Synthesis and Characterization of a Walnut PeptidesâZinc Complex

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Synthesis and Characterization of Walnut Peptides-Zinc Complex and Its Antiproliferative Ability against Human Breast Carcinoma Cells through Induction of Apoptosis Wenzhen Liao, Ting Lai, Luying Chen, Junning Fu, Sreeprasad T Sreenivasan, zhiqiang yu, and Jiaoyan Ren J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04924 • Publication Date (Web): 01 Feb 2016 Downloaded from http://pubs.acs.org on February 1, 2016

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Journal of Agricultural and Food Chemistry

Synthesis and Characterization of Walnut

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Peptides-Zinc Complex and Its Antiproliferative Activity

3

against Human Breast Carcinoma Cells through Induction

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of Apoptosis Wenzhen Liao†, Ting Lai†, Luying Chen†, Junning Fu‡, Sreeprasad T

5

⊥

Sreenivasan§, Zhiqiang Yu *, Jiaoyan Ren†*

6

7

†

8

Guangzhou 510640, China

9

‡

Department of Food Science and Technology, South China University of Technology,

Department of Food Science and Engineering, Jinan University, Guangzhou,510623，

10

China

11

§

12

Street, Houston, TX, 77005, USA

13

Department of Civil and Environmental Engineering, Rice University, 6100 Main

⊥

School of Pharmaceutical Science, Guangdong Provincial Key Laboratory of New

14

Drug Screening, Southern Medical University, Guangzhou, Guangdong 510515,

15

China

16

*

17

*

18

of Technology, Wushan Road 381, Guangzhou, Guangdong, China.

19

Tel: (+86)20-87112594; Fax: (+86)20-38897117; E-mail: [email protected]

Corresponding authors:

Jiaoyan Ren, Department of Food Science and Technology, South China University

1

ACS Paragon Plus Environment


20

*

21

Laboratory of New Drug Screening, Southern Medical University, Guangzhou,

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Guangdong 510515, China; E-mail: [email protected]

Zhiqiang Yu, School of Pharmaceutical Sciences, Guangdong Provincial Key

23

2


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ABSTRACT

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The walnut peptides and zinc ions were combined to generate walnut peptides-zinc

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complex (WP1-Zn) with enhanced antiproliferative ability as well as reduced toxicity.

27

The result indicated that Zn ions were successfully combined with WP1 through Zn-N

28

and Zn-O covalent bonds. WP1-Zn compounds exhibited strong antiproliferative

29

ability against the selected human cell lines, especially MCF-7 cells, whose survival

30

rate reduced to 20.02% after exposure to 300 µg/mL of WP1-Zn for 48

31

h.WP1-Zninhibited MCF-7 cells proliferation through inducing cell apoptosis and cell

32

cycle arrest.The results indicated that WP1-Zninduced MCF-7 cell apoptosis via ROS

33

triggered mitochondrial-mediated pathway and cell surface receptor-mediated

34

pathway. Our work is the first attempt to elucidate the synergic effect of novel walnut

35

peptides and Zn and with the hope of better understanding the antiproliferative action

36

of bioactive peptides and Zinc Complex and support the potential application of

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WP1-Zn as functional food ingredient or complementary medicine.

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KEYWORDS: walnut peptides-zinc complex, biosynthesis, antitumor, apoptosis

39

40

3



41

INTRODUCTION

42

Bioactive peptides are the protein fractions showing positive physiological effects on

43

human body, such as mineral binding, immunoregulation, antitumor, antioxidant,

44

hypocholesterolemic, antihypertensive, antimicrobial as well as antithrombotic

45

effects.1-6 In normal conditions, bioactive peptides maintain inactive in the sequence

46

of their parent proteins; however, with appropriate enzymatic hydrolysis, they will be

47

released and activated. Due to their multiple biological functions, bioactive peptides

48

have attracted significant attention in recent years.

49

Walnut (JuglansregiaL.), a member of Juglandaceae family,7 are good sources of

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proteins, essential fatty acids, and other nutrients.8 Numerous studies have

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demonstrated that walnuts exhibited excellent pharmacological properties including

52

antihypertensive, antioxidant, stress signals decreasing and neuronal signaling

53

enhancing abilities.8-12 Walnut peptides, which can be obtained by the enzymatic

54

hydrolysis of walnut proteins, have much better bioactivity compared with their

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parent proteins.13-15 However, the antitumor activity of walnut peptides has not been

56

fully studied and the relevant mechanism is not fully understood. Thus, to explore

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walnut sole or synergistic effect for tumor treatment as and have a deep understanding

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about mechanism involved may advance the understanding of bioactive peptides as

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anti-tumor agents.

