Tea Polysaccharides Inhibit Colitis-Associated Colorectal Cancer via

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Bioactive Constituents, Metabolites, and Functions

Tea Polysaccharides Inhibits Colitis-associated Colorectal Cancer via Interleukin-6/STAT3 Pathway Li-Qiao Liu, Mingyue Shen, Shao-Ping Nie, Jie-Lun Hu, Qiang Yu, Deming Gong, and Ming-Yong Xie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00710 • Publication Date (Web): 15 Apr 2018 Downloaded from http://pubs.acs.org on April 15, 2018

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1 2 3

Tea Polysaccharides Inhibit Colitis-associated Colorectal

4

Cancer via Interleukin-6/STAT3 Pathway

5 6

Li Qiao Liu,a, b Shao Ping Nie,* a Ming Yue Shen, a

7

Deming Gong, a, c

Jie Lun Hu, a

Qiang Yu, a

Ming Yong Xie* a

8 9

a

State Key Laboratory of Food Science and Technology, Nanchang University, Nan-

10

chang, Jiangxi 330047, China; b Basic Medical College, Nanchang University, Nan-

11

chang, Jiangxi 330047, China; c New Zealand Institute of Natural Medicine Research,

12

Auckland, 2104, New Zealand

13 14 15 16 17 18

Corresponding authors

19

* Professor Shao Ping Nia. State Key Laboratory of Food Science and Technology,

20

Nanchang University, 235 Nanjing East Road, Nanchang 330047, China. Tel/Fax: +86

21

791-88304452. E-mail address: [email protected]

22

* Professor Ming Yong Xie. State Key Laboratory of Food Science and Technology,

23

Nanchang University, 235 Nanjing East Road, Nanchang 330047, China. Tel/Fax: +86

24

791-83969009. E-mail address: [email protected]



25

ABSTRACT:

26

The interleukin-6 (IL-6)/signal transducer and activator of transcription (STAT)-3

27

signaling pathway regulates proliferation and survival of intestinal epithelial cells, and

28

has profound impact on the tumorigenesis of colitis-associated cancer (CAC). Tea

29

polysaccharides (TPS) are the major nutraceutical component isolated from tea-leaves

30

and is known to possess antioxidant, anti-inflammatory and antitumor bioactivities.

31

Here, we investigated the antitumor activities of TPS on CAC using the

32

azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model and IL-6-induced

33

colorectal cancer cell line (CT26), and determined whether TPS exerted its antitumor

34

effects through the IL-6/STAT3 pathway. Results demonstrated that TPS significantly

35

decreased the tumor incidence, tumor size, and markedly inhibited the infiltration of

36

pro-inflammatory cells and the secretion of pro-inflammatory cytokines via balancing

37

cellular microenvironment. Furthermore, we found that TPS suppressed the activation

38

of STAT3 and transcriptionally regulate the expressions of downstream genes includ-

39

ing MMP2, cyclin Dl, survivin and VEGF, both in vivo and in vitro. Thus, it is con-

40

cluded that TPS attenuate the progress of CAC via suppressing IL-6/STAT3 pathway

41

and downstream genes’ expressions, which indicated that TPS may be a hopeful anti-

42

tumor agent for the prevention and treatment of colon cancer.

43

KEYWORDS: Tea polysaccharides (TPS), colitis-associated cancer (CAC), inter-

44

leukin-6 (IL-6), signal transducer and activator of transcription-3 (STAT3)


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INTRODUCTION

46

Colorectal carcinoma (CRC) is the third most common malignancy in the devel-

47

oped countries, with the change of people’s eating habits and lifestyle, the morbidity

48

of patients with colon cancer has been rising steadily in the developing countries in

49

recent years.1-3 Inflammatory bowel disease (IBD) sufferers are at increased risks for

50

developing ulcerative colitis-associated colorectal cancer (CAC).4-5 One of the im-

51

portant underlying etiologies of carcinogenesis in the colon is chronic inflammation,6

52

which can cause the gradual loss of the host’s antitumor immune effect, and might

53

promote the growth of malignant tumor.7

54

The chronic intestinal inflammation is also considered as one of the main influ-

55

ence factors in the progress of CAC by facilitating pro-inflammatory cytokine pro-

