleno-amino Acid Derivative Antagonizes Cisplatin-induced Nephrotoxicity Through Inhibition of ROS-mediated Signaling Pathways Xiaoling Li, Haobin Zhang, Leung Chan, Chang Liu, and Tianfeng Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01876 • Publication Date (Web): 20 May 2018 Downloaded from http://pubs.acs.org on May 20, 2018

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Journal of Agricultural and Food Chemistry

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Nutritional Available Selenocysteine Derivative Antagonizes

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Cisplatin-induced Toxicity in Renal Epithelial Cell Through

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Inhibition of ROS-mediated Signaling Pathways

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Xiaoling Liab, Haobin Zhang c, Leung Chan c, Chang Liuc, Tianfeng Chen bc*

6 7

a

Institute of Food Safety and Nutrition, Jinan University, Guangzhou, China

8

b

The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China

9 10

c

Department of Chemistry, Jinan University, Guangzhou 510632, China.

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* Corresponding authors. Department of Chemistry, Jinan University, Guangzhou

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510632, China. Tel.: +86 20 85225962.

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E-mail addresses: [email protected].

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ACS Paragon Plus Environment


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ABSTRACT:

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Discovery of nutritionally available agents that could antagonize cisplatin-induced

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nephrotoxicity is of great significance and clinical application potential.

26

3,3′-Diselenodipropionic acid (DSePA) is a seleno-amino acid derivative that exhibits

27

strong antioxidant activity. Therefore, this study aimed to examine the protective

28

effects of DSePA on cisplatin-induced renal epithelial cell as well as the molecular

29

mechanisms. The results revealed that DSePA effectively inhibited cell apoptosis

30

induced by cisplatin, through suppressing the caspases activation and PARP cleavage.

31

In addition, DSePA blocked the cisplatin-induced mitochondrial dysfunction, as

32

evidenced by loss of mitochondrial membrane potential and reduction of

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mitochondrial mass. The results of Western blot analysis showed that DSePA reversed

34

the expression level of Bcl-2 family proteins altered by cisplatin. Cisplatin-activated

35

AKT pathway was also modulated by DSePA. Moreover, these results indicate that

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DSePA could protect HK-2 cells from cisplatin-induced toxicity in renal epithelial cell

37

by inhibiting intracellular ROS-mediated apoptosis, while showed un-obvious effect

38

on its anticancer efficacy. Taken together, this study demonstrates that selenocysteine

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could be further developed as novel nutritionally available agents to antagonize

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cisplatin-induced nephrotoxicity during cancer therapy.

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KEYWORDS: Selenium, Cisplatin, 3,3′-Diselenodipropionic acid, Nephrotoxicity,

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Mitochondria

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INTRODUCTION

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Cisplatin is one of the most potent chemotherapeutic drugs, which is widely used for

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treatment of many cancers, including testicular, head and neck, ovarian, cervical,

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non-small cell lung carcinoma, and many other types of cancer1, 2. However, its

49

clinical use has been limited by serious side effects and toxicity, especially

50

nephrotoxicity. Studies have showed that, about 30% of the patients administered with

51

cisplatin developed a mild and partly reversible decline in renal function3, 4. Although

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the underlying mechanisms of cisplatin-induced nephrotoxicity are still not clear, it

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has been suggested that oxidative stress injury and apoptosis probably explain part of

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this injury. Previous studies also indicated that cytochrome P450 2E1 (CYP2E1), an

55

active producer of reactive oxygen species (ROS), can enhance cisplatin-induced

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cytotoxicity5. ROS influence the function of cells by directly affecting cell

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components, including lipids, proteins, and DNA, and destroy their structure6. Also,

58

several studies have demonstrated that antioxidants have benefit in preventing the

59

cisplatin-induced

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N-acetylcysteine, glutathione and ebselen7-12. Therefore, the search for novel

61

nutritionally available antioxidants could be a good way to discover nephroprotective

62

agents.

nephrotoxicity,

such

as

melatonin,

vitamin

C

and

E,

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The trace element selenium (Se) is an essential nutrient of fundamental importance

64

to human and animal13. A variety of Se-enriched biological products, such as garlic,

65

yeast, and Se-containing protein have been developed and commercialized as diet

66

supplements. A lot of potent organoselenium compounds, such as seleno-amino acids, 3



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ebselen, selenocyanate, selenobetaine, have been found and identified to show

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effective chemopreventive and antioxidant activities with minimal side effects14-17.

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Several mechanisms have been postulated to elucidate the function of Se, which

70

includes induction of cell apoptosis, maintenance of glutathione peroxidase activity,

71

modulation

72

3,3′-Diselenodipropionic acid (DSePA, Figure 1A), a simple, stable and water-soluble

73

derivative of selenocystine, has been synthesized and examined for antioxidant

74

activity, glutathione peroxidase (GPx) activity, and cytotoxicity18. Previously, DSePA

75

has also been reported to protect human red blood cells (RBCs) from

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free-radical-induced hemolysis19,

77

suggests a potential application in antagonism of cisplatin-induced nephrotoxicity.

of

redox

state

and

stimulation

of

the

immune

system.

20

. The strong antioxidant activity of DSePA

78

ROS is actively involved in the pathogenesis of cisplatin-induced acute kidney

79

injury. PI3K/AKT pathway is the major oxidative stress sensitive signal transduction

80

pathways in most cell types21,

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PI3K/AKT pathway lessens apoptosis and plays a critical role in the maintenance of

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renal function in cisplatin-induced acute kidney injury23. Therefore, in the present

83

study, human proximal tubular epithelial (HK-2) cells have been used as an

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experimental model to investigate the protective effects of DSePA against

85

cisplatin-induced damage, and the underlying molecular mechanisms were also

86

elucidated. The results showed that cisplatin significantly inhibited the growth of

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HK-2 cells through induction of ROS-mediated apoptosis, while DSePA could

88

effectively reverse the damage caused by cisplatin through inhibition of ROS

22

. Recent study suggests that the activation of

4


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generation, caspase activation and modulation of mitochondrial-mediated and AKT

90

pathway. Taken together, this study demonstrates that seleno-amino acid could be

91

further

92

cisplatin-induced nephrotoxicity.

developed

as

novel

nutritionally

available

agents

to

antagonize

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MATERRIALS AND METHODS

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Materials and Reagents. Cisplatin, 3,3′-Diselenodipropionic acid (DSePA),

96

propidium

97

2′,7′-dichlorofluorescein diacetate (DCF-DA) and 5-chloromethylfluorescein diacetate

98

(CMFDA ) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO)..