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Zinc ion (Zn2+), an essential trace element for living organisms, is well

61

acknowledged as a key component of approximately 300 enzymes and involved in 4


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numerous enzymatic and metabolic processes in human organism.16, 17 Zinc also plays

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an important role in many intracellular processes such as DNA metabolism and repair,

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oxidative defense, cell proliferation and cellular signaling pathways, etc.18,

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Additionally, zinc deficiency can result in serious health problems, including growth

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retardation, cognitive impairment and immune dysfunction.20-22 Disturbed zinc

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homeostasis was also found in many types of cancer.23-25 Previous studies suggested

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that zinc can prevent cancer by decreasing angiogenesis and inducing apoptosis in

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cancer cells,26-31 which opens the door to make use of zinc for antiproliferative

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treatment. However, overdose accumulation of zinc ion could induce toxicity and side

71

effects.32, 33 Thus, the dose control of zinc is crucial for its clinic application. To

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overcome such drawback, combination of zinc with bioactive compounds specific

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attracted great attention. Considerable studies have shown that these zinc complexes

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display significant antiproliferative abilities against human cancers, exhibiting

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promising cancer treatment effect.34-41

19

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The hybrid walnut peptides-zinc complex (WP1-Zn) with enhanced overall

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antiproliferative ability as well as reduced toxicity of zinc was successfully

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synthesized in the present study. In addition, the antiproliferative activities of the

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walnut peptides-zinc complex were evaluated against several human cancer cells and

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the underlying mechanisms through which the zinc-peptide complex induced cancer

81

cell death were investigated in detail. Our study suggested that WP1-Zn could be

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developed as an active ingredients in functional foods. 5

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MATERIALS AND METHODS

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Materials

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Walnut meals (protein content 42%, w/w) were obtained from Huizhi source co., Ltd.

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(Yunnan, China). Trypsin (E.C. 3.4.21.4., 46 million U/g proteins) was obtained from

88

Shisheng technology co., Ltd. (Hangzhou, Zhejiang, China). Zinc chloride was

89

purchased

90

China).Phosphate-buffered saline (PBS, pH 7.4), Dulbecco’s modified Eagle’s

91

medium (DMEM), penicillin and streptomycin were purchased from Gibco Life

92

Technologies (Grand Island, NY, USA). Fetal bovine serum (FBS) was obtained from

93

Hyclone (Australia origin). Substrates for caspase-3/7 (Ac-DEVD-AMC), caspase-8

94

(Ac-IETD-AFC) and caspase-9 (Ac-LEHD-AFC) were purchased from Calbiochem

95

(La Jolla, CA, USA). Bicinchoninic acid (BCA) kit 2’,7’-dichlorofluorescein

96

diacetate (DCFH-DA) and thiazolyl blue tetrazolium bromide (MTT)were purchased

97

from Sigma Company (St. Louis, MO, USA).Ultrapure water was prepared using a

98

Milli-Q water purification system (Millipore, Germany).The human breast carcinoma

99

MCF-7 cell line, the human pulmonary carcinoma A549 cell line, the human cervical

100

carcinoma Hela cell line, and the human prostate carcinoma PC3 cell line were

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obtained from the American Type Culture Collection (ATCC, Rockville, Md,

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USA).The human gastric carcinomaSGC-7901 cell line and the human normal liver

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HL-7702 (LO2) cell line were purchased from the Medical College of Sun Yat-Sen

104

University (Guangzhou, Guangdong, China).All the other chemical reagents used in

from

Tianjin

Fuchan

Chemical

Reagents

6


Factory

(Tianjin,

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the present study were of analytical grade.

106

Preparation of Walnut Peptides (WP1)

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Walnut meals were mixed and homogenized with a 2-fold-volume of deionized water

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and walnut proteins were extracted by alkali solution and acid precipitation. The

109

obtained protein was then hydrolyzed by adding1.0% trypsin (w/w, trypsin

110

weight/protein weight, %) in a shaking water bath at 55 °C for 22 h. At the end of the

111

incubation, the protein hydrolysate was heated in boiling water for 15 min to

112

terminate further hydrolysis. After that, the protein hydrolysate was cooled to room

113

temperature and centrifuged at 8,000gfor 20min. The obtained supernatant was

114

collected and then filtrated by a membrane filter (Vivaflow 200, Vivascience,

115

Sartorius, Goettingen, Germany) with molecular weight cutoff (MWCO) of 10 kDa.

116

Filter residue was then lyophilized and collected as walnut peptides (WP1) sample.

117

Biosynthesis of Walnut Peptides-Zinc Complex (WP1-Zn)

118

Aqueous solution of 5 mg/mL walnut peptides was mixed with 6 mg/mL freshly

119

prepared zinc chloride solution (2:5 (v/v))under magnetic stirring. Then, 10%

120

ammonia was added to the mixture to adjust the pH to 5.0.Afterincubated in a shaking

121

water bath at 50 °C for 48 h, the complex were precipitated with 75%ethanol

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(1:3,v/v)at 4 °C overnight. The synthesized WP1-Zn was washedin75% ethanol

123

several times, lyophilized and stored at -20 °C for further use.

124

Characterization of WP1-Zn 7



125

Morphology analysis using Scanning Electron Microscopy (SEM)

126

The morphology of WP1-Zn was observed by a model 1530VP SEM (LEO,

127

Oberkochen, Germany).The powder sample of WP1-Zn was spread on a glass slide

128

fixed on the specimen holder by double-sided adhesive tapes and observedby SEM.

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Elemental composition analysisusing Energy Dispersive X-ray (EDX)

130

The elemental composition of WP1-Zn was analyzed by an energy dispersive X-ray

131

spectrometer (EDX, EX-250, Horiba Ltd., Tokyo, Japan). The powder sample of

132

WP1-Zn was applied to a glass slide, fixed on the specimen holder by double-sided

133

adhesive tapes and observed using EDX spectrometer.