56

duction, cell proliferation, alterations in immune responses and thereby promoting

57

tumorigenesis.8-9 Therefore, the anti-inflammatory therapy may be an effectively way

58

to reduce the incidence of CAC, and many epidemiological studies have found

59

chemoprevention effects of natural or synthetic compounds in preventing, or revers-

60

ing the process of carcinogenesis.10-11

61

One of the cytokines, IL-6, a critical tumor-associated inflammatory promoter

62

during early CAC tumorigenesis, was mostly mediated by the signal activator and

63

regulator of transcription STAT3 protein overexpressed and persistently activated in

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CAC.12 Transcription factors STAT3 are originally located in the cytoplasm in their

65

inactive state. After “receives” those extracellular signals, including cell growth fac-

66

tors, cytokines, and hormones, the activated Janus kinases (JAKs) subsequently



67

phosphorylates STAT3 on Tyr 705. Activated STAT3 protein dimerizes and translo-

68

cates from the cytoplasm into nucleus, where they binding to specific recognition sites

69

and regulate the expression of downstream target genes involved in proliferation, sur-

70

vival, migration and invasion.13-14 The constitutive activation of STAT3 proteins was

71

constantly detected in clinical tumor specimens from an extensive range of human

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tumor, especially gastrointestinal carcinoma.15 Hence, STAT3 has been considered as

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a hopeful pharmacological target of cancer chemoprevention and therapy.

74

China is the hometown of tea (Camellia sinensis L.O. Kuntze), and it is the larg-

75

est tea producer in the world.16 In 2015, the cultivated area of tea trees in China

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reached up to 2.87 million hectares. Tea polysaccharides (TPS) are a kind of bioactive

77

compound with great potential for exploitation. Along with the increase of people’s

78

living standard, the coarse leaves are unsalable for their worse taste so that it causes a

79

huge waste of nature resources. Extracting TPS from coarse tea leaves can not only

80

utilize the tea leaves resources sufficiently but also is significant to promote human

81

health.17 While the low-grade/coarse leaves was discovered to contain more polysac-

82

charides when compared with the high-grade/tender tea leaves.18 The monosaccharide

83

compositions of TPS were confirmed to be rhamnose, arabinose, ribose, xylose,

84

mannose, galactose, glucose and so on.19 It has been reported that TPS had diverse

85

biologically activities such as anti-tumor, hypolipidemic, hypotensive, antioxidant and

86

anti-virus activities, and enhanced body immunity function.20

87

The current clinic treatment of CRC is not satisfactory, and the common treat-

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ment may lead to the body immunological system damages.21 Fortunately, the


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food-borne TPS has a specific feature of low toxicity and immunoregulation function,

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without above-mentioned drawbacks.22 Recent numerous studies have described the

91

antitumor activity of polysaccharides in tumor-bearing mice and tumor cell lines.23-28

92

These researches mainly focused on the chemical constitution and structure of poly-

93

saccharides, and explored its immunoregulatory and antitumor functions. However,

94

the details of the underlying molecular mechanisms behind these biological functions

95

have remained unclear. In this study, we used the purified TPS whose structure was

96

preliminarily identified by our group,29-30 and investigated the anti-tumor effects of

97

TPS on CAC mice colon carcinogenesis and mouse colon tumor cell line CT26 under

98

inflammatory conditions. We have, for the first time, found that TPS may exhibit an

99

antitumor effect via inhibiting STAT3 activation through IL-6/STAT3 signaling path-

100

way, and offered new perspective into the potential treatment of regulating the in-

101

flammatory cell infiltration and STAT3 activation by TPS.

102

MATERIALS AND METHODS

103

Reagents and chemicals

104

Extraction and purification of TPS from the low-grade tea leaves were collected

105

in Wuyuan County, Jiangxi Province, China. This specie was identified by Prof.

106

Zhi-hong Fu, Jiangxi University of Traditional Chinese Medical University (Nan-

107

chang, China). A voucher specimen (Wu tea No.1) were deposited at the State Key

108

Laboratory of Food Science and Technology, Nanchang University, Jiangxi Province,

109

China.