99

DMEM medium and fetal bovine serums (FBS) were purchased from Hyclone (Logan, US).

iodide

(PI),

Caspase-3

thiazolyl

substrate

blue

tetrazolium

(Ac-DEVD-AMC),

bromide

100

Utah,

101

(Ac-IETD-pNA) and Caspase-9(Ac-LEHD-AFC) substrate were purchased from

102

Biomol(Germany). MitoTracker Red CMXRos, 10-n-nonyl acridine 104 orange

103

(NAO) and tetramethylrhodamine methyl ester (TMRM) 105 were purchased from

104

Invitrogen, Molecular Probes (Eugene, OR, 106 USA), AKT inhibitor (LY 294002)

105

were obtained from Calbiochem (San Diego,CA). All of the antibodies used in this

106

study were purchased from Cell Signaling Technology (Beverly, MA), and

107

bicinchoninic acid kit for protein concentration measurement was purchased from

108

Beyotime (Shanghai, China). The water used for all experiments was ultrapure,

109

supplied by a Milli-Q water purification system from Millipore.

110

ABTS•+ Free Radical Scavenging Assay. ABTS•+ free radical scavenging activities 5


Caspase-8

(MTT),

substrate


111

of antioxidants were measured according to the method previously described24.

112

Briefly, 20 µL of the tested samples mixed with 200 µL of ABTS reagent with

113

absorbance of 0.40 ± 0.02 at 734 nm, and then the mixture was measured at the

114

absorbance of 734 nm in the followed 30 min.

115

Plasma Oxidation Assay. Plasma samples were obtained after centrifugation of the

116

heparinized blood from healthy volunteers at 1500 rpm for 10 min. Aliquots were

117

stored at 4 °C until used. Lipid oxidation was carried out at 37 °C by treated plasma

118

samples with 200 µM CuCl2 as oxidant for the indicated time. To evaluate the

119

protective role of the DSePA, samples were pre-incubated with DSePA at different

120

concentrations (0- 30 µM) for 15 min. The copper-induced oxidation in 40-fold

121

diluted plasma samples was monitored by recording the formation of diene at 245 nm

122

for 2 h at 37 °C.

123

Cell Culture and Drug Treatment. HK-2 (human kidney proximal tubular) and

124

HepG2 (human liver hepatocellular carcinoma) cell lines were obtained from

125

American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in

126

Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine

127

serum (FBS), 100 U/mL penicillin and 100 µg/mL of streptomycin in a humidified

128

incubator (5% CO2, 37 °C). The numbers of living or dead cells were counted under

129

microscopy by staining trypan blue solution. And then, for the drug treatments, HK-2

130

cells and HepG2were seeded at 6×104 cells/mL and cultured for 12, and then

131

pretreated with15 µM of DSePA for 12 h，after that treated with 8 µg/mL of cisplatin

132

for a period of time.

133

MTT Assay. Cell viability was determined by measuring the ability of the cells 6


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transform MTT to a purple formazan dye25, 26. HK-2 and HepG2 cells were seeded in

135

96-well microplate and cultured at 37 °C in a humidified atmosphere for 12 h, and

136

then the cells were exposed to different treatments for different periods of time. After

137

incubation, 20 µL/well of MTT solution (5 mg/mL in PBS buffer) was added and then

138

incubated for 5 h. The medium was aspirated and replaced with 150 µL/well of

139

DMSO to dissolve the formazan salt. The color intensity of the formazan solution,

140

which reflects the cell growth condition, was measured at 570 nm using a microplate

141

spectrophotometer (Moleclure Devices).

142

Detection of Intracellular GSH. Cellular GSH levels were analyzed using CMFDA

143

(Molecular Probes Ex/Em = 522 nm/595 nm) as previously described27. In brief,

144

treated cells were washed with PBS and incubated with 5 µM of CMFDA at 37 ℃

145

for 30 min according to the instructions of the manufacturer. Cytoplasmic esterases

146

convert nonfluorescent CMFDA to fluorescent 5-chloromethylfluorescein, which can

147

then reacted with the thiol group of GSH. CMF fluorescence intensity was determined

148

using a FACStar flow cytometer (Becton Dickinson). And 10,000 events per sample

149

were recorded for each experiment.

150

Flow Cytometric Analysis. Flow cytometric analysis was carried out according to

151

our previous method28-30. Briefly, after exposed to the complexes, the cells were

152

harvested by centrifugation and washed with PBS. Cells were stained with PI (1.21

153

mg/mL of Tris, 700 U/mL of RNase, 50.1 µg/mL of PI, pH 8.0) for 30 min in

154

darkness and then fixed with 70% ethanol at - 20 ◦C overnight. The analysis of DNA

155

content was accomplished by flow cytometer. Apoptotic cells with hypodiploid DNA 7



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contents were measured by quantifying the sub-G1 peak. And 10,000 events per

157

sample were recorded for each experiment.

158

Determination of Caspase Activity. After exposed to the complexes the HK-2 cells

159

were harvested. Total cellular proteins were extracted by incubating cells in lysis

160

buffer. For determination of caspase-3/8/9 activity, cell lysates were placed in 96-well

161

plates and then added specific caspase substrate (Ac-DEVD-AMC for caspase-3,

162

Ac-IETD-AMC for caspase-8 and Ac-LEHD-AMC for caspase-9, respectively).Then

163

the 96-well plates were incubated at 37 ◦C for 1 h and caspase activity was determined

164

by fluorescence intensity with appropriate excitation and emission wavelengths.

165

Measurement of ROS and O2− Generation. A fluorometric assay (DCFH-DA assay)

166

was used to determine the relative levels of ROS25, 29. Treated cells were harvested

167

washed, and then incubated with 10 µM of DCFH-DA at 37 ◦C for 30 min.