134

Fourier Transform Infrared Spectroscopy(FTIR)

135

The chemical binding of zinc to walnut peptide in WP1-Zn was characterized by

136

Fourier transform infrared spectrophotometer (FT-IR, Bruker, Ettlingen, Germany) at

137

the wave number range from 400 to 4000 cm-1.

138

Antiproliferative ability of WP1-Zn

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Cell Culture

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Six different cell lines including MCF-7, SGC-7901, PC3, A549,Hela and LO2 were

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grown and maintained in DMEM medium supplemented with 10% FBS, 100 U/mL of

142

penicillin and 100 µg/mL of streptomycin at 37 °C and 5% CO2.

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Cell viabilities analysis by MTT 8


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Cell viabilities after treated with WP1-Zn and WP1 were assessed using the MTT

145

assay. Briefly, MCF-7, SGC-7901, PC3, A549,Hela and LO2 cell lines were seeded

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separately at a density of 5 × 104 cells/ mL into a 96-well microplate. After 24 hours

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post seeding, the growth medium was removed and the wells were washed with PBS.

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Triplicate wells were treated with 200 µL of WP1-Zn (37.25, 75, 150 and 300 µg/mL)

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and 200 µL of WP1(250, 500 and 1000 µg/mL), respectively, for 48 h. After that, 20

150

µL of 5 mg/mL MTT was added to each well and incubated for 4 h. The medium was

151

then removed and replaced with 100 µL of DMSO. Finally, the 96-well microplate

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was placed in a microplate spectrophotometer (Versamax, Molecular Devices,

153

Sunnyvale, CA, USA) and the absorbance was measured at 570 nm. The cell viability

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was calculated by the absorbances of the treated cells and the control cells,

155

respectively.

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Apoptosis Analysisby Flow Cytometry

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The apoptosis of MCF-7 cells was measured by an Annexin V-FITC/ PI apoptosis

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detection kit (Bipec Biopharma Corporation, City, MA. USA) and analysed by a flow

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cytometry FACS™ Universal Loader (BD Becton Dickinson, Franklin Lakes, NJ,

160

USA). MCF-7 cells were harvested after treatment with WP1-Zn at various

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concentrations (75, 150 and 300 g/mL)for 48 h. After incubation, the cells were

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washed three times with PBS and digested by 500 µL of trypsin at 37 °C for 3 min.

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Then the cells were suspended in the binding buffer provided by the kit and the

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suspension was divided into two parts. One part was dyed by FITC-labeled Annexin 9



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V and PI in the dark for 15 min while the other part used as the control. The dyed

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cells were analyzed by a flow cytometer with the excitation wavelength at 488 nm.

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The signals from FITC and PI were read at 516 nm and 560 nm, respectively. The

168

obtained data was analyzed using the Cell Quest Research Software(Becton

169

Dickinson).

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Cell Cycle Analysis by Flow Cytometry

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The cell cycle of MCF-7 cells was monitored using a flow cytometer through PI

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staining method. MCF-7 cells were treated with WP1-Zn for 48 h and washed three

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times in PBS. Cells were fixed with 70% ethanol at -20°C overnight, then stained by

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10µL RNase(100 µg/mL) and PI mixture in the dark for 30 min. The dyed cells were

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washed three times in PBS and analyzed by a flow cytometer. Cell cycle distribution

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of MCF-7 cells was measured by the fluorescence intensity and the data was analyzed

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using the Cell Quest Research Software.

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Nuclear Morphology Analysis

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The nuclear morphology of MCF-7 cells was observed using a fluorescence

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microscope (Nikon Eclipse 80i,Nikon). MCF-7 cells were seeded at the density of 5 ×

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104 cells/well into 6-well culture plates. WP1-Zn at different concentrations(75, 150

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and 300 µg/mL) were added to each well and incubated for 48 h. The cells were then

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washed in PBS three times and digested by 500 µL of trypsin at 37 °C for 3 min. After

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fixed with 4% methanol at 4 °C for 10 min, cells were centrifuged at 2,000 g for 10 10


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min. The harvested cells were suspended with 30 µL of PBS and placed on a glass

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slide which was previously air-dried and stained with 100 µL of Hoechst33258 for 10

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min. Cells were then washed with PBS three times and observed with the excitation

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and emission actively participate by activating at 350nm and 460 nm, respectively.

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Separation of DNA by Gel Electrophoresis

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The nuclear DNA was extracted from MCF-7 cells and the DNA fraction was

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separated by electrophoresis. Briefly, MCF-7 cells were treated with WP1-Zn at

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different concentrations (75, 150 and 300 µg/mL) at 37 °C for 48 h. The treated cells

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were then washed in PBS for three times and lysed in the lysis buffer (0.5% Triton

194

X-100, 10 mM EDTA and 10 mM Tris-HCl, pH 7.5) for 25 min. The cell-lysates were

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incubated at 37 °C for 30 min and for another 1 h at 55 °C. After incubation, the

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cell-lysates were dissolved in a proteinase K(0.25 mg/mL) and RNase (0.03 mg/mL)

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mixture. Phenol chloroform was used to extract the nuclear DNA of the MCF-7 cells

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and the extraction was then purified with isopropyl alcohol and incubated at -20 °C

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for 20 min. The nuclear DNA was washed with for three times and suspended in

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DEPC treated water. After that, the DNA sample was dyed by 1 mg/mL ethidium

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bromide

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electrophoretograms were captured and collected by a gel image processing system

203

(Tanon 1600,Tanon, Shanghai, China).