110

The best condition was 80% aqueous alcohol, 1:10 as the ratio for 24 h. The res-

111

idue could be placed in boiling water for 4 h. Extracted twice, use 80% alcohol to

112

precipitate the crude polysaccharides. Use Sevag method to remove protein, and this

113

deproteinization step was repeated 3 times. The crude polysaccharides were extracted

114

in distilled water for 72 h, concentrated by vacuum and then freeze dried.30

115

Azoxymethane (AOM) was from Sigma-Aldrich Corporation (St. Louis, MO,

116

United States). Dextran sulfate sodium (DSS) was from MP Biomedicals Inc. (Irvine,

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CA, United States). Anti-mouse CD68 PE, anti-mouse CD3 FITC, and Anti-mouse

118

Gr-1 FITC were purchased from eBioscience (San Diego, CA, United States). IL-6

119

recombinant protein was obtained from R&D Systems (Abingdon, Oxon, United

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Kingdom). Primary antibodies against t-STAT3 and p-STAT3 were obtained from

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Abcam (Cambridge, UK); Antibodies against cyclin D1, survivin and IL-6 were ob-

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tained from Cell Signaling Technology Inc. (Danvers, MA, USA); Antibodies against

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MMP-2 and VEGF were obtained from R & D Systems (Minneapolis, MN, USA);

124

Antibody against β-actin was purchased from ZSGB-BIO (Beijing, China). The sec-

125

ondary antibodies were purchased from ZSGB-BIO (Beijing, China).

126

Cell culture and conditioned culture

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Mouse colon carcinoma cells (CT26) were obtained from Type Culture Collec-

128

tion of Chinese Academy of Sciences (Shanghai, China). RAW 264.7 cells were pur-

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chased from ATCC (Rockville, MD, United States). CT26 cells and RAW 264.7 cells

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were grown in RPMI-1640 medium with 10% fetal bovine serum (FBS). Cells were

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grown in the carbon dioxide incubator at 37 °C. After 25 ng/ml IL-6 were added into


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the RAW 264.7 cells culture medium or 12 h, the culture media were removed and

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and replaced with free-serum media for the next 12 h, then the culture supernatant of

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RAW 264.7 cells was collected by centrifugation at 3,000 rpm/min for 8 min. In the

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co-culture system, cells were placed in the culture supernatant in the with or without

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of TPS for 24 h (RAW 264.7 and CT26 in a ratio of 1:4).

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Cell proliferation assays

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CT26 cells’ survival ability were measured by the Cell Counting Kit 8 (CCK8;

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Dojindo Lab, Kumamoto, Japan). CT26 cells were seeded in 96-well plates exposed

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to the conditional culture in the absence or presence of TPS for 24 h. After 24 h, 10 µl

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of CCK8 reagent was dripped into and the CT26 cells were cultivated at the carbon

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dioxide incubator for 1 h. The optical density (OD) was measured with a microplate

143

reader (Thermo, Shanghai, China) at 450 nm immediately.

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Cell matrigel invasion assay

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CT26 cells’ migration rate was investigated by using transwell chambers assays.

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Briefly, the experiment was operated after CT26 cells were treated with IL-6-activated

147

RAW264.7 conditioned medium absence or presence TPS for 24 h. In the upper

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chamber, approximately 1 × 104 tumor cells were seeded with serum-free culture me-

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dium, while culture medium including 10% FBS was added into the lower chamber.

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The culture medium containing TPS (40-160 µg/ml) was added into the upper cham-

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ber. About 24 hours later, crystal violet staining was used to observe the invaded cells.

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The number of invaded cells was calculated under microscope.