168

Intracellular ROS generation was monitored by measuring the fluorescence intensity

169

of cells with a BioTek microplate reader, with excitation and emission wavelengths

170

set at 488 and 525 nm, respectively. The superoxide anion radicals were generated by

171

the

172

2,4-iodiphenyl-3,4-nitrophenyl-5-phenyltetrazolium chloride to form formazan, a

173

colored compound which can be spectrophotometrically quantified at 300 nm. The

174

production of formazan is proportional to the level of superoxide anion radicals in the

175

tested samples.

176

Determination of ∆ψm. The ∆ψm was determined using TMRM fluorescent probe in

177

this assay and TMRM assay was performed as described previously31. Briefly, cells

xanthine/xanthine

oxidase

system

8


and

reacted

with

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178

were plated in a 6-well plate, the treated cells were incubated with 50 nM of TMRM.

179

After incubated for 30 min in the dark at 37 ◦C, the staining solution was removed and

180

the cells were re-suspended in PBS and then examined with fluorescence microscope

181

immediately (Nikon Eclipse 80i).

182

Measurement of Mitochondrial Mass. The mitochondrial mass was determined

183

using the fluorescent dye NAO as described previously31. Briefly, the treated cells

184

were trypsinized and re-suspended in PBS buffer containing 10 µM of NAO for 15

185

min in the darkness at 37 ◦C. After washed with PBS for three times, the number of

186

viable staining cells was counted with a hemocytometer. The intensity of NAO in

187

staining cells was then analyzed by fluorescence intensity with appropriate excitation

188

and emission wavelengths.

189

Western Blot Analysis. Cytosolic extracts were prepared by incubating the cells on

190

ice in hypotonic buffer (20 mM of Hepes, 10 mM of KCl, 1.5 mM of MgCl2, 1mM of

191

EDTA, 1 mM of EGTA, 250 of mM sucrose, 1 mM of dithiothreitol, 2 mg/mL of

192

aprotinin, leupeptin and pepstatin, respectively, pH = 7.5) for 30 min32. The

193

homogenates were collected and centrifuged at 12 000 g for 30 min at 4 ◦C to separate

194

the mitochondria and cytosol fraction. Total cellular proteins were extracted by

195

incubating cells in lysis buffer obtained from Cell Signaling Technology. SDS-PAGE

196

was done in 10% tricine gels, each lane was loading equal amount of proteins. After

197

electrophoresis, separated proteins were transferred to nitrocellulose membrane and

198

then blocked with 5% non-fat milk in TBS buffer for 1 h. After that, the membranes

199

were incubated with primary antibodies at 1:1000 dilutions in 5% BSA overnight at 4 9



200

◦

201

temperature. Secondary antibodies could conjugate with horseradish peroxidase.

202

Protein bands were visualized on X-ray film using an enhanced chemiluminescence

203

system (Kodak). To assess the presence of a comparable amount of proteins in each

204

lane, the membranes were stripped finally to detect the β-actin.

205

Statistical Analysis. Experiments were carried out at least in triplicate, and results

206

were expressed as means ± SD. Statistical analysis was performed using SPSS

207

statistical package (SPSS 13.0 for Windows; SPSS, Inc., Chicago, IL). The difference

208

between two groups was analyzed by two-tailed Student’s t-test, and that between

209

three or more groups was analyzed by one-way analysis of variance multiple

210

comparisons. Differences with P < 0.05 (*) were considered statistically significant.

C, and then the membranes were incubated with secondary antibodies for 1 h at room

211 212

RESULTS

213

ABTS•+ Free Radical Scavenging Activity of DSePA. The antioxidant activity

214

of DSePA was evaluated by ABTS•+ free radical scavenging assay. In this assay, the

215

radicals produced by the relatively long-lived ABTS•+ free radicals and the direct

216

oxidation of manganese dioxide could be decolorized upon reaction with antioxidants.

217

As shown in Figure 1B, concentration response curves were obtained at

218

concentrations ranging from 0 to 30 µM of DSePA, and the results showed that

219

DSePA efficiently inhibited ABTS oxidation, suggesting the strong antioxidant

220

activity of DSePA under the hydrophilic condition. For instance, after incubation for 5

221

min, the OD734 decreased from 0.40 to 0.39, 0.31, 0.30 and 0.25 at 0, 7.5, 15, 30 µM 10


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of DSePA, respectively. Correspondingly, the OD734 decreased from 0.40 to 0.39, 0.18,

223

0.16 and 0.11 after incubation for 20 min, respectively. These results suggested that

224

DSePA could scavenge ABTS•+ free radical in a time and concentration depend way.

225

DSePA inhibited AAPH-Induced Oxidative Hemolysis. The inhibition effect of

226

DSePA on AAPH-induced hemolysis was examined in erythrocytes. Intracellular ROS

227

was detected by using a fluorescein-labeled dye, DCF-DA. As shown in Figure 1C,

228

the hemolysis inhibition rate of erythrocytes was 42.9%. Whereas, pre-incubated with

229

DSePA, the inhibition rate was increased in a dose dependence way. The inhibition

230

rate increased to 79.8% when pre-incubated with 30 µM of DSePA. Furthermore, no

231

significant differences were found in erythrocytes treated with 30 µM of DSePA

232

compared with control erythrocytes. These results indicated that DSePA protected

233

human erythrocytes from AAPH-induced hemolysis through inhibition of ROS

234

generation.

235

DSePA Reversed Cisplatin-induced Cytotoxicity in HK-2. The protective effects of

236

DSePA against cisplatin-induced cytotoxicity in HepG2 cells and HK-2 cells were

237

measured by MTT reduction assay, respectively. As shown in Figure 2B, cisplatin

238

alone showed significant antitumor effect on HepG2 cells. When pretreated with 15

239

µM of DSePA, the anticancer effect on HepG2 cells was not changed when treated

240

with 16 µg/mL or 32 µg/mL of cisplatin. Interestingly, the cell viability was decrease

241

to 66% when treated with 8 µg/mL of cisplatin, while the viability of HepG2 cells was

242

82% when treated with 8 µg/mL of cisplatin alone. Furthermore, Vitamin E, as a

243

negative control, was used to treat HepG2 cells combined with cisplatin, and the 11



244

anticancer activity of cisplatin was significantly decreased with the cell viability

245

increased to 74.1%.