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Mitochondrial Membrane Potential (MMP) Assay

and

separated

by

electrophoresis

(2.0%

11


agarose

gel).The


205

The mitochondrial membrane potential (MMP) was measured and analyzed by a flow

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cytometer, fluorescence microscopy and Fluoroskan ascent microplate fluorometer

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(Thermo Electron Corporation, Vantaa, Finland). Briefly, MCF-7 cells were treated

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with different concentrations of WP1-Zn (75, 150 and300 µg/mL) for 48 h in 6-well

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plates and tryptic digested by 500 µL of trypsin for 3 min at 37 °C. The treated cells

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were then washed three times in PBS and dyed using 10 mg/mL JC-1 for 10 min in

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the dark at 37 °C. The dyed cells were washed and suspended in PBS and the MMP of

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cells was analyzed using flow cytometry. The excitation wavelength for flow

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cytometric analysis is 488 nm and the emission wavelengths are 525 nm for green and

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590 nm for red. The fluorescence intensity of the dyed cells was determined using the

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fluorometer with the excitation and emission wavelengths at 590 nm and 525 nm,

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respectively. Further, to observe the fluorescence image, the dyed cells were spread

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on a glass slide and observed using a fluorescence microscope with Argon-ion 488

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nm laser excitation (Nikon Eclipse 80i).

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Determination of Reactive Oxygen Species (ROS) Generation

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The intracellular ROS level of MCF-7 cells was determined using method previously

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described with some modification.42 MCF-7 cells were seeded on 96-well plate and

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washed three times in PBS. Then, 10 µM DCFH-DA were added to the cells and

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incubated at 37°C for 30 min. After incubation, the cells were washed again with PBS

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three times and treated with WP1-Zn at different concentrations (75, 150 and 300

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µg/mL) at 37°C for 30 min. The intracellular ROS production was measured by the 12


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fluorometer with the excitation and emission wavelength at 488 nm and 525 nm,

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respectively. The fluorescence intensity of cells was also observed by the fluorescence

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microscope with the excitation and emission wavelength at 495 nm and 525 nm,

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respectively.

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Determination of Caspase Activity

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MCF-7 cells were treated with WP1-Zn at different concentrations (75, 150 and 300

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µg/mL) for 48 h and suspended in lysis buffer on ice for 1 h. After incubation, the cell

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lysates were divided into two parts. One part of cell lysates were centrifuged at 11,000

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× g for 30 min. Supernatants were collected the protein concentration was determined

235

using a BCA assay kit. The other part of cell lysates were treated with the specific

236

caspase substrates (Ac-DEVD-AMC for caspase-3, Ac-IETD-AMC for caspase-8,

237

andAc-LEHD-AMC for caspase-9) in 96-well plates at 37 °C for 1 h. The caspase

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activity was measured by a fluorometer with the excitation and emission wavelength

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at 380 nm and 440 nm, respectively.

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Statistical Analysis

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All experiments were repeated at least three times. Statistical analysis was done using

242

SPSS 19.0 software (IBM Corporation, NY, USA). The differences between the

243

control and the experimental groups were analyzed using the two-tailed Student’s

244

t-test. The difference between three or more groups was analyzed by one-way

245

ANOVA for multiple comparisons. Interactions with P < 0.05 were determined to be 13



246

statistically significant. If there is statistical significance between two groups,

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different letters (as a1, a2, a3, a4...or a, b, c, d, etc etc.) were used to label these

248

groups in figures. Otherwise, the same letter was used to label no statistical

249

significance between two groups.

250

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14


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RESULTS AND DISCUSSION

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Synthesis and Characterization of WP1-Zn

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The combination of WP1-Zn is achieved through ionic reactions between Zn2+ and

255

COO- in walnut peptide molecule. Initially, 10 % ammonia was added to the mixture

256

to react with the H+ and promoted the ionization of COO- in walnut peptides molecule.

257

It should be noted the pH could not exceed 6.0 to avoid the formation and

258

precipitation of Zn(OH)2.

259

The surface morphology of WP1-Zn and WP1 was shown in Figure 1A and Figure

260

1B. It could be seen clearly that WP1-Zndisplayed a nubbly appearance whereas WP1

261

showed a powdery morphology. The surface elemental composition of WP1-Zn and

262

how Zn incorporated with WP1 were investigated by SEM-EDX and FT-IR.

263

SEM-EDX results showed that the WP1was mainly composed of three elements, C

264

(69.71%), N (6.62%) and O (19.46%) (Figure 1C). After conjugation with Zn, the

265

surface elemental composition of WP1-Zn has changed to C (57.83%), N (2.77%), O

266

(14.63%) and Zn (24.77%) (Figure 1D), indicating that Zn has been successfully

267

incorporated with WP1 and the conjugation rate was 24.77%. Further, FT-IR was used

268

to characterize the structure of WP1-Zn, as illustrated in Figure 1E.In the FT-IR

269

spectrum of WP1, the absorbance peak at 3384 cm-1 was attributed to the stretching

270

vibrations of –N–H group. The peaks at 2961 cm-1 and 1661 cm-1were assigned to

271

–O–H group and C=C group, respectively. However, in the spectrum of WP1-Zn, the

272

peak belonged to –N–H group shifted from 3384 cm-1 to 3427 cm-1 and the 15



273

transmittance λ also increased from 28% to 47%, indicating the absorption intensity

274

of the peak decreased, which may derive from replacement of Zn to H. Besides, the

275

peak assigned to –O–H group shifted from 2963 cm-1 to 2926 cm-1 and the peak

276

assigned to C=C group shifted from 1661 cm-1 to 1642 cm-1. The decreased

277

absorption intensities of these two peaks suggested that Zn atoms concatenated with O

278

atoms by replacing H atoms and also linked with C atoms through breaking

279

carbon-carbon double bonds. All these data demonstrated that the conjugation of

280

WP1with Zn ions was achieved through Zn-N and Zn-O bonds.