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AOM/DSS-induced CAC carcinogenesis and TPS treatment

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6-8 weeks BALB/c mice were from Medical Laboratory Animal Center, Medical

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College, Nanchang University. All animals used in this experiment were kept for ac-

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cording to the guidelines published in the Guide for the Care and Use of Laboratory

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Animals (NRC 2011), and this experiment gained admission from Nanchang Univer-

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sity Animal Ethnics Committee, Nanchang University, China. The AOM/DSS mice

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model was widely used in the study of the occurrence and progression of colon cancer

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and a powerful model for chemopreventive intervention studies.31 The AOM/DSS

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mice were intraperitoneally (ip) injected with AOM (10 mg/kg, dissolved in saline)

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and maintained on a regular food and water for 7 days. 2% DSS drinking water for a

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further 7 days. Then all experimental animals were given regular water continued for

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14 days and suffered 2 more 2% DSS water cycles. The CAC mice were randomly

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divided into 4 groups (n = 10). TPS-treated animals received TPS intervention every

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day via gastric incubation from Week 4 to the end of the study. Whereas the model

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control group was given 0.9% saline. The body weights of mice were monitored 3

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times per week. On Day 112, all experimental animals were sacrificed and the colon

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tissues, spleen samples, and thymus samples were obtained. The colon samples were

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opened longitudinally, and counted the number of megascopic tumors with a caliper.

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and then stored at −80 °C for further detection.

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Immunofluorescence of colon tissue

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The macrophage (CD68+), the neutrophil (Gr-1+) and B lymphocyte (CD3+) in-

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filtration analyses were performed on paraffin section of mice colon tissue.32-33 Briefly,


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the thin-tissue sections were deparaffinized, rehydrated and washed with PBS. Then

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the sections were treated with H2O2 and blocked with 5% BSA reacted with an-

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ti-CD68+, anti-Gr-1+ and anti-CD3+ antibodies (1:100) for 1 h. The slides were

178

co-incubated with DAPI about 30 min. Images were observed with a fluorescent mi-

179

croscope (Olympus, Lake Success, NY).

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Immunohistochemistry of colon tissue

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The protein expression levels of STAT3, pSTAT3, IL-6, and cyclin-Dl in the co-

182

lonic tissues were judged by immunohistochemistry. The thin-tissue sections were

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rehydrated, in sequence, from xylene to graded ethanol solutions. The paraffin sec-

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tions were blocked with TBST containing 5% BSA for 2 h, and the sections were then

185

co-incubated with first antibodie at a dilution of 1:100 overnight. The tissue sections

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were co-incubated with the matched secondary antibodie for 2 h. With the manufac-

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turer’s instructions, the slides co-incubated with DAB kit for 10 min. All immuno-

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histochemical staining was assessed by two pathologists blinded to the specimen in-

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formation.

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Cytokine Assay

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The supernatant of the colonic tissue homogenate was harvested and purified.

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The concentrations of IL-2, IL-6, IL-10, TNF-α and IFN-γ were determined by ELISA

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kits (Boster Biotechnology, Wuhan, China) according to the manufacturers’ instruc-

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tions.

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Western blot analysis



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The expression levels of STAT3, pSTAT3, Cyclin-Dl, MMP2, survivin and

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VEGF were determined by Western blotting analysis. Aim proteins were separated by

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SDS-PAGE and were transferred to a PVDF membrane. Blots were blocked with

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TBST containing 5% BSA for 2 h and further incubated overnight at 4 ℃ with the

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first antibodies. After washed with TBST 3 times, the PVDF membranes were

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co-incubated with the secondary antibody RT for 1.5 h. After a further 3 times wash-

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ing, and the PVDF membranes was visualized by using the enhanced chemilumines-

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cence detection system (Bio-Rad, CA, USA).

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Statistical analysis

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Data were expressed as means ± SD. One-way ANOVA was used to determine

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the difference between various groups. All statistical results were analyzed by SPSS

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17.0 software. P values less than 0.05 was deemed to be statistically significant.

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RESULTS

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TPS inhibited the growth of CT26 cells exposed to the conditional culture me-

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dium from IL-6-activated RAW 264.7 cells

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IL-6 was reported to be a determining factor of the alternative activation of

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macrophages and was associated with poor survival.34-35 The growth and invasive ca-

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pacity of CT26 cells were also detected in vitro. The non-contact culture system was

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used in this study.36 The CT26 cells were exposed to culture medium from

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IL-6-stimulated RAW 264.7 cells to determine the bioactivity of TPS on the devel-

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opment of inflammation-associated cancer. It was found that the conditional culture


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medium from IL-6-stimulated macrophages promoted the growth of CT26 cells (Fig-

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ure 1A). Conversely, the treatment with TPS significantly slowed the growth of CT26

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cells in a dose-dependent manner (Figure 1A). Similar to the results from cell prolif-

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eration assay, matrigel invasion assay showed that the invasion ability of CT26 cells

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was significantly decreased by treatment with TPS at concentrations 40-160 µg/ml for

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24 h (Figure 1B and C).