246

Furthermore, HK-2 cells treated with 8 µg/mL of cisplatin for 12 h reduced cell

247

viability to approximately 72% relative to control cells as Figure 2A shown. However,

248

this cytotoxic effect was significantly attenuated by pretreatment with 15µM of

249

DSePA for 12 h, with the cell viability increased to 89.7%. Moreover, 15 µM of

250

DSePA alone showed no cytotoxicity on HK-2 cells. These data suggested that DSePA

251

could reduce cisplatin-induced nephrotoxicity while didn’t affect the antitumor

252

activity of cisplatin.

253

Detection of Intracellular GSH. Intracellular glutathione (GSH) plays an

254

important role in the regulation of redox reactions. As shown in Figure 2C&E, the

255

GSH level in HepG2 cells was increased when treated with cisplatin. Meanwhile, the

256

GSH level was also significantly increased when HepG2 cells pretreatment with

257

DSePA compared with cisplatin group. However, as shown in Figure 2D&F, the GSH

258

level was increased when treated with cisplatin for HK-2 cells, while it was

259

significantly decreased when pretreated with 15µM of DSePA for 12 h. Besides,

260

DSePA alone did not alter GSH levels in HepG2 or HK-2 cells. All these indicated

261

that DSePA could protect HK-2 cells from cisplatin-induced toxicity by reducing the

262

level of GSH.

263

DSePA Decreased Cisplatin-induced Apoptosis. The protective effects of

264

DSePA against cisplatin-induced apoptosis in HK-2 cells were further examined by

265

optical microscope and flow cytometric analysis. As shown in Figure 3A, DSePA 12


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effectively attenuated cisplatin-induced cell morphological changes, such as cell

267

shrinkage, cell rounding, and the appearance of apoptotic bodies. Furthermore, flow

268

cytometric analysis showed that cisplatin increased the HK-2 cell number in sub-G1,

269

which reflected that cisplatin could induce apoptosis. However, proportion of sub-G1

270

significantly decreased after pretreatment with 15µM of DSePA. Moreover, HK-2

271

cells treated with 15µM of DSePA alone had no effect on the sub-G1 peak or cell

272

cycle distribution of HK-2 cells.

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DSePA Suppresses Cisplatin-induced Caspase Activation and PARP

274

Cleavage. Caspases, a family of cysteine acid proteases, are known to act an essential

275

role in apoptosis regulation. PARP is also a maker of apoptosis, which is a

276

downstream of caspase-3 in the apoptosis pathways. From the results of flow

277

cytometric analysis, cisplatin-induced cell apoptosis was the major mode of HK-2 cell

278

death. To investigate the underlying mechanisms of protective function of DSePA

279

against cisplatin-induced apoptosis, we examined whether activation of caspases and

280

cleavage of PARP were involved in this process. Based on the results of fluorescence

281

and Western blotting analysis, it was found that treatments of HK-2 cells with

282

cisplatin caused activation of caspase-3, caspase-8, caspase-10, and cleavage of PARP

283

(Figure 3C, D&E). Interestingly, cisplatin-induced activation of caspase-10 was

284

effectively attenuated by pretreatment with DSePA. However, the protein expression

285

of caspase-3 and caspase-8 was cleaved after pre-treatment and quantified in Figure

286

3D at 19/17 and 18 kD, respectively. For instance, the activity of caspase-3 decreased

287

from 1285% to 791%, and the activity of caspase-8 decreased from 295% to 211% 13



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when HK-2 cells were pretreated with 15 µM of DSePA, respectively. These results

289

were further confirmed by Western blot analysis. As shown in Figure 3D, it was able

290

to attenuate cisplatin-induced activation of caspase-3, caspase-8, caspase-10, and

291

cleavage of PARP when HK-2 cells pretreated with DSePA. These results clearly

292

demonstrated that DSePA could protect HK-2 cells against cisplatin-induced death by

293

suppressing caspase-mediated apoptosis.

294

DSePA Prevented Cisplatin-induced Cytotoxicity by Inhibiting ROS Generation.

295

ROS generation is a biomarker of oxidative stress. Intracellular ROS was detected by

296

using a fluorescein-labeled dye, DCFH-DA. As show in Figure 4A, compared with

297

control group, the ROS of HK-2 cells showed a significant increase when treated with

298

cisplatin. However, pretreatment with DSePA significantly reduced the ROS

299

generation in HK-2 cells before exposed to cisplatin. For instance, 15 µM of DSePA

300

reduced the intracellular ROS from 144 % to 109 % of control cells. Furthermore,

301

there was no significant difference in HK-2 cells treated with 15 µM of DSePA alone

302

compared with control.

303

Treatment with cisplatin also increased the level of O2− in HK-2 cells in a

304

time-dependent manner (Figure 4B). Pretreatment with DSePA significant reduced

305

the levels of O2− in cells treated with cisplatin. DSePA had no effect on the free radical

306

accumulation in HK-2 cells.

307

DSePA Prevented

Cisplatin-induced

DNA Damage.

Generally,

excess

308

intracellular ROS could cause DNA damage and evidences have been shown that

309

cisplatin could defect DNA and arrest the synthesis of DNA33. To confirm this 14


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310

hypothesis, the p53 expression and phosphorylation in HK-2 cells treated with

311

cisplatin or DSePA and cisplatin. As shown in Figure 4C, Western-blot showed that

312

DNA damage marker Ser139-Histone H2A was up-regulated in HK-2 cells treated

313

with DSePA. Furthermore, p-H2A up-regulated the expression of total and

314

phosphorylated p53 gene (Figure 4C), which is a tumor suppressor gene that can

315

directly or indirectly induce cell apoptosis through both the extrinsic and intrinsic

316

apoptosis pathways.

317

To further examine the important role of DSePA on decrease cisplatin-induced ROS

318

generation, we next investigate the effects of GSH on overall apoptotic cell death.

319

Data shown in Figure 4D revealed that individual GSH has un-obvious cytotoxicity

320

to HK-2 cells, but 4 mM of GSH blocked the decrease of cell viability induced by

321

cisplatin (Figure 4E and F). These results showed that the role of GSH was quite

322

consistent with that of DSePA. All these indicated that DSePA protected HK-2 cells

323

from cisplatin-induced cell apoptosis through inhibition of ROS generation.