281

Antiproliferative Activity of WP1-Zn

282

The cytotoxicity of WP1-Zn on MCF-7,SGC-7901, A549, Hela, PC3 and LO2 cells

283

was assessed by MTT assay. As observed in Figure 2A, WP1-Zn showed significant

284

antiproliferative capacities on the tested cells in a dose-dependent manner, indicating

285

that WP1-Zn exhibited a broad spectrum of inhibition on human cancer cells. Among

286

all the cells examined, the growth of MCF-7 cells was most susceptible to WP1-Zn.

287

The survival rate of MCF-7 cells decreased to 20.0% with 300 µg/mL of WP1-Zn.

288

The antiproliferative ability of WP1-Zn on human normal hepatic LO2 cells was also

289

measured to evaluate the toxicity effect of WP1-Zn. Compared with WP1-Zn, little

290

antiproliferative effect of WP1 was found on the selected cancer cells as shown in

291

Figure 2B, implying that the antitumor ability of WP1-Zn was much higher than that

292

of WP1 and the synthesis of WP1 and Zn contributed much to the antiproliferative

293

activity. Since MCF-7 cells were specifically sensitivity to WP1-Zn, this cell line was 16


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chosen for further investigation on the revealing mechanisms involved the antitumor

295

activity of WP1-Zn.

296

Induction of Cell Apoptosis byWP1-Zn

297

There are two forms of cell death, namely apoptosis and necrosis. Apoptosis, as the

298

dominant form of programmed cell death, plays an important role in cellular

299

homeostasis. The distinct characterizations of apoptosis are the shrinkage of the cell,

300

hyper condensation of chromatin, cleavage of chromosomes into nucleosomes, and

301

formation of membrane-enclosed apoptotic bodies packaging cellular content.

302

43

303

apoptosis in cultured human cancer cells.41, 44In contrast, necrosis is caused by cell

304

injury and is often detrimental by initiating inflammation of surrounding tissues.45, 46

Previous reports suggest that zinc complex can induce proteasome inhibition and

305

To investigate whether WP1-Zn could induce apoptosis or necrosis mediated cell

306

death, Annexin V-FITC/PI staining assay was performed. Early stage apoptotic cells

307

and living cells can be distinguished by Annexin V because the phosphatidylserine

308

(PS) in apoptotic cells exposes at the outer leaflet of the plasma membrane, which can

309

be identified by Annexin V. When combined with PI, the double labeling procedure

310

allows a further distinction of necrotic (Annexin V+/PI+) and early stage apoptotic

311

(Annexin V+/PI−) cells.47 In Figure 3A, the lower left quadrant(LL), the lower right

312

quadrant (LR), the upper left quadrant (UL), and the upper right quadrant (UR)

313

represent normal cells, early apoptotic cells, necrotic cells and late apoptotic cells,

314

respectively. The augmentation of the concentration of WP1-Zn results in a 17



315

remarkable decrease in normal cells. The ratio of normal cells in the control group is

316

94.40%, which decreased to 14.78% after being treated with 300 µg/mL WP1-Zn. The

317

treatment with WP1-Zn also caused a dose-dependent increase in early stage apoptotic

318

cells, from 0.03% to 19.06% when exposed to 300 µg/mL WP1-Zn. Besides, the late

319

apoptotic cells increased drastically, from 0.65% to 61.01%, when the concentration

320

of WP1-Zn rose from 150 to 300 µg/mL. The necrotic cells also increased from 0.2%

321

(150 µg/mL) to 5.15% (300 µg/mL). However, there was no significant increase in the

322

early stage apoptotic cells when the concentration rose from 150 µg/mL to 300 µg/mL.

323

These results indicated that low concentration of WP1-Zn (< 150 µg/mL) mainly

324

induced early-stage apoptosis, while higher concentration of WP1-Zn caused mainly

325

late stage-apoptosis and necrosis. In early-stage apoptosis, the targeted cells activates

326

a cascade of biochemical reactions that lead to natural cell death. In contrast, the

327

process of necrosis releases cellular content and subsequently induces an

328

inflammatory response in the effected tissue. Thus, to avoid the inflammatory effect

329

induced by necrosis, the concentration of WP1-Zn used in cancer treatment should be

330

less than 300 µg/mL.