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TPS inhibited IL-6-activated STAT3 pathway in CT26 cells

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Dysregulation of IL-6/STAT3 pathway has a important role in the development

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of colorectal cancer.37-38 Western blotting analysis showed that TPS suppressed the

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conditional medium-induced activation of STAT3 in a concentration dependent man-

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ner (Figure 2). Furthermore, the conditional medium induced the protein expression

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levels of STAT3 downstream proteins, including MMP2, cyclin-Dl, survivin and

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VEGF. Oppositely, TPS reduced the expression levels of these proteins (Figure 2). In

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conclusion, TPS inhibited the activation of STAT3 pathway stimulated by the condi-

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tional medium from IL-6-induced RAW264.7 cells in murine colon carcinoma cells.

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TPS inhibited the occurrence and progression of CAC

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We used the AOM/DSS-induced colonic carcinogenesis BALB/c mouse model to

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determine the anti-tumor effect of TPS on CAC tumorigenesis (Figure 3 A). We found

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that TPS was well tolerated among the BALB/c mice, and no toxic effect and adverse

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reaction were observed in any organ examined of all the mice in experimental groups

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until 112 days (Table 1). Compared to the AOM/DSS group, TPS significantly in-



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creased the survival rate of CAC mice throughout the experiment (Figure 3B). As

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shown in Figure 3C, TPS at high-dose markedly increased the body weight of CAC

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mice, and the body weights among all intervention groups were higher than the

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AOM/DSS group at the end of the study. Also, as shown in Figure 4A, the size of tu-

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mor was smaller and the number was lower in the TPS-intervention groups, and the

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colons were longer in TPS-treated groups than the AOM/DSS group at Day 112. The

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statistical analysis of tumor size, tumor number and colon length showed that TPS

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dramatically attenuated the development of CAC in the AOM/DSS mice (Figure

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4B-D).

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The HE staining technique was used to assess the inflammation status in colonic

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tissue slice (Figure 4E). Macroscopically, multiple nodular-like colonic adenocarci-

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noma with heterogeneous population of cells and cellular cleavage were discovered in

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the distal colon of CAC mice. All findings are consistent with a previous report.39

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Surprisingly, TPS at a high dose was found to inhibit AOM/DSS induced chronic co-

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litis and extensive infiltration of inflammatory cells (Figure 4E). In conclusion, these

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results indicated that TPS relieved colitis-associated colorectal carcinogenesis, and

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thereby attenuating tumor progression in the CAC mice.

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TPS relieved the extent of inflammatory cells infiltration

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Inflammation, especially the chronic inflammation of tumor microenvironment

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can cause the gradually losing of the host’s antitumor immune effect, and might pro-

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mote the growth of malignant tumor.40 To evaluate the pathological degree and sever-

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ity of colonic inflammation of AOM/DSS mice, we stained the section to test the de-


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gree of macrophage (CD68+), neutrophil (Gr-1+) and B lymphocyte (CD3+) infiltra-

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tion. A higher macrophages, neutrophils, and B lymphocytes were accumulated in the

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tumor regions of AOM/DSS mice. Obviously, TPS reduced the clustering of the in-

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flammatory cells into the tumor tissue regions compared with those in the AOM/DSS

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mice (Figure 5A-F). It’s worth noting that high-dose TPS group showed more signifi-

265

cantly than the other two TPS- treated groups in inhibiting the inflammatory cellular

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infiltration.