324

DSePA Inhibits Cisplatin-induced Mitochondrial Dysfunction. Mitochondria

325

play an important role in the regulation of apoptosis, in which mitochondrial mass and

326

∆Ψm are important indexes. In this section we studied whether cisplatin-induced

327

apoptosis occurred with the involvement of mitochondrial dysfunction. Cisplatin

328

markedly reduced intracellular TMRM fluorescence intensity as Figure 5A shown.

329

On the other hand, cisplatin-induced decrease in TMRM fluorescence intensity was

330

effectively attenuated by pretreatment with DSePA. Moreover, the results of NAO

331

fluorescence analysis also revealed that DSePA could prevent cisplatin-induced 15



332

mitochondrial mass reduction in HK-2 cells (Figure 5B).

333

Mitochondria could integrate the apoptotic signals originating from both the

334

extrinsic and intrinsic apoptosis pathways34. Bcl-2 family members have been

335

described as the central regulator of mitochondrial permeability and caspase

336

activation. Therefore, the effects of cisplatin on the expression levels of pro-survival

337

and pro-apoptotic Bcl-2 family proteins and activation of caspase in HK-2 cells were

338

examined by Western blotting. As shown in Figure 5C, 8 µg/mL of cisplatin

339

incubation up-regulated the expression of pro-apoptosis protein, such as Bax, but

340

down-regulated the expression of pro-survival proteins, such as Bcl-2 and Bcl-xl.

341

However, the expression levels of Bcl-2 family proteins were obviously reversed after

342

pretreatment with DSePA for 12 h. Furthermore, we demonstrated that cisplatin

343

induced the truncation of Bid in HK-2 cells. Moreover, cleaved caspase-7/-9 were

344

increased after incubated with 8 µg/mL of cisplatin for 12 h (Figure 5D &E),

345

indicating that cisplatin induced the activation of initiated caspase of intrinsic

346

mitochondrial apoptosis pathway. On the other hand, the expression levels of

347

caspase-7/-9 were suppressed by pretreatment of 15 µM of DSePA for 12 h. Taken

348

together, these results confirmed that DSePA protected HK-2 cells against

349

cisplatin-induced apoptosis through inhibition of mitochondrial-mediated apoptosis

350

and caspases activation.

351

DSePA Regulated Cisplatin-induced AKT Inhibition. ERK and AKT signaling

352

pathways have been confirmed to paly important role in cell apoptosis and cell

353

proliferation. AKT promotes growth and proliferation of cancer cells by inhibiting the 16


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354

expression of pro-apoptosis proteins, such as caspases-9 and p5335,17. Therefore, in

355

this study, we tried to elucidate whether DSePA regulate cisplatin-induced AKT

356

inhibition or not. As shown in Figure 6A, there was a decrease in the expression of

357

phosphorylated AKT in HK-2 cells which exposed to 8 µg/mL cisplatin. However, the

358

pretreatment of HK-2 cells with DSePA restored cisplatin-induced decease in

359

expression phosphorylated AKT and the protein expression level significantly

360

decreased compared with control group. In contrast, the phosphorylation of AKT was

361

significantly increased after treatment with DSePA compared with control group (3.5

362

times). Moreover, the phosphorylation of AKT was decreased remarkably when HK-2

363

cells treated with cisplatin after pretreated with DSePA compared with the group

364

treated with DSePA alone. To further determine whether AKT was important for

365

cisplatin-induced apoptosis, specific inhibitor of AKT was used to examine their

366

effects on cisplatin-induced cytotoxicity. The cells were pretreated with LY294002

367

(PI3K inhibitor) before addition of cisplatin and then the cell viability was measured

368

by MTT assay. As shown in Figure 6B, LY294002 significantly decreased the cell

369

viability of HK-2 cells exposed to cisplatin. Besides, LY294002 also decrease the cell

370

viability to 89% contrast to control. These results indicated the important roles of

371

AKT pathways in cisplatin-induced apoptosis.

372 373

DISCUSSION

374

3,3’-Diselenodipropionic acid (DSePA), a derivative of selenocystine, has been

375

synthesized and examined for antioxidant activity, glutathione and glutathione 17



376

peroxidase (GPx) activity. Kunwar A et.al studied the cytotoxicity of DSePA in

377

lymphocytes and mouse lymphoma EL4 tumor cells and the results showed that

378

DSePA was not nontoxic to these cells at the concentration of 125 µM18. Kunwar A

379

et.al also studied the in vivo radioprotection effect of DSePA against whole-body

380

γ-radiation and the probable mechanisms of action involve the maintenance of

381

antioxidant enzymes, prophylactic action through the attenuation of the DNA damage,

382

and the inhibition of apoptosis36.

383

In the present study, we demonstrated the protective effects of DSePA on HK-2

384

proximal tubular cells against cisplatin-induced apoptosis through inhibition of ROS

385

generation and modulation of AKT pathway for the first time. Our results showed that

386

cisplatin inhibited HK-2 cell growth through induction of apoptosis, with the

387

involvement of caspase activation, PARP cleavage and reduction of mitochondrial

388

mass, mitochondrial fragmentation and loss of ∆Ψm and changes in expressions of

389

Bcl-2 family members. Additionally, there was cross-talk between the extrinsic and

390

intrinsic apoptotic pathways as demonstrated by cleavage of Bid by caspase-8 in the

391

apoptotic process triggered by cisplatin. Especially cisplatin would induce ROS

392

overproduction, p53 phosphorylation and AKT phosphorylation. Interestingly, these

393

cisplatin-induced changes were significantly blocked by pre-treatment with DSePA.

394

The hypothetical mechanisms were mainly due to attenuation of oxidative stress,

395

reduction of p53 induction and modulation of the AKT signaling pathways.