331

To further confirm thatWP1-Zn induced cell death through apoptosis, the nuclear

332

morphological change of MCF-7 cells incubated withWP1-Zn was examined using

333

the Hoechst 33258 assay. Hoechst 33258 is a cell-permeant, minor groove binding

334

DNA stain which fluoresces bright blue when binding with DNA. As shown in Figure

335

3B, round-shape nuclei with homogeneous fluorescence was observed in the control 18


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336

group. After being incubated with WP1-Zn, the nucleus appeared slightly smaller than

337

normal nucleus in the control group. Chromatin condensed and assembled at the

338

nuclear membrane as revealed by a bright fluorescence at the periphery. These small

339

rounded masses around the nuclear membrane indicated the formation of apoptotic

340

bodies (arrow in Figure 3B).The DNA fragmentation can be clearly observed in the

341

nuclear morphology of MCF-7 cells treated with 150 µg/mL and 300 µg/mL WP1-Zn.

342

Since these nuclear morphologies were indicators of apoptosis, these results further

343

confirmed that the death of MCF-7 cells induced by WP1-Zn was through apoptosis.

344

Agarose gel electrophoresis was performed to assess nuclear DNA strand breaks

345

after being treated with WP1-Zn.(Figure 3C) Specially, Lane (a) was a DNA marker

346

lane which was used to determine the molecular weight of each DNA fragmentation.

347

As observed in lane (b), DNA was intact and no DNA ladder was found in the control

348

group. In contrast, DNA ladder formation, which is one of the characteristics for

349

apoptosis, was detected in the WP1-Zn treated group. Besides, the DNA fragments

350

increased whenWP1-Zn concentration augmented. Therefore, it is reasonable to

351

assume that the mode of MCF cells death induced by WP1-Zn was apoptosis.

352

Effect of WP1-Zn on Cell Cycle

353

PI staining assay was used to determine whether the antiproliferative effect of

354

WP1-Zn was induced by apoptosis or the combination of apoptosis and cell cycle

355

arrest. Cell cycle phase is identified by the DNA content of cells which is determined

356

by PI staining. Although PI does not enter untreated cells with great efficiency, PI 19



Page 20 of 44

357

cannot effectively enter untreated cells. But after ethanol fixation, the membrane

358

permeabilization increased significantly which enable the PI to enter the nucleus,

359

resulting in quantitative DNA staining.48 Cell cycle distribution of MCF-7 cells

360

treated with WP1-Znis revealed in Figure 4A and Table 1. The ratio of S phase

361

arrested cells was observed in a dose-dependent increase manner, which increased

362

from 35.70% (control) to 70.19% (300 µg/mL) upon the WP1-Zn concentration

363

increase. The increase ofWP1-Zn concentration also led to the reduction of the

364

proportion of G0/G1 phase arrested cells, from 51.49% (control) to 18.62% (300

365

µg/mL). The S phase of the cell division cycle is the period where cells replicate their

366

DNA (and thus the DNA content of S phase is intermediate between that of G0/G1

367

and G2/M cells).49 The accumulation of S phase arrested cells resulted in the

368

retardation of cell mitosis and inhibition of cell proliferation. Therefore, conclusion

369

could be drawn that WP1-Zn-induced cell growth inhibition was the result of both

370

apoptosis and S phase cell cycle arrest.

371

Mitochondrial Dysfunction Induced by WP1-Zn

372

It is known that two pathways could lead to apoptosis, initiated by mitochondria

373

(intrinsic

374

Mitochondria-mediated apoptosis occurs in response to internal stimuli such as

375

irreparable genetic damage, extremely high concentrations of cytosolic Ca2+ or severe

376

oxidative stress. The internal stimuli increase the permeability of the mitochondrial

377

membrane, which can be measured by mitochondrial membrane potential. In this

pathway)

or

the

cell

surface

receptors

20


(extrinsic

pathway).

Page 21 of 44


378

study,

mitochondrial

membrane

potential

(△Ψm)

379

mitochondrial-targeted fluorescent probe JC-1 and determined by flow cytometry.

380

Normal cells with functional mitochondria showed red JC-1-aggregates,while

381

apoptotic cells with impaired mitochondria contained green JC-1 monomers. In

382

Figure 4B, the UR and LR represent the red fluorescence (aggregated form of JC-1)

383

and green fluorescence (monomeric form of JC-1), respectively. The cells treated with

384

WP1-Zn for 48 h showed a significant decrease in△Ψm evidenced by a shift in JC-1

385

fluorescence from red to green. There were 81.11% depolarized cells at 300µg/mL as

386

compared to the control cells (1.26%).The fluorescence intensity quantify the

387

fluorescence change. In Figure 4C, the ordinate was expressed as the percentage of

388

red/green Fluorescence intensity compared to the control group. The red/green

389

fluorescence intensity at 300 µg/mL dropped to 58.27% compared with the control

390

group, suggesting that the depolarized cells were 1.71 times more than normal cells

391

when treated with 300 µg/mL WP1-Zn. When examined by fluorescent microscopy

392

(Figure 4D), the control cells displayed an intense red fluorescence and weak green

393

fluorescence. However, the cells treated with WP1-Zn exhibited brighter green

394

fluorescence with a remarkable reduction in red fluorescence implying a loss of MMP,

395

which is extremely obvious at 300 µg/mL. The loss of mitochondrial membrane

396

potential is a biomarker of increased permeability of mitochondrial membrane.

397

According to previous reports, increased permeability of mitochondrial membrane

398

will promote the release of certain mitochondrial proteins, most notably cytochrome c

21


was

detected

by

the


399

which help form procaspase-9.50 Caspase-9 is an initiator caspase that activates

400

downstream executioner caspases and finally bring about apoptosis.51 Therefore, the

401

mitochondrial-mediated pathway was confirmed in the WP1-Zn-induced cell

402

apoptosis.