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TPS changed cytokine secretion in the colon tissues

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The immunosuppressive cytokines produced by the tumor cells were closely re-

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lated to the occurrence and development of inflammatory-associated cancer.41-42 TPS

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treatment was found to change the secretion cytokines in the colon tissue, such as

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IL-2, IL-6, IL-10,TNF-α and IFN-γ (Table 2). Significant decreases in the levels of

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IL-2, IL-6, IL-10,TNF-α and IFN-γ were found in TPS-treated CAC mice, and the

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contents of IL-10 were significantly increased in the 100 mg/kg and 200 mg/kg TPS

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groups compared with the AOM/DSS group on Day 112 (P < 0.05). There was no ob-

275

vious differences of TNF-α, IFN-γ and IL-10 secretion between 50 mg/kg TPS treat-

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ment group and AOM/DSS group at Day 112.

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TPS inhibited STAT3 activation via regulating IL-6/STAT3 pathway

278

IL-6/STAT3 cascade is an important regulator of the proliferation and deprava-

279

tion of tumor-initiating intestinal epithelial cells.43 In our study, we determined

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whether TPS might be able to inhibit the activation STAT3. We measured the levels of



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phosphorylated STAT3 [p-STAT3 (Tyr705)] and total STAT3 (t-STAT3) in the

282

AOM/DSS and TPS-treated groups. It was found that TPS inhibited the phosphoryla-

283

tion of STAT3. Moreover, we examined the expression levels of the downstream pro-

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teins of STAT3, including survivin, cyclin D1, MMP-2, and VEGF by western blots.

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The data showed that TPS significantly decreased the levels of pSTAT3, Cyclin D1,

286

survivin, MMP-2 and VEGF (Figure 6). In addition, the protein levels of STAT3,

287

pSTAT3, Cyclin D1 and IL-6 in mouse colon tissues were examined by immuno-

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histochemical staining. TPS was found to inhibit the expression of phosphorylated

289

STAT3 in colonic tumor tissues of CAC mice (Figure 7). These results suggested that

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TPS may suppress colon tumorigenesis, at least in part, via inhibiting the activation of

291

the IL-6/STAT3 signaling pathway.

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DISCUSSION

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The colorectal cancer is one of the most common malignant cancers, whose

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global incidence rates have been increased gradually. Aside from hereditary factors,

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environmental factors may play key roles in colon carcinogenesis.44 Chronic inflam-

296

mation is closely related to the progression of the carcinogenesis of colon. Patients

297

with colon carcinoma were mainly treated by surgery and assisted with some other

298

ways, such as chemotherapy, radiotherapy and so on.45-46 However, the chemotherapy

299

drugs are limited and have apparent toxic side effects which may cause severe damage

300

to the body's immune system and hematopoietic system.47 So it is necessary to devel-

301

op novel chemopreventive agents with high selectivity and minor toxic side effects.

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Many clinical studies have reported the pharmacological bioactivities of tea, in-


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cluding anti-inflammatory, anti-oxidant, hypolipidemic effects, anti-tumor and im-

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munomodulating effects.48-53 Tea polysaccharides and polyphenols are the major bio-

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active components and may have a bright future in the application in medicine.54 Most

306

of natural polysaccharides are extracted from Chinese herbal plants, such as ginseng

307

and edible fungi, such as Ganoderma lucidum, but they are expensive. More and more

308

researchers are studying the use of tea polysaccharides extracted from the low-grade

309

tea in food and medicine industries. Our previous studies have reported that polysac-

310

charides might be beneficial for the colon health in mice.55-57

311

In this study, we established the AOM/DSS mice model and macrophages-colon

312

cancer cells co-culture model to determine the anti-tumor bioactivity of TPS. Our data

313

showed that TPS substantially improved the survival rate of AOM/DSS mice (Figure

314

3B). Besides, it played a noticeable function to attenuate intestinal chronic inflamma-

315

tion and inhibit carcinogenesis caused by AOM/DSS without side effects (Table 1).