396

Importantly, DSePA didn’t affect the effect of cisplatin on cancer cells, while the

397

actual mechanism is still not clear. 18


Page 18 of 41

Page 19 of 41


398

Glutathione redox cycle is the most important intracellular antioxidant system

399

which maintains cell integrity and participation in the cell metabolism37. The role of

400

glutathione in cisplatin-induced nephrotoxicity is not clear. Some investigators have

401

indicated that nephrotoxicity of cisplatin is not necessarily associated with depletion

402

in renal glutathione38, 39. However, there was an increase in glutathione level of

403

HepG2 and HK-2 cells treated with cisplatin compared to control group in our study,

404

which was similar to previous study40. The cisplatin-induced glutathione up regulation

405

might be due to the enhancement of GSH synthesis under conditions of oxidative

406

stress or glutathione depletion. In our study, DSePA pretreatment produced a

407

significant increase in GSH levels in HepG2 cells and a significant decrease in GSH

408

levels in HK-2 cells compared to cisplatin group. According to the results, we

409

supposed that DSePA may amplify the oxidative stress in cisplatin treated HepG2

410

cells and attenuate the oxidative stress in cisplatin treated HK-2 cells, but the

411

underlying mechanism was not clear.

412

During the past few decades, emerging evidences demonstrated that apoptosis is

413

one of the major mechanisms of nephrotoxicity induced by cisplatin. Generally,

414

apoptosis consists of two major pathways, the extrinsic pathway mediated by death

415

receptors with the involvement of caspase-8 activation and the intrinsic pathway

416

centered on mitochondria with the participation of caspase-9 activation41. Moreover,

417

the intrinsic mitochondrial pathway displays central role in integrating the apoptotic

418

signals originated from both the intrinsic and extrinsic apoptosis pathways. Evidence

419

demonstrated that intrinsic or mitochondrial pathway exerted as the major apoptotic 19



420

pathway in cisplatin-induced nephrotoxicity42. In this present study, we found that

421

cisplatin exerted significant suppression effects towards HK-2 cells, as indicated by

422

the MTT assay and flow cytometry assay (Figure 2). Further investigation

423

demonstrated that the proliferation inhibition effect of HK-2 induced by cisplatin was

424

the results of apoptosis, as revealed by the accumulation of sub-G1 peaks population,

425

which is further confirmed by caspase-8/-9 and caspasr-3 activation and PARP

426

cleavage. Moreover, these results also were confirmed by the morphology of cell

427

shrinkage and appearance of apoptotic bodies (Figure 3). These results suggested that

428

apoptosis was the major mechanism induced by cisplatin in HK-2 cells. However,

429

pretreatment with DSePA dramatically attenuated the apoptosis inducing capacity of

430

cisplatin. Taken together, our results suggest that the anti-apoptotic effects of DSePA

431

involve the suppression of both extrinsic and intrinsic apoptosis pathways.

432

Oxidative stress injury is actively involved in the pathogenesis of cisplatin-induced

433

kidney injury, ROS directly act on cell components, including lipids, proteins, and

434

DNA, and destroy their structure. Oxidative stress injury is actively involved in the

435

pathogenesis of cisplatin-induced kidney injury43. Excess intracellular ROS may

436

attack cellular, and which leads to the generation of a variety of ROS-mediated

437

modified products like DNA strand breaks and DNA-protein crosslink. The

438

phosphorylation of histone H2A is a very early cellular event induced by DNA strand

439

breaks. In this study, we found that cisplatin significantly increased the level of

440

phosphorylated H2A and p53. The activation of p53 has been demonstrated to be

441

involved in cisplatin-induced nephrotoxicity44. However, the activation of p53 was 20


Page 20 of 41

Page 21 of 41


442

suppressed by pre-treatment with DSePA. Therefore, it was suggested that DSePA

443

decreased cisplatin-induced apoptosis through the inhibition of ROS-mediated p53

444

pathway in HK-2 cells. Furthermore, we found that GSH, which have been reported to

445

act not only as free radical scavengers, but also by replenish-intracellular stores of

446

endogenous antioxidants, or as thiol-reducing agents45, effectively reduced

447

cisplatin-induced apoptotic cell death, activation of caspase-3 and cleavage of PARP

448

in HK-2 cells. Taken together, these results suggest the possibility that DSePA

449

effectively scavenged free radical and protect HK-2 cells from cell apoptosis induced

450

by cisplatin.

451

Mitochondrial pathway has emerged as the major apoptotic pathway in cisplatin

452

nephrotoxicity46, 47. Our results showed that cisplatin-induced HK-2 cells apoptosis

453

was associated with disruption of mitochondrial function, as evidenced by reduction

454

of mitochondrial mass, mitochondrial fragmentation and loss of ∆Ψm. Moreover,

455

changes in expressions of Bcl-2 family members, activation of caspase-7/-9 were

456

observed in HK-2 cells after cisplatin treatment. Intriguingly, pre-treatment of DSePA

457

significantly attenuated these changes induced by cisplatin, which suggested the

458

capacity of DSePA to prevent cisplatin triggered mitochondrial dysfunction and

459

modulate mitochondria-mediated apoptotic pathways. Additionally, there was

460

crosstalk between the extrinsic and intrinsic apoptotic pathways as demonstrated by

461

cleavage of Bid by caspase-8 in the apoptotic process triggered by cisplatin. The

462

extrinsic cell apoptosis pathway induced by cisplatin was also blocked by DSePA.

463

These results suggested that DSePA exhibited protective effect on HK-2 cells through 21



464

reversing extrinsic apoptosis and intrinsic apoptosis inducing capability of cisplatin by

465

protecting the integrity of mitochondria membrane.

466

PI3K/AKT pathways are the major oxidative stress-sensitive signal transduction

467

pathways in most cell types48. The phosphorylation of AKT showed a marked

468

induction in both time- and dose-dependent manner during the course of cisplatin

469

injury49. In agreement with these findings, our results showed that exposure of HK-2

470

cells cisplatin resulted in a decrease of phosphorylated AKT. Furthermore, we found

471

that DSePA was able to restore cisplatin-induced decease in expression

472

phosphorylated AKT. To further determine the role of AKT for cisplatin-induced

473

apoptosis, specific inhibitor of PI3K (LY294002), one upstream kinase for AKT

474

kinase activation, was used to examine the effects on cisplatin-induced cytotoxicity.

475

The results of MTT assay showed that LY294002 significantly decreased the cell

476

viability of HK-2 cells exposed to cisplatin. These data indicated that AKT pathway

477

played an important role in cisplatin-induced cytotoxicity.

478

Overall, the results showed that cisplatin significantly inhibited the growth of HK-2

479

cells through induction of ROS-mediated apoptosis, while DSePA could effectively

480

reverse the damage caused by cisplatin through inhibition of ROS generation, caspase

481

activation and modulation of mitochondrial-mediated and AKT pathway. Taken

482

together, this study demonstrates that seleno-amino acid could be further developed as

483

novel nutritionally available agents to antagonize cisplatin-induced nephrotoxicity.