403

Reactive Oxygen Species (ROS) Generation Induced by WP1-Zn

404

Since the loss of mitochondrial membrane potential is usually triggered by ROS

405

generation, the ROS level in MCF-7 cells was examined by DCFH-DA assay. As

406

shown in Figure 5A, the DCF fluorescence value increased upon a dose-dependent

407

manner. The ROS generation in the MCF-7 cells exposed to WP1-Zn(300µg/mL)

408

increased by 184.98% compared to those in the control group. The fluorescence

409

intensity change of dyed ROS was also observed under a fluorescence microscopy

410

(Figure 5B). The control cells showed a weak green fluorescence. However, the green

411

fluorescence in the cells treated with WP1-Zn became brighter, indicating a higher

412

production of ROS. Zinc could impair antioxidative glutathione (GSH) system and

413

upregulate the production of reactive oxygen species (ROS).52, 53 Besides, previous

414

studies on the ZnO nanoparticles have clarified that dissolved zinc ions played an

415

important role in the toxicity of ZnO nanoparticles.54, 55 Therefore, the overproduction

416

of ROS induced by WP1-Zn may mainly attribute to the release of zinc ions.

417

Induction ofCaspase-activation Apoptosis by WP1-Zn

418

Caspases are involved in both the mitochondria-mediated and cell surface 22


Page 22 of 44

Page 23 of 44


419

receptor-mediated pathways of apoptosis.43 All caspases are generated as catalytically

420

inactive zymogens and must be proteolytic activated during apoptosis. Caspases

421

participated in the apoptosis are generally divided into two categories, the initiator

422

capsases, including caspase-2, -8, -9, and -10, and the effector capsases including

423

caspase-3, -6, and -7.56 The activation of an effector caspase (such as caspase-3 or -7)

424

is performed by an initiator caspase (such as caspase-9) through cleavage at specific

425

point. To determine whether caspase activation is involved in the WP1-Zn induced

426

cell death, the activities of the initiator caspases (caspase-8and caspase-9) and effector

427

caspases-3 were analyzed. Figure 5C shows that the activities of caspase-3,caspase-8

428

and caspase-9 remarkably increased in a dose-dependent way after treated with

429

WP1-Zn. When exposed to WP1-Zn at concentration of 300 µg/mL, the activities of

430

caspase -3,caspase-8 and caspase-9 were upregulated by 100%, 75%and 120%,

431

respectively. Since caspases participated in both the mitochondria-mediated and cell

432

surface receptor-mediated pathways, the conclusion can be made that both these two

433

pathways were involved in the WP1-Zn induced apoptosis.

434

Based on the above results, we can draw the conclusion that the induction of

435

MCF-7 cell apoptosis by WP1-Zn may mainly through two signaling pathways: cell

436

surface receptor-mediated and mitochondrial-mediated pathways. For the cell surface

437

receptor-mediated pathway, WP1−Zn promotes the activation of caspase-8, which

438

consequently activates the downstreamcaspase-3. For the mitochondrial-mediated

439

pathway, WP1−Zn induced the dysfunction of mitochondrion, leading to the 23



Page 24 of 44

440

activation of caspases-9 and -3. The two pathways finally resulted in MCF-7 cell

441

apoptosis (Figure 6).

442

In conclusion, WP1-Zn hybrid drug delivery system was successfully fabricated

443

and the conjugation rate was 24.77%. The combination of WP1 and Zn ions were

444

mainly achieved through Zn-N and Zn-O bonds. WP1-Zn could remarkably inhibit

445

the proliferation of MCF-7, SCG-7901, A549, Hela and PC3 cells especially for

446

MCF-7 cells. WP1-Zn could induce cell cycle arrest in S phase, which directly

447

inhibited cell proliferation as well as caused apoptosis. The pathways of WP1-Zn

448

induced cell apoptosis were also revealed in this study. Mitochondrial disruption was

449

detected in the MCF-7 cells treated with WP1-Zn, which was caused by ROS

450

overproduction.

451

mitochondrial-mediated and cell surface receptor-mediated pathways, were activated

452

with the treatment of WP1-Zn. Hence, WP1-Zn induced carcinoma cell apoptosis via

453

both ROS triggered mitochondrial-mediated and cell surface receptor-mediated

454

pathways. In a word, WP1-Zncomplex exhibits great potential as functional

455

ingredients in food and nutraceuticals.

Furthermore,

caspase-3,

-8

and

-9,

456

24


involved

in

both

Page 25 of 44


457

ACKNOWLEDGEMENTS

458

This study was supported by the Guangdong Natural Science Funds for Distinguished

459

Young Scholars (No.S2013050013954), Program for New Century Excellent Talents

460

in University (NCET-13-0213), Guangdong Province Funded Research Projects

461

(2013B010404001), and Key Laboratory of Aquatic Product Processing, Ministry of

462

Agriculture, P.R. China

(NYJG201402).

463

Also, we appreciate the kind help from Guangdong Provincial Key Laboratory of

464

Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital,

465

Sun Yat-Sen University, Guangzhou, Guangdong, China.

466

Conflict of Interest

467

All authors declare no conflict of interest.