316

TPS significantly reduced the tumor number, tumor size, and occurrence of large size

317

colonic adenomas in the CAC mice (Figure 4). Exploring the molecular mechanism

318

by which TPS exhibits anti-tumor activity is very important. Tumor occurrence and

319

development were driven in various occasions by inflammatory immune cells, which

320

produced cytokines and chemokines that stimulate the development and survival of

321

tumor cells.12 Furthermore, several cytokines, chemokines and tumor growth factors

322

secreted by inflammatory cells and tumor cells exerts critical pro-tumorigenic effects

323

to support the growth of colorectal cancer cells.58 There are increasing experimental

324

evidence and clinical data showing that carcinoma cells, immunosuppressive cells and



325

immune effector cells within the tumor microenvironment control antitumor immuni-

326

ty and immune escape.59-61

327

In order to confirm our visual observations, we carried out a series of quantita-

328

tive analyses in the study. TPS was found to remarkably alleviate DSS-induced exten-

329

sive infiltration of inflammatory cells as determined by HE and immunofluorescence

330

staining (Figure 4E and 5). A significant decline in the number of macrophages

331

(CD68+), neutrophils (Gr-1+) and B lymphocytes (CD20+) infiltration into the tumor

332

sites was observed in the TPS-treated groups (Figure 5). Furthermore, ELISA analysis

333

showed that TPS effectively reduced the levels of pro-inflammatory cytokines, IL-2,

334

IL-6, INF-γ and TNFα in the distal colon (Table 2). It was noteworthy that TPS in-

335

creased the production of anti-inflammatory cytokine, IL-10.

336

We further explored the molecular mechanism underlying the anti-tumor activity

337

of TPS. In this experiment, we examined the effect of TPS on IL-6/STAT3 pathway

338

under inflammatory conditions in vivo and in vitro. STAT3 is in the intersection of

339

some distinct but cross-linked pathways, and has appeared as a potential target for

340

tumor immunotherapy.62 The aberrantly activation of STAT3 was reported to promote

341

further release of pro-inflammatory cytokines, formed the vicious cycle of cytokine

342

crosstalk, and accelerate the growth of malignant tumors.63 In vivo results confirmed

343

that TPS inhibited the activation of STAT3 and the levels of downstream proteins’ ex-

344

pression (Figure 7). In order to verify our speculation, we established the non-contact

345

co-cultured cell system in vitro (CT26 cells exposed to the conditioned culture media

346

from IL-6-activated RAW264.7). The CCK8 assay and western blotting analysis in-


Page 24 of 38

Page 25 of 38


347

dicated TPS significantly inhibited the proliferation of CT26 cells in the co-cultured

348

cell system (Figure 1). Further, TPS reduced the activation of STAT3 and the expres-

349

sion of STAT3 downstream proteins, such as MMP2, cyclin-Dl, survivin and VEGF

350

(Figure 2).

351

Taken together, this study has shown that TPS inhibited the progression of CAC

352

and regulated the biological behaviors of CT26 cells via inhibiting STAT3 activation.

353

Therefore, TPS might be a promising tumor chemopreventive agent for treating in-

354

flammation-associated colon cancer in the future.

355

Funding Sources

356 357

The Excellent Youth Foundation of National Natural Science Foundation of China（No.31422042）

358

Key Program of National Natural Science Foundation of China (No.31130041)

359

National Natural Science Foundation of China (No: 31560438)

360

Natural Science Foundation of Jiangxi Province, China (20161BAB214163)

361 362

363

Notes The authors declare that there are no conflicts of interest.

Acknowledgements

364

The financial support for this study by National Key Technology R& D Program

365

of China, Key Program of National Natural Science Foundation of China, the Pro-

366

gram for New Century Excellent Talents in University and Research Project of State

367

Key Laboratory of Food Science and Technology, is gratefully acknowledged.



Page 26 of 38

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541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561

56. Hu, J. L.; Nie, S. P.; Xie, M. Y., High pressure homogenization increases antioxidant capacity and

562

Figure captions

563

Figure 1. TPS inhibited the cell proliferation and invasion ability of CT26 cells

564

cultured in the conditioned medium from recombinant IL-6-activated RAW

565

264.7 cells.

566

(A) CCK8 assays of CT26 cells were performed to evaluate the bioactivity of TPS on

567

the cell proliferation. (B) Transwell migration assays were performed to evaluate the

568

effect of TPS on the cell invasion ability. (C) Quantification of volume of the

569

Transwell migration assay.

570

experiments performed. *P

Tea Polysaccharides Inhibit Colitis-Associated Colorectal Cancer via

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