484 485

AUTHOR INFORMATION 22


Page 22 of 41

Page 23 of 41


486

Corresponding Authors

487

Funding

488

This work was supported by National High-level personnel of special support

489

program (W02070191), YangFan Innovative & Entepreneurial Research Team Project

490

(201312H05), Fundamental Research Funds for the Central Universities, National

491

Key Scientific Instrument and Equipment Development Project (2017YFF0104904)

492

and Natural Science Foundation of Guangdong Province (2017A030313091).

493 494

Notes

495

The authors declared no competing financial interest.

496 497

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655 656 657 658 659 660 661 30


rat alveolar

Page 31 of 41


662

Figure Legends

663 664

Figure 1. The structure and Antioxidant activities of DSePA. (A) The chemical

665

structure of DSePA. (B) The antioxidant activities of DSePA determined by ABTS

666

assay. (C) The antioxidant activities of DSePA determined by erythrocyte hemolysis

667

assay at different concentration. n=3, P < 0.05.

668 669

Figure 2. DSePA suppressed cisplatin-induced toxicity and GSH depletion. (A)

670

DSePA attenuated cisplatin-induced cytotoxicity in HK-2 cell by MTT assay. (B)

671

DSePA showed no effect on the anticancer efficacy of cisplatin against HepG2 cells

672

by MTT assay. (C, E) Flow cytometry-based GSH profiles and quantification of

673

intracellular GSH intensity in HepG2 cells. (D, F) Flow cytometry-based GSH

674

profiles and quantification of intracellular GSH intensity in HK-2 cells. Cells were

675

pretreated with or without DSePA for 12 h and then cultured in the presence or

676

absence of 8 mg/mL of cisplatin for 12 h. n=3, P < 0.05.

677 678

Figure 3. DSePA protected HK-2 cells from cisplatin-induced apoptosis. (A)

679

Morphology of HK-2 cells under different treatments. (B) Cell apoptosis was

680

determined by flow cytometry analysis. (C) Caspase-3 activity and (E) Caspase-8

681

activity were measured by specific fluorescent substrates, respectively. (D) Inhibition

682

of caspases and PARP cleaved in the apoptosis pathway by Western blot analysis. n=3,

683

P < 0.05. 31



684

Figure 4. (A) ROS generation was determined by fluorescence intensity of

685

DCFH-DA. Cells pretreated with or without DSePA for different time before treated

686

with cisplatin, and then cytosol fraction were collected by centrifuged at 12 000 g for

687

30 min at 4 °C.. (B) DSePA suppressed cisplatin-induced accumulation of superoxide

688

radicals in HK-2 cells. (C) Western blot analysis of expression levels of p53,

689

phosphorylation of p53 and histone H2A in HK-2 cells. (D) Effects of GSH on

690

cisplatin-induced cytotoxicity in HK-2 cells. (E) Western blot analysis of protein

691

levels of activated caspase-3 and cleavaed PARP in HK-2 cells. (F) GSH prevented

692

cisplatin-induced activation of caspase in HK-2 cells. Cells were pretreated with or

693

without DSePA for 12 h, and cells were treated with 4 mM of GSH for 2 h followed

694

by cultured in the presence or absence of 8µg /mL of cisplatin for 12 h in D, E and F.

695

n=3, P < 0.05.

696 697

Figure 5. DSePA suppressed cisplatin-induced mitochondrial dysfunction. (A)

698

Depletion of ∆Ψm as monitored by fluorescence microscopy. The cells treated with

699

DSePA and cisplatin were stained with 50 nM TMRM, and then examined under a

700

fluorescence microscope. (B) DSePA prevented cisplatin-induced mitochondrial mass

701

reduction in HK-2 cells. Mitochondrial mass was determined by Fluorescence

702

Microplate after staining the cells with NAO. (C) The expression of Bcl-2 protein

703

family. (D) Activation of caspase-7/-9 in cisplatin-induced apoptosis. (E) Caspase-9

704

activity was measure by fluorescent substrate for caspase-9. Cells were pretreated

705

with or without DSePA for 12 h and then cultured in the presence or absence of 8µg 32


Page 32 of 41

Page 33 of 41


706

/mL of cisplatin for 12 h, and then mitochondria were collected by centrifuged at 12

707

000 g for 30 min at 4 °C. n=3, P < 0.05.

708 709

Figure 6. DSePA and cisplatin impacted on AKT signaling pathway. (A) DSePA

710

blocked cisplatin-induced decease in expression phosphorylated AKT. (B) Effect of

711

AKT inhibitors on cell viability of cisplatin-treated HK-2 cells. Cells were pretreated

712

with or without DSePA for 12 h, and cells were treated with 10 µM of LY294002

713

(PI3K inhibitor) for 2 h followed by cultured in the presence or absence of 8 µg/mL of

714

cisplatin for 12 h, and then cytosol fraction were collected by centrifuged at 12 000 g

715

for 30 min at 4 °C. n=3, P < 0.05.

716 717

Figure 7. Signaling pathways regulated by DSePA and cisplatin in HK-2 cells. Cisplatin

718

directly act on cell DNA, leading to the phosphorylation of Histone H2A and

719

activation of p53. P53 regulates the expression of Bcl-2 family proteins, resulting in

720

mitochondrial dysfunction and finally cell apoptosis. P53 also regulates death receptor

721

pathway and finally results in apoptotic cell death. Excess intracellular ROS generated

722

from mitochondrial may attack cellular DNA and modulation of AKT pathway.