468

25



469

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34


Page 34 of 44

Page 35 of 44


Table 1. Effect of WP1-Zn on the cell cycle of MCF-7 cells

WP1-Zn (µg/mL)

Dip of HEPG2 Control Cells (%)

75

150

300

G0/G1

51.49

45.36

21.62

18.62

S

35.70

39.25

57.51

70.19

G2/M

12.81

15.39

20.88

11.20

635

35



636

FIGURE LEGENDS

637

Figure 1. Characterization of WP1-Zn. SEM images of WP1 (A) and WP1-Zn (B).

638

Surface elemental compositions of WP1 (C) and WP1-Zn (D) obtained from EDX

639

analysis. FTIR spectrum of WP1 and WP1-Zn (E).

640

Figure 2. Antiproliferative activity of WP1-Zn. Cytotoxic effect of WP1-Zn (A) and

641

WP1 (B) on selected human carcinoma and normal cell lines.

642

Figure 3. Flowcytometric analysis of MCF-7 cells treated with WP1-Zn at 75 µg/mL

643

(A2), 150 µg/mL (A3) and 300 µg/mL (A4) for 48 h. The control group was treated

644

with PBS (A1). The lower left quadrant(LL)represents normal cells; the lower right

645

quadrant (LR) represents early apoptotic cells; the upper left quadrant (UL)represents

646

necrosis cells and the upper right quadrant (UR) represents late apoptosis cells. (B)

647

Nuclear morphology of MCF-7 cells treated with WP1-Zn (75, 150 and 300 µg/mL)

648

for 48 h. (C) DNA fragmentation of MCF-7 cells treated with PBS (lane b) and

649

WP1-Zn at 75 µg/mL (lane c), 150 µg/mL(lane d) and 300 µg/mL (lane e) for 24 h.

650

Lane a served as a DNA marker.

651

Figure 4. (A) Cell cycle distribution of MCF-7 cells treated with WP1-Zn at different

652

concentrations (75, 150 and 300 µg/mL). (B) Mitochondrial membrane potential

653

(MMP) changes inMCF-7 cells induced by WP1-Zn at 75 µg/mL (B2), 150 µg/mL

654

(B3) and 300 µg/mL (B4). The control group was treated with PBS (B1). The UR and

655

LR represent the red fluorescence(aggregated form of JC-1) and green fluorescence 36


Page 36 of 44

Page 37 of 44


656

(monomeric form of JC-1), respectively. (C) Red/green fluorescence intensity of

657

changes in MMP induced by WP1-Zn (75, 150 and 300 µg/mL). (D) Fluorescence

658

images of changes in MMP induced by WP1-Zn (75, 150 and 300 µg/mL).

659

Figure 5. (A) Intracellular ROS level induced by WP1-Zn at different concentrations

660

(75, 150 and 300 µg/mL) in MCF-7cells. (B) Fluorescence images of intracellular

661

ROS generation induced by WP1-Zn (75, 150 and 300 µg/mL). (C) Activity of

662

caspase-3, caspase-8 and caspase-9 in MCF-7 cells after treated with WP1-Zn (75,

663

150 and 300 µg/mL) for 48 h.

664

Figure 6. Possible mechanisms of WP1-Zn-induced apoptosis of MCF-7 cells.

37



A

Page 38 of 44

B

C

D

WP1 WP1-Zn

100

888

E

4000

1054

1402

1053

1374

1642 2963

3500

1661

20

3427

40

1453

60

3384

τ(λ) /%

2926

1424

80

3000

2500

2000

1500 -1

Wavenumber /cm

38


1000

500

Page 39 of 44


Figure 1

A

LO2 MCF-7 SCG-7901 A549 PC3 Hela

140

Survival rate (%)

120 100

a6 a1 a3 a4a5 a2

a1 b6 b5

80

b1 b3 b2 b4

60

c1 c3 c2 c4c5c6

40 20

d2d3d4 d5d6

0

37.25

150

75

300

Concentration of WP1-Zn (µg/mL)

140

B

a5 a3

120

ab1 bc2

Survival rate (%)

a5a6 c4

b1

a2

a6

bc4

ab3

a1

b5

ab4

a4 a6

a1

ab2

b3

100

LO2 MCF-7 SCG-7901 A549 PC3 Hela b5

c2 b3

b6

80 60 40 20 0

125

250

500

1000

Concentration of WP (µg/mL)

Figure 2

39



Page 40 of 44

A

B

Control

75µg/mL

150µg/mL

C

Figure3

40


300µg/mL

Page 41 of 44


A

Control

75µg/mL

150µg/mL

300µg/mL

B

a 100

Red/green Fluorescence Intensity (% of control)

C

b

80

c d

60

40

20

0

Control

125

250


D

41


500


Control

75 µg/mL

150 µg/mL

A

Page 42 of 44

300 µg/mL

DCF Fluorescence (% of control)

Figure 4 d

300

250

c 200

b 150

a

100

50

0

control

75

150

300


B

Control

75 µg/mL

150 µg/mL

300 µg/mL

C 300

Control 75 µg/mL 150 µg/mL 300 µg/mL

Caspase Activity (% of Control)

250

a

a

200

a b

b

150

b

c c

c d

d

d

Caspase-3

Caspase-8

Caspase-9

100 50 0

Figure 5 42


Page 43 of 44


Figure 6

43



665

Table of Contents Graphic

44


Page 44 of 44

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