723

However, DsePA pretreatment inhibited the cisplatin-induced caspase-8/-10 activation

724

in HK-2, and blocked mitochondrial membrane potential depletion by suppressing the

725

truncation of Bid translocated to mitochondria. Additionally, DsePA activated AKT

726

pathway and inhibited cisplatin-induced nephrotoxicity by inhibiting intracellular

727

ROS-mediated apoptosis, while showed un-obvious effect on its anticancer efficacy. 33



Page 34 of 41

728 729

Figure 1

A

O Se HO

OH

Se

O

DSePA

B

C 100 0.4 0.3 0.2 ABTS ABTS + 30 uM DSePA ABTS + 15 uM DSePA ABTS + 7.5 uM DSePA

0.1 0

730

Inhibition (%)

Absorbance 734 nm

0.5

10

Time

20

30

a

a d

75 50

b

e

c

25 0

AAPH (100 mM) DSePA (μM) -

+ -

+ 7.5

+ 15

+ 30

30

731

Figure 1. The structure and Antioxidant activities of DSePA. (A) The chemical

732

structure of DSePA. (B) The antioxidant activities of DSePA determined by ABTS

733

assay. (C) The antioxidant activities of DSePA determined by erythrocyte hemolysis

734

assay at different concentration. n=3, P < 0.05.

735 736 737 738 739 740 34


Page 35 of 41


741

Figure 2

742 743

Figure 2. DSePA suppressed cisplatin-induced toxicity and GSH depletion. (A)

744

DSePA attenuated cisplatin-induced cytotoxicity in HK-2 cell by MTT assay. (B)

745

DSePA showed no effect on the anticancer efficacy of cisplatin against HepG2 cells

746

by MTT assay. (C, E) Flow cytometry-based GSH profiles and quantification of

747

intracellular GSH intensity in HepG2 cells. (D, F) Flow cytometry-based GSH

748

profiles and quantification of intracellular GSH intensity in HK-2 cells. Cells were

749

pretreated with or without DSePA for 12 h and then cultured in the presence or

750

absence of 8 mg/mL of cisplatin for 12 h. n=3, P < 0.05.

751 752 35



753

Figure 3.

754 755

Figure 3. DSePA protected HK-2 cells from cisplatin-induced apoptosis. (A)

756

Morphology of HK-2 cells under different treatments. (B) Cell apoptosis was

757

determined by flow cytometry analysis. (C) Caspase-3 activity and (E) Caspase-8

758

activity were measured by specific fluorescent substrates, respectively. (D) Inhibition

759

of caspases and PARP cleaved in the apoptosis pathway by Western blot analysis. n=3,

760

P < 0.05.

761 762 763 36


Page 36 of 41

Page 37 of 41


764

Figure 4

765 766

Figure 4. DSePA prevents cisplatin-induced ROS-mediated cell apoptosis. (A)

767

ROS generation was determined by fluorescence intensity of DCFH-DA. Cells

768

pretreated with or without DSePA for different time before treated with cisplatin, and

769

then cytosol fraction were collected by centrifuged at 12 000 g for 30 min at 4 °C. (B)

770

DSePA suppressed cisplatin-induced accumulation of superoxide radicals in HK-2

771

cells. (C) Western blot analysis of expression levels of p53, phosphorylation of p53

772

and histone H2A in HK-2 cells. (D) Effects of GSH on cisplatin-induced cytotoxicity

773

in HK-2 cells. (E) Western blot analysis of protein levels of activated caspase-3 and

774

cleavaed PARP in HK-2 cells. (F) GSH prevented cisplatin-induced activation of

775

caspase in HK-2 cells. Cells were pretreated with or without DSePA for 12 h, and

776

cells were treated with 4 mM of GSH for 2 h followed by cultured in the presence or

777

absence of 8µg /mL of cisplatin for 12 h in D, E and F. n=3, P < 0.05. 37



778

Figure 5

779 780

Figure 5. DSePA suppressed cisplatin-induced mitochondrial dysfunction. (A)

781

Depletion of ∆Ψm as monitored by fluorescence microscopy. The cells treated with

782

DSePA and cisplatin were stained with 50 nM of TMRM, and then examined under a

783

fluorescence microscope. (B) DSePA prevented cisplatin-induced mitochondrial mass

784

reduction in HK-2 cells. Mitochondrial mass was determined by Fluorescence

785

Microplate after staining the cells with NAO. (C) The expression of Bcl-2 protein

786

family. (D) Activation of caspase-7/-9 in cisplatin-induced apoptosis. (E) Caspase-9

787

activity was measure by fluorescent substrate for caspase-9. Cells were pretreated

788

with or without DSePA for 12 h and then cultured in the presence or absence of 8µg

789

/mL of cisplatin for 12 h, and then mitochondria were collected by centrifuged at 12

790

000 g for 30 min at 4 °C. n=3, P < 0.05.

791 38


Page 38 of 41

Page 39 of 41


792

Figure 6

793 794

Figure 6. DSePA and cisplatin impacted on AKT signaling pathway. (A) DSePA

795

blocked cisplatin-induced decease in expression phosphorylated AKT. (B) Effect of

796

AKT inhibitors on cell viability of cisplatin-treated HK-2 cells. Cells were pretreated

797

with or without DSePA for 12 h, and cells were treated with 10 µM of LY294002

798

(PI3K inhibitor) for 2 h followed by cultured in the presence or absence of 8 µg/mL of

799

cisplatin for 12 h, and then cytosol fraction were collected by centrifuged at 12 000 g

800

for 30 min at 4 °C. n=3, P < 0.05.

801 802 803 804 805 806 807 808 809 39



810

Figure 7

811

812 813

Figure 7. Signaling pathways regulated by DSePA and cisplatin in HK-2 cells. Cisplatin

814

directly act on cell DNA, leading to the phosphorylation of Histone H2A and

815

activation of p53. P53 regulates the expression of Bcl-2 family proteins, resulting in

816

mitochondrial dysfunction and finally cell apoptosis. P53 also regulates death receptor

817

pathway and finally results in apoptotic cell death. Excess intracellular ROS generated

818

from mitochondrial may attack cellular DNA and modulation of AKT pathway.

819

However, DsePA pretreatment inhibited the cisplatin-induced caspase-8/-10 activation

820

in HK-2, and blocked mitochondrial membrane potential depletion by suppressing the

821

truncation of Bid translocated to mitochondria. Additionally, DsePA activated AKT

822

pathway and inhibited cisplatin-induced nephrotoxicity by inhibiting intracellular

823

ROS-mediated apoptosis, while showed un-obvious effect on its anticancer efficacy. 40


Page 40 of 41

Page 41 of 41


824 825

TOF graphic

826

827 828

41


Nutritional Available Selenocysteine Derivative Antagonizes Cisplatin